AU627150B2 - Novel proteins with factor viii activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them - Google Patents

Description

AU-AI-1809 7 /88 pC-Ur WORLD INTIELLECTUAL PROPERTY ORiA NI ION INTERNATIONAL APPLICATION PULSuDUDReau C OPERTIk,!R Y(I (51) International Patent Classifitation 4 C12N 15/00, A6fK 37/02 C07K 13/00, Cl2P 21/02 C12N 1/00 (11) International Publication Number: Al (43) International Publication Date: 151 WO088/ 09813 )ecember 1988 (15.12.88) (21) International Application Number: (22) International Filing Date: 13 (31) Priority Application Number: (32) Priority Date: 12 PCT/NL88/00028 June 1988 (13.06.88) 87201121.8 June 1987 (12.06.8?)

NL

(74) Agents: HUYGENS, Arthur, Victor et al.; Gist-Brocades Patents and Trade Marks Department, Wateringseweg 1, P.O. Box 1, NL-2600 MA Delft

(NL).

(81) Designated States: AU, BB, BG, BJ (QAPI patent), BR, CF (QAPI patent), CG (QAPI patent), CM (OAPI patent), DK, Fl, GA (OAPI patent), HU, JP, KP, KR, LK, MC, MG, ML (OAPI patent), MR (QAPI patent), MW, NO, RO, SD, SN (QAPI patent), SU, TD (QAPI patent), TG (QAPI patent), US.

(33) Priority Country: (71) Applicant (for all designated States except US): GIST- RROGAPB-gN. [NL/NLI, Wateriigeweg-I--ft0- Box 1, N-L 2600 MA-Dzlf (NL).

(72) Inventors; and Inventors/Applicants (for US only) VAN OOYEN, Albert, Johannes, Joseph [NL/NL]; Laan van Heldenburg 23, NL-2271 AR Voorburg PANNEKO- EK, Hans [NL/NL]; Centraal Laboratorium van de Bloedtransfusiedienst, Plesmanlaan 125, NL-1066 CX Amsterdam VERBEET, Martinus, Philippus [NL/NL]; Centraal Laboratorium van de Bloedtransfusiedienst, Plesmanlaan 125, NL-1066 CK Amsterdam (NLQ. VAN LEEN, Robert, Willem [NL/NL]; Heeskesacker 23-23, NL-6546 JW Nijmegen (NT~.

T(lL cir-Aaa,. tai& lo66 C4. Q4.eA+no,,, -e-J~ Published With international search report.

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AUSTRALIAN

4 JAN 1989 PATENT OFFICE IL54) Title: NOVEL PROTEINS WITH FACTOR VIII ACTIVITY: PROCESS FOR THEIR PREPARATION USING GEN ETICALLY-ENGINEERED CELLS AND PHARMACEUTICAL COMPOSITIC'-NS CONTAINING

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2332 ainino ocift I-GACACcNAMuCIA lu 3 pro ar Va al@ 1577 acids Ittl amin~o acids (57) Abstract Novel polypeptides having Factor ViII activityare provided as well as compositions and methods for their preparation. The polypeptides comprise derivatives and fragments of Factor Vill and have sequences substantially similar to por tions of natu rrly occuring Factor ViII. The polypeptides find use in treatment of Hemophilia A.

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p -4 S88/03 PCT/NL88/00028 W. 88/09813 NOVEL PROTEINS WITH FACTOR VIII ACTIVITY: PROCESS FOR THEIR PREPARATION USI'N4G GENETICALLY-ENGINEERED CELLS AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

INTRODUCTION

Technical Field The invention relates to novel proteins having Factor VIII activity and methods for their preparation using genetically engineered cell-lines and microorganisms.

Background Hemophilia A is a sex-linked bleeding disorder characterized by a deficiency in Factor VIII, an essential element in the blood coagulation cascade. The disease occurs in about 0.01% of the male population. Hemophilia A can be treated by administering Factor VIII-containing blood plasma obtained from healthy donors. This treatment has several disadvantages however. The supply of Factor VIII is limited and very expensive; the concentration of Factor VIII in blood is only about 100 ng/ml and the yields using current plasma a fractionation methods are low. Since the source of Factor VIII is pooled donor blood, the recipient runs a high risk of acquiring various infectious diseases, including those caused by hepatitis non-A, non-B, hepatitis B or AIDS viruses which may be present in the donor blood. In addition, recipients may develop antibodies against the exogenous Factor VIII, which can greatly reduce its effectiveness.

IIf 1 Z8 PCT/NL88/G00 WO 88/09813 2- Factor VIII comprises three regions, an N-terminal region, the so-ca led "AlK2-domain"; a central region, the socalled "B domain"; and a C-terminal region comprising the A3, Ci and C2 domains. The A1A2-domain and the C-terminal region are believed essential for clotting activity. Factor VIII circulates in the blood combined with a protein, the von Willebrand factor (vUf), which is believed to protect the sensitive Factor VIII against early degradation.

Factor VIII is obtained in unsatisfactorily low yields when produced by known recombinant DNA processes.

Moreover the proteins appear not to be present as an intact chain, hence products are difficult to isolate and to purify and consequently the costs are high. It is therefore desirable to develop an efficient way to produce large quantities of compounds having Factor VIII activity, the compounds preferably having decreased immunogenic activity.

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ii Relevant Literature Molecular cloning of Factor VIII cDNA obtained from human mRNA and the subsequent production of proteins with Factor VIII activity in mammalian, yeast and bacterial cells has been reported. See WO 85/01961, EP 0160457, EP 0150735, EP 0253455. A method for producing proteins with Factor VIII activity using transformed microorganisms is disclosed in EP 0253455. European patent applications EP 0150735 and EP 0123945 and Brinkhous et al. (1985) disclose Factor VIII activity in proteolytic cleavage products of Factor VIII. A complex of two proteolytic cleavage products of Factor VIII, a 92 kDa and an 80 kDa polypeptide exhibits enhanced Factor VIII activity. (Fay et al., Biochem. Biophys. Acta (1986) 871:268-., 278; Faton et al Biochemistry (1986) 25:505-512) .V Eaton et al., Biochemistry (1986) 25:8343-8347 disclose that a polypeptide in which 766 amino acids (797- 1562) have been deleted from the central region retains Factor KVIII activity. Moreover, mammalian cells transformed with a i i i 4

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a ii i/ WO 88/09813 PCTINL88/00028 I r? y Li i' i 16 amplification will be employed.

S' o88/09813 PCT/NL88/00028 -3vector comprising DNA encoding this deletion polypeptide had a higher production level than cells transformed with a vector comprising DNA encoding the full length polypeptide.

PCT application WO 86/06101 discloses that recombinant Factor VIII proteins with deletions of up to 880 amino acids in the central region exhibit Factor VIII activity. The largest deletion stretches from Thr-760 through Asn-1639 (numbering according to Figure The host cells for preparation of the recombinant Factor VIII included mammalian cells, including Chinese hamster ovary cells.

SUMMARY OF THE INVENTION i Novel compositions, together with expression vectors and methods for their preparation are provided, which comprise derivatives and fragments of Factor VIII. The compositions are prepared by transforming a host cell, preferably a mammalian cell, with an expression vector comprising a DNA sequence encoding a Factor VIII congener, growing the transformed host cell to express the exogenous DNA sequence, and. recovering the resultant Factor VIII derivative or fragment from a cell lysate or from conditioned growth medium. The compositions may have enhanced Factor VIII activity and/or decreased immunogenicity. Uses of the compositions include treatment of Hemophilia A.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the Factor VIII cDNA insert of pCLI389 including the amino acid sequence Ala-1 through Tyr- 2332 of Factor VIII and its signal sequence M(-19) through with the correspondin sequence of nucleotides. F-973 is encoded by the codon TTC.

Figure 2 shows the structure of expression vector pCLB201 encoding the full-length Factor VIII protein.

3- .i:y WO 88/09813 PCT/NL88/00028 y 4 The circular plasmid pCLB201 is shown beginning with the EcoRI position within the 4217 bp HindIII-BglII fragment derived from plasmid pSV2 (Gorman, 1985 DNA Cloning (Ed. Glover) IRL Press, pp. 143-169; Mulligan and Berg, Proc. Natl. Acad. Sci.

USA (1981) 78:2072). The early SV40-promoter region is indicated: SVep. The vector also contains a NdeI-HindIII fragment derived from plasmid pRSV-neo (Gorman, 1985, supra) bearing the Rous Sarcoma Virus-Long Terminal Repeat (LTR) inserted into a SalI site. The full-length Factor VIII is encoded by a SalI-HpaI fragment of 7440 bp and indicated: Factor VIII cDNA (H HindIII, Sal SalI). The restriction endonuclease cleavage sites lost during construction of pCLB201 are enclosed in parenthesis. The size of the plasmid is given in kilobase pairs (kb).

Figure 3 shows vectors for transient expression of deletion mutant Factor VIII proteins: a. The landmarks of the pSV2-derived vector: two tandemly situated promoters: the SV40 early transcription promoter (SVep) and the Rous Sarcoma Virus-Long Terminal Repeat (RSV-LTR); the capping site (Capsite) and 5' end of the messenger RNA (mRNA); the cDNA insert bearing the full-length Factor VIII coding region with the start codon (ATG), the open IK Jreading frame and the stop codon (TGA); the 3' non-coding region of the mRNA with a short intron and the polyadenylation signal (polyA) derived from SV40 DNA (compare to Figure 2).

b. The 7440 bp (base pairs) fragment SalI- ,j HpaI bearing the full-length Factor VIII coding cDNA is depicted. The start and stop codons of the reading frame are indicated. Restriction endonuclease sites within the fulllength cDNA involved in the mutagenesis for the construction of pCLB202 and pCLB203 are shown.

c. Map of the Factor VIII protein. The 19 amino acid long signal peptide and the domain or repeat structure of plasma Factor VIII are shown. Al, A2 and A3 are homologous amino acid sequences. B is a unique region, whereas Cl and C2 are again homologous (Vehar et al., 1984). The amino acid positions bordering the A and C repeats are given. Below

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r Wp 88/09813 PCT/NL88/00028 The circular plasmid pCLB201 is shown beginning with the EcoRI position within the 4217 bp HindIII-BglII fragment derived from plasmid pSV2 (Gorman, 1985 DNA Cloning (Ed. Glover) IRL Press, pp. 143-169; Mulligan and Berg, Proc. Natl. Acad. Sci.

USA (1981) 78:2072). The early SV40-promoter region is indicated: SVep. The vector also contains a NdeI-HindIII fragment derived from plasmid pRSV-neo (Gorman, 1985) bearing the Rous Sarcoma Virus-Long Terminal Repeat (LTR) inserted into a SalI site. The full-length Factor VIII is encoded by a SalI-HpaI fragment of 7440 bp and indicated: Factor VIII cDNA (H HindIII, Sal Sal). The restriction endonuclease cleavage sites lost during construction of pCLB201 are enclosed in parenthesis. The size of the plasmid is given in kilobase pairs (kb).

Figure 3 shows vectors for transient expression of deletion mutant Factor VIII proteins: a. The landmarks of the pSV2-derived vector: two tandemly situated promoters: the SV40 early transcription promoter (SVep) and the Rous Sarcoma Virus-Long Terminal Repeat (RSV-LTR); the capping site (Capsite) and 5' end of the messenger RNA (mRNA); the cDNA insert bearing the full-length Factor VIII coding region with the start codon (ATG), the open reading frame and the stop codon (TGA); the 3' non-coding region of the mRNA with a short intron and the polyadenylation signal (polyA) derived from SV40 DNA (compare to Figure 1).

b. T e 7440 bp (base pairs) fragment Sall- I HpaI bearing the full-length Factor VIII coding cDNA is depicted. The start and stop codons of the reading frame are indicated. Restriction endonuclease sites within the fulllength cDNA involved in the mutagenesis for the construction of pCLB202 and pCLB203 are shown.

c. Map of the Factor VIII protein. The 19 amino acid long signal peptide and the domain or repeat structure of plasma Factor VIII are shown. Al, A2 and A3 are homologous amino acid sequences. B is a unique region, whereas Cl and C2 are again homologous (Vehar et al., 1984). The amino acid positions bordering the A and C repeats are given. Below S1 WO 88/09813 .PCT/NL88/00028

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6 1 L the map the cleavage sites of the proteolytic enzymes, that process Factor VIII, activated protein C (APC), thrombin activated coagulation factor X and a trypsin-like protease (indicated with are indicated by arrows. Factor Xa is thought to cleave the IIa- and APC-cleavage sites also shown (Eaton et al., Biochemistry (1986) 25:8343-8347; Fay et al., Biochem. Biophys. Acta (1986) 871:268-278). Their cleavage sites are given. The B region contains multiple cleavage sites.

d. The subunit structure of activated Factor VIII.

Factor VIIIa subunits of 92 kDa and 80 kDa are indicated as 92k and 80k, respectively. Their amino terminal and carboxy terminal amino acid positions within the full-length sequence are indicated.

e. The structure of the cDNA and protein of pCLB202 containing a direct fusion between the PstI and BamHI site indicated in b. The resulting protein is indicated containing a peptide bond between Ala-867 and Asp-1563. The total length of the protein is 1637 amino acids. (The landmarks of the repeat border positions and specific proteolytic cleavage sites are as depicted; see above.) f. The structure of the cDNA and protein pCLB203 containing a MluI-linker sequence coding for four extra amino acids that bridge the MaeIIIsite and HgiAT site, as indicated under b. The total length of the protein is 1411 amino acids.

(.The landmarks of the repeat border positions and specific proteolytic cleavage sites are depicted; see above.) Figure 4 shows the results of a molecular weight K (size) determination of several deletion mutant Factor VIII proteins using immunoprecipitation and electrophoresis.

The autoradiograph shows the lanes for the proteins made with the vectors pCLB202 (lane 2) and pCLB203 (lane The molecular weight markers are as indicated.

a 1 mm S WO 88/0 9813 PCT/NL88/00028 -7 4 S' DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, novel DNA constructs and novel compositions comprising host cells producing polypeptides having Factor VIII activity are provided. The polypeptides having Factor VIII activity include deletion mutant proteins of Factor VIII in which substantially all of the central region or "B domain" as well as a portion i of the 92 kilo-Dalton (kDa) region has been deleted. Plasmid constructs comprising a DNA sequence encoding deletion polypeptides having Factor VIII activity are used to transform a host cell. The host cell is then grown to express the gene.

The host cell may be either a eukaryotic or a prokaryotic cell.

Human Factor VIII has the sequence shown in Figure 1. Single letter abbreviations for the amino acids are used, and have the following meaning: A alanine; R arginine; N asparagine; D aspartic acid; C cysteine; Q glutamine; E glutamic acid; G glycine; H histidine; I isoleucine; L leucine; K lysine; M methionine; F phenylalanine; P proline; S =.serine; T threonine; W tryptophan; Y tyrosine; and V valine. The numbering of the amino acid sequence starts with A-i, the first amino acid after the 19' amino acid signal sequence. The last amino acid of Factor VIII is Y-2332. This numbering is used throughout the specification. Factor VIII-like materials, including Factor VIII fragments, mutants of the polypeptide as well as fusion peptides comprising functional portions of Factor VIII having the biological activity of the intact factor VIII, including blood clotting activity are also provided.

The polypeptides of this invention include congeners of Factor VIII, namely compounds having at least one biological activity of Factor VIII and having at least one amino acid sequence having substantially the same amino acid sequence,as Factor VIII. The congener may be of greater or lesser amino acid sequence than Factor VIII. Biological activity includes the ability, when administered to patients with Hemophilia A, to \i WO 88/09813 PCT/NL88/0028 I ii a correct the clotting defect and/or immunological crossreactivity with naturally occuring Factor VIII. The correction thus may be effected either by direct action in the clotting cascade or by binding to antibodies against Factor VIII so that the biological activity of subsequently administered Factor VIII is less affected. By "immunological crossreactivity" is meant that an antibody induced by a novel polypeptide of this invention will cross-react with intact Factor VIII. The polypeptide will have at least one biologically active sequence, for example immunological or epitopic, and may have more than one biologically active sequence, where such sequence may compete with a naturally occuring product for the biological property.

Novel polypeptides of interest will for the most part have a formula comprising an N-terminal region, NR, a linking region,.L R and a C-terminal region, CR. N R is characterized as having an amino acid sequence substantially corresponding to a consecutive sequence found in amino acid sequence A-1 through R-740 of the full-length Factor VIII (the "A1A2 domain" or 92kDa polypeptide), wherein no more than 45%, i usually no more than 20%, preferably no more than 10%, most preferably no more than 5% of the amino acids in the AlA2i domain have been deleted. Preferred sequences correspond substantially to the sequences A-l through D-712 or A-1 through R-740.

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1 may be a sl ort linking group- of from 1 to amino acids, a bond or any sequence of amino acids, usually not substantially similar to the sequence of the "B domaih" of the full-length Factor VIII protein. It may also comprise sequences corresponding substantially to consecutive sequences in the B domain up to the complete sequence S-741 to S-1637, or any portion thereof. Of particular interest are compositions wherein L R comprises at least one of the sequences S-741 through A-867 and D-1563 through S-1637.

CR is characterized as having an amino acid sequence subsrantially similar to a consecutive sequence found in the sequence of Factor VIII which includes anino acids Gin- 1638 through Tyr-2332, wherein no more than 25%, usually no more than 200, preferably no more than 10% of the amino s

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1' WO 88/09813 PCT/NL88/00028 -9 Q-1633 through Y-2332 have been deleted. The sequence preferably includes sequences corresponding substantially to the Factor VIII sequences Q-1638 through Y-2332, E-1649 through Y-2332, S-1669 through Y-2332 and S-1690 through Y- 2332, more preferably Q-1638 through Y-2332.

The polypeptides of interest may be fragments of Factor VIII wherein up to 75% of the amino acids have been deleted from the full length Factor VIII, or fusion proteins wherein the 92 kDa protein or a fragment thereof is fused to the 80kDa polypeptide or a fragment thereof. The polypeptide usually will have no more than about 75% of the amino acids deleted, usually no more than about 45% of the amino acids deleted, more usually no more than about 40% of the amino acids deleted.

To provide for immunogenicicity, the Factor VIII congeners can be joined covalently to a large immunogenic polypeptide entity. Exemplary of such immunogenic entities are bovine serum albumin, keyhole limpet hemocyanin (KLH) and the like.

These conjugated polypeptides will be useful for inducing antibodies in an appropriate host organism. The antibodies can be used to determine the presence or absence and/or i concentration of Factor VIII in a bodily fluid, the absence of which may indicate Hemophilia A. Preparation of Congeners of Factor VIII Factor VIII congeners may be obtained in a variety of ways. They may be obtained by proteolytic cleavage of the full-length Factor VIII into three regions, preferably by cleavage before Arg-740 and Gln-1638 followed by truncating the amino or carboxyl terminus of the Ala-1 through Pro-739 sequence at least one amino acid at a time and/or truncating the amino or carboxyl terminus of the sequence Gln-Z,638 through Tyr-2332 at least one amino acid at a time. The polypeptide fragments obtained may then be fused directly or via a central linking group, or the fragments may be combined in a composition to provide for Factor VIII activity.

Factor VIII congeners, including fusion proteins in which the N R and CR are fused, can also be prepared by recombinant DtA techniques. Techniques used in isolating the i i. i.r o- WO 88/09813 PCT/NL88/00928 i 10 Factor VIII gene are known in the art, including synthesis, isolation from genomic DNA, preparation from cDNA, or, combinations thereof. The various techniques for manipulation of the genes are well known, and include restriction, digestion, resection, ligation, in vitro mutagenesis, primer repair, and poly linkers and adapters, and the like. See Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.

Generally the method comprises preparing a genomic library from cells which synthesize Factor VIII. Tc enhance the likelihood of identifying the correct sequence, a cDNA library from cells which do not produce Factor VIII may be used to cross-hybridize. An assay for the expression of Factor VIII using restriction fragments inserted into a prokaryotic expression vector such as pTZ 18 or 19 and screening with antibodies for Factor VIII to detect a cross-reactive peptide fragment or the like can be used.

Once a complete gene has been identified, either as cDNA or chromosomal DNA, the desired deletions in the structural gene can be made in several ways. Deletions may be made by enzymatically cutting the full length Factor VIII cDNA followed by modification and ligation of the purified fragments or by site-directed mutagenesis, especially by loopout mutagenesis as described by Kramer et al., Nucl. Acids Res. (1984) 12:9441-9456. The gene thus obtained may then be manipulated in a variety of ways well known in the art to provide for expression.

Both prokaryotic and eukaryotic hosts may be employed, which may include bacteria, yeast, mammalian cells, for example, CHO cells, C127 cells, human "293" cells, myeloma cells, or specialized cells such as liver cells, and COS cells.

Therefore where the gene is to be expressed in a host which recognizes the wild-type transcriptional and translational regulatory regions of Factor VIII, the entire gene with its wild-type and 3'-regulatory regions may be introduced into an appropriate expression vector. Various expression vectors exist employing replication systems from mammalian viruses, a r i!; i iij i it Iii F t i.:ci1; ;t ~1 1 i :i i i I

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i :ii i i! i r -r i WO 88/09813 PCT/NL88/00028 i 11 such as simian virus 40, Epstein-Barr virus, bovine papalloma virus, vaccinia virus, etc.

Where the gene is to be expressed in a host which does not recognize the naturally occurring wild-type transcriptional and translational regulatory regions, further manipulation will be required. Conveniently a variety of 3'transcriptional regulatory regions are known and may be inserted downstream from the stop codons. The non-coding region upstream form the structural gene may be removed by endonuclease restriction, Bal31 resection, or the like.

Alternatively, where a convenient restriction site is present near the 5' terminus of the structural gene, the structural gene may be restricted and an adapter employed for linking the structural gene to the promoter region, where the adapter provides for the lost nucleotides of the structural gene.

Various strategies may be employed for providing a foreign expression cassette, which in the 5'-3'-direction of transcription has a transcriptional regulatory region and a translational initiation region, which may also include regulatory sequences allowing for the induction of regulation; an open reading frame encoding the full length Factor VIII or a congener of Factor VIII, including the deletion mutant proteins, desirably including a secretory leader sequence recognized by the proposed host cell; and translational and transcriptional termination regions. The expression cassette may additionally include at least one marker gene. The initiation and termination regions are functional in the host cell, and may be either homologous (derived from the original host), or heterologous (derived from a foreign source) or synthetic DNA sequences. The expression cassette thus may be wholly or partially derived from natural sources, and either wholly or partially derived from sources homologous to the host cell, or heterologous to the host cell. The various DNA constructs (DNA sequences, vectors, plasmids, expression cassettes) of the invention are isolated and/or purified, or synthesized and thus are not "naturally occurring".

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4 ,i 1 r WO 88/09813 PCT/NL88/00928 12 For optimal gene expression, the nucleotide sequences surrounding the translational initiation codon ATG have been found to be important in animal cells. For example, Kozak, Microbiol. Reviews (1983) 47:1-45, has studied extensively the effect of these regions on the expression of polypeptides such as insulin in COS cells. Thus it may be necessary to modify the nucleotide sequences surrounding the initiation codon. This can bed done by site-directed mutagenesis or by fusing the exogenous gene to the initiation region of a highly expressed gene.

Illustrative transcriptional regulatory regions or promoters include, for bacteria, the beta-gal promoter, amylase promoter, lambda left and right promoters, trp and lac promoters, trp-lac fusion promoter, etc.; for yeast, glycolytic enzyme promoters, such as ADH-1 -2 promoters, PGK promoter, and lactase promoter and the like; for mammalian cells, SV40 early and later promoters, cytomegalovirus (CMV) promoter, beta-actine promoter, adenovirus major late promoters and the like.

In eukaryotic cells, regulatory sequences can include, for example, the cytomegalovirus enhancer sequence which can be fused to a promoter sequence such as the SV40 promoter, forming a chimeric promoter, or inserted elsewhere in the expression cassette, preferably in close proximity to the promoter sequence.

Expression of the structural gene can also be amplified by, for example, ligating in tandem a gene for a dominant amplifiable genetic marker 5' or 3' to the structural gene and growing the host cells under selective conditions. An example of an amplifiable gene is the gene for dihydrofolate reductase (dhfr), the expression of which may be increased in cells rendered resistant to methotrexate (mtx), a folate antagonist.

Of interest for expression in mammaliaci cells such as COS cells are expression cassettes capable of expressing Factor VIII or congeners thereof which enploy a metallothionein promoter, or the SV40 early transcription unit transcription promoter (SVep), particularly the Rous Sarcoma Virus-Long Terminal Repeat (RSV-LTR) promoter in tandem with the SVep ;ii I il~n o

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0 13l WO 88/09813: PCT/NL8/00028 promoter. Examples of promoters and promoter/enhancer combinations which can be used in C127 cells in combinaticn i with expression vectors according to example 7 has been described in e.g. Hurwitz et al., Nucl. Acids Res. (1987) 15:7137-7153. For optimal expression the introduction of an intron into the expression cassette, may be beneficial. An intron in the 5'-part of the mRNA is preferred.

In addition, a fused gene may be prepared by providing a 5'-sequence to the structural gene which encodes a secretory leader and a processing signal. For secretion of Factor VIII polypeptides the naturally occurring Factor VIII leader sequence is preferably used, but other signal sequences including secretory leaders of penicillinase, alfa-factor, immunoglobulin, T-cell receptors, outer membrane proteins, serum albumin, tissue plasminogen activator, insulin, digestive tract enzymes, and the like may also be used. By fusion of a secretory leader in proper reading frame with a structural gene for Factor VIII or a congener therof, the mature Factor VIII may be secreted into the culture medium.

The termination region may be derived from the 3' region of the gene from which the initiation region :s :as obtained or from a different gene. A large number of termination regions are known and have been found to be satisfactory in a variety of hosts from the same and different genera arid species. The termination region may include sequences for the proper processing of the mRNA in a mammalian cell: a small intron and a polyadenylation signal.

During the construction of the expression cassette, the various DNA fragments will usually be cloned in an appropriate cloning vector, which allows for expansion of the DNA, modification of the DNA or manipulation by joining or removing of the sequences, linkers, or the like. Normally, the vectors will be capable of replication in at least a relatively high copy number in E. coli. A number of vectors are readily available for cloning, including such vectors as pBR322, pML2, pUC7-pUC19, pSP64, pSP65, pSPI8, pSP19, pTZi8 and pTZl8R, pTZ19 and pTZl9R and the like.

t: f GACCCACCSTGCCAA lug 2332 1411 omanw oods (57) Abstract Novel polypeptides having Factor VIII activity are provided as well as compositions and methods for their preparation. The polypeptides comprise derivatives and fragments of Factor VIII and have sequences substantially similar to por tions of naurclly occuring Factor VIII. The polypeptides find use in treatment of Hemophilia A.

WO 88/09813 i t PCT/NL88/00028. 14: The cloning vectors are characterized as having an efficient origin of replication at least functional in E.

coli. Also the cloning vector will have at least one unique restriction site, usually a plurality of unique restriction sites and may also include multiple restriction sites, particularly two of the same restriction sites for substitution. In addition, the cloning vector will have one or more markers which provide for selection for transformation.

The markers will normally provide for resistance to cytotoxic agents such as antibiotics,'heavy metals, toxins or the like, complementation of an auxotrophic host, or immunity to a phage. By appropriate restriction of the vector and cassette, and, as appropriate, modification of the ends, by chewing back or filling in overhangs, to provide for blunt ends, by addition linkers, by tailing, complementary ends can be provided for "igation, and joining of the vector to the i expression cassette or a component thereof.

In some instances, a shuttle vector will be employed where the vector is capable of replication in different hosts requiring different replication systems.

Preferably a simian virus 40 (SV40) origin of replication is employed which allows the plasmid to be propagated in COS-1, while the Bovine Papi2loma Virus (BPV-1) genome is used for the episomal maintenance of expression vectors in C127 cells (see for example Howley et al., Meth. Enzymol. 101: 387-402, 1983).

The expression cassette may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome. The manner of transformation of the host organism with the various DNA constructs is not critical to this invention. The DNA may be introduced into the host in accordance with known techniques, such as transformation, using calcium phosphate precipitated DNA, electroporation, transfection by contacting the cells with a virus, microinjec ion of the DNA into cells or the like.

i il may be present in the donor blood. In addition, recipients may develop antibodies against the exogenous Factor VIII, which can greatly reduce its effectiveness.

1 ^l :I: i IWO 088/09813 PCT/NL88/00028 15 As a host organism, normal primary cells or cell lines derived from cultured primary tissue may be used, as well as microbial cells. The host cell is preferably a mammalian cell line, preferably hamster CHO cells, mouse C127 cells or human "293" cells, but microbial cells, for example yeast, preferably a Kluyveromyces species, or bacteria, preferably a Bacillus species, may also be used.

For stable transformation of a mammalian cell line with the expression vector bearing the Factor VIII congener coding sequence and subsequent amplification of the vector inserted in the host chromosome, Chinese hamster ovary cells (CHO) are especially suitable. Transformation involves transfection of the host cell with the expression vector together with a selectable marker gene such as dhfr or a G418 (neo-) resistance gene or the like, and subsequent integration into the chromosome and selection for stable transformation.

Another method for stable transformation for host cell lines uses expression vectors containing BPV-1 sequences.

C127 cells are well suited for this purpose. Transformation involves transfection of the host cell with the expression vector conta iing-BPV-1 sequences. Transformants are recognized by the formation of foci. Stable transformants are established and screened for Factor VIII activity using the Kabi Coatest according to examples 6 and 7. In contrast, when the expression is carried out in a transient transformation system such as COS-1 cells with pSV2-derived expression vectors, there is no selection step. Once the structural gene has been introduced into the appropriate host, the host may be grown to express the structural gene. The host cell may be grown to high density in an appropriate medium.

Ihere the promoter is inducible, such as in a prokaryotic system, permissive conditions will then be employed, for example temperature change, exhaustion, or excess of a metabolic product or nutrient, or the like. In a mammali'n system, where an amplifiable gene is used in tandem with the structural gene, the appropriate means for c Eaton et al., Biochemistry (1986) 25:8343-8347 disclose that a polypeptide in which 766 amino acids (797- i 1562) have been deleted from the central region retains Factor I v VIII activity. Moreover, mammalian cells transformed with a .01{ I 1 S WO 88/09813 PCTNL88/00028 I -16 amplification will be employed.

Where secretion is provided for, the expression product, either fus&'3 or unfused, may be isolated from the growth medium by conventional means. Where secretion is not provided for, the host cells may be harvested and lysed in accordance with conventional methode. The desired product is then isolated and purified by conventional techniques, for example affinity chromatography with immobilized antibodies, chromatography on aminohexyl-sepharose or the mixed polyelectrolyte method.

The recombinant products may be glycosylated or non-glycosylated, having the wild-type or other glycosylation.

The amount of glycosylation will depend in part upon the sequence of the particular peptide, as well as the organism in which it is produced. Thus, expression of the product in E.

coli cells will result in an unglycosylated product, and expression of the product in mammalian cells will result in a product with glycosylation similar to the wild-type peptide.

Uses of Factor VII'I Congeners The subject compounds can be used in a wide i variety of ways, both in vivo and vitro. The subject compounds may be used as the active component of pharmaceutical preparations for treating patients exhibiting symptoms of Hemophilia A. By a pharmaceutical composition is meant any preparation to be administered to mammals. Thus, a pharmaceutical preparation with Factor VIII activity is a preparation which can be administered to a mammal to alleviate symptoms associated with Hemophilia A, the inability to properly clot blood. In preparing the pharmaceutical composition, generally the subject polypeptides are admixed with parenterally acceptable vehicles and other suitable excipients in accordance with procedures known in the art. The pharmaceutical preparations may conveniently be a sterile lyophilized preparation of the subject polypeptide which may o If I L encoded by the codorn TTC.

Figure 2 shows the structure of expression vector pCLB201 encoding the full-length Factor VIII protein.

I.

IMWimilliall :1 i W,I88/09813 PCT/NLF2/OOO28 -17 be reconstituted by addition of a sterile solution suitable for producing solutions, preferably isotonic with the blood of the recipient. The pharmaceutical preparation may be presented in single unit or multi-dose containers, for example in sealed ampoules or vial. Their use would be analogous to that of known human Factor VIII polypeptide, appropriately adjusted for potency. The subject polypeptide may be administered in vivo, for example by injection, intravenously, peritoneally, subcutaneously, or the like.

The subject compounds additionally can be used for making antibodies to the subject compounds, which may find use in vivo or in vitro. The antibodies can be prepared in conventional ways, either by using the subject polypeptide as an immunogen and injecting the polypeptide into a mammalian host, for example mouse, cow, goat, sheep, rabbit, etc., particularly with an adjuvant, for example complete Freunds adjuvant, aluminum hydroxide gel, or the like. The most may then be bled and the blood employed for isolation of polyclonal antibodies, or in the cases of the mouse, the peripheral blood lymphocytes or splJrnic lymphocytes (B cells) employed for fusion with an appropriate myeloma cell to immortalize the chromosomes for monoclonal expression of antibodies specific for the subject compounds.

Either polyclonal or monoclonal antibodies may be prepared, which may then be used for diagnosis or detection of the presence of the subject polypeptide in a sample, such as cells or a physiological fluid, for example blood. Failure to detect the subject polypeptide may indicate the condition of hemophilia A.

Probes comprising sequences complementary to Factor VIII mRNA may also be prepared and used as a diagnostic aid, for example, the presence and/or amount of Factor; VIII mRNA in a cell may be used for determining whether the patient is making Factor VIII but has developed anicbo es which Orevent its activity. A tesS sample comprising a cell, tissue sample or bodily fluid believed to contain hybridizing t r r i -1 i v f 1: 1 i1 jf ;i iii ii

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c -i amino acid long signal peptide and the domain or repeat structure of plasma Factor VIII are shown. Al, A2 and A3 are homologous amino acid sequences. B is a unique region, whereas b C1 and C2 are again homologous (Vehar et al., 1984). The amino acid positions bordering the A and C repeats are given. Below WO 88/09813 PCT/NL88/00,028 18 sequences can be treated so as to iyse any cells, then treated with a denaturing agent such as guanidine hydrochloride to release single-stranded mRNA. The probe labeled with, for example 32p or biotinylated nucleotides, can then be hybridized to the cellular mRNA to form a double-stranded complex which may be detected by means of the label. For some purposes it may be desirable to quantitate the amount of Factor VIII mRNA. This is done by comparing the amount of label detected in reference samples containing known amounts of single-stranded Factor VIII mRNA with the amount of label detected in the test sample.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

General cloning techniques were used as described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, CSH, NY, 1982. All DNAmodifying enzymes were obtained from commercial suppliers.

They were used according to manufacturers instructions.

Materials and apparatus for DNA purification and separation were used according to instructions from the supplier.

Example 1 Construction of Expression Vector PCLB201 pCLB201 is an expression vector comprising the RSV-LTR promoter in the right orientation for transcriptional initiation, and the full-length Factor VIII cDNA (see Figure It is derived from the pSV2-vector of Mulligan and Berg, Proc. Natl. Acad. Sci. USA (1981) 78:2072.

The unique HindIII site of the plasmid pSV2.tPA (Van Zonneveld et al., Proc. Natl. Acad. Sci. USA (1986) 83:4670-4674) was modified into a SalI site by making blunt the HindIII sticky-ends and ligating SalI-linkers i, i i 1 S WO 88/09813 PCT/NL88/00028 19 CGTCGACG-3') to the blunt ends. A plasmid containing a SalI site was selected and identified as pCLB91. This plasmid is the same as pSV2.tPA except 'that it contains a Sall linker inserted into the HindIII site.

The isolation of Factor VIII mRNA from human liver, and the preparation, purification and identification of its cDNA and its assembly in the plasmid pEP121 resulting inI plasmid pCLB89 have been described in patent application EP 0253455, which disclosure is hereby incorporated by reference. A 7440 bp long SalI-HpaI fragment from pCLB89 containing the factor VIII cDNA (see Figure 1) was purified and inserted into pCLB91 digested with Sail and BglII. The resulting expression vector pCLB200 contained an intact SalI site with the HpaI site ligated to the BglII site which had been filled in.

Plasmid pRSVneo (Gorman, DNA Cloning, 1985, ed., D. Glover, IRL-press, Washington, Oxford, pp. 143-169) was digested with NdeI and HindIII and incubated with the Klenow fragment of DNA po.ymerase to create blunt ends and the 0.6 kb I fragment containing a Rous Sarcoma Virus-Long Terminal Repeat (RSV-LTR) sequence was isolated and inserted into the Sall site of pCLB200 which had similarly been made blunt-ended to form expression vector pCLB201 (see Figure 2).

Example 2 Construction of pCLB202 A deletion mutant of Factor VIII having a deletion from the PstI site at nucleotide sequence position 2660 (see I Figure 3b) to the BamHI site at postition 4749 (see Figure 3b) was constructed in plasmid pGB860 as described in patent application EP 0253455. The Factor VII, DNA in pGB860 has a deletion of the DNA coding for 695 amino acids in the central region.

Expression vector pCLB202 was derived from pGB860 i and pCLB201. Both plasmids were digested with KpnI and Xbal restriction enzymes. The '3.1 kb KpnI-XbaI fragment in pCG860 was ligated to the 7 kb KpnI-XbaI fragment from pCLB201. The (lane The molecular weight markers are as indicated.

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_e WO 88/09813 PCT/NL88/00028 r 20 resulting plasmid pCLB202 has intact KpnI and XbaI sites. Its structure is depicted in Figure 3e.

Example 3 Construction of pCLB203 pCLBl00: The SalI-HpaI 7440 bp fragment (see Figure 2) from pCLB89 was inserted into plasmid pSP64 (Melton et al., Nucleic Acids Res. (1984) 12:7035-7056) which had been cleaved with SalI and SmaI. The resulting plasmid, pCLB100, contained an intact SalI-site and the HpaI-end linked to the SmaI-end.

pCLB101: (numbers in parentheses refer to Figure 1) Plasmid pCLB00 was digested with KpnI and TthlllI, generating a 631 bp fragment: KpnI (1817) to TthlllI (2447). The 631 bp fragment was purified, then out with MaeIII (2199). The MaeIJI sticky end was filled in resulting in a 383 bp fragment.

Plasmid pCLB100 was digested with Apal and BanI. The 669 bp Apal (6200)-BanI (5532) fragment was isolated.

Plasmid pCLB100 was digested with HgiAI, producing a HgiAI-fragment of 634 bp. The fragment incubated with T4 DMA polymerase to make blunt ends, then digested with BanI, to produce a 565 bp fragment.

Plasmid pCLB10O was digested with Apal and KpnI, generating a 5,9 kb long fragment (Apal (6200) to KpnI (1817)) containing vector sequences for maintenance and 30 propagation in E. coli.

The fragments obtained in steps through were purified and equimolar amounts of the fragments and a ten-fold molar excess of MluI-linker (CCACGCGTGG) were ligated, giving rise to plasmid pCLB101. Plasmid pCLBlO1 contained a MluI-linker between the MaeIII and HgiAI sites (see Figure 3b) in addition to KpnI, BanI and Apal sites. Four ii i a x 1 :i i: ,li Fli ii i i :i: i j i i l j jjli:I~d ii" iii i:.i io i, iS

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i of Factor VIII and having at least one amino acid sequence havinj substantially the same amino acid sequence as Factor VIII. The congener may be of greater or lesser amino acid sequence than Factor VIII. Biological activity includes the ability, when administered to patients with Hemophilia A, to 88/09813 PCT/NL88/00028 21 extra amino acids between amino acids D-712 and Q- 1638 (Figure 1) are shown in Figure 3f. pCLB203: pCLBl01 and pCLB201 were digested with enzymes KpnI and XbaI. The 2.4 kb KpnI-XbalI fragment from pCLB101 was ligated to the 7.0 kb KpnI-XbaI fragment derived from pCLB200.

The resulting plasmid pCLB203 possessed intact KpnI and XbaI sites. Its structure is depicted in Figure 3f.

Example 4 Construction of pCLB212 pCLB212 was constructed using the loop-out mutagenesis technique as described in Kramer et al., (1984) Nucleic Acids Res. 12:9441-9456. The deletion mutagenesis of the central region of Factor VIII cDNA was performed using the following oligonucleotide: Primer IV 3' TTC.CAA.AGA.TCA.ACA.CTG.GTT.TTG.GGT.GGT.CAG.AAC to produce in factor VIII cDNA internal deletion ,of the sequences coding for amino acids Lys 713 through Ser-1637.

The corresponding protein containis an internal I deletion of 925 amino acid residues compared with the full length Factor VIII protein.

To obtain a target fragment for loop-out mutagenesis a HindIII-PstI fragment of 1.4 kb derived from pCLB203 (Example 3) was selected. The nucleotide position (Figure 1) of the HindIII-site is at 1025 in the full length Factor VIII sequence upstream of the region to be modified, the PstI site is at position 5165 downstream of the region.

These sites are indicated in Figure 2. The 1i4 kb fragment was subcloned in M13mp9 followe by the loop-out mutagenesis.

in i i. L i WO 88/09813 PCT/NL PCL881000;8 22 After selecting the fragment with the precise deletion, this was followed by the insertion of the KpnI-PstI part of the HindIII-PstI fragment containing the desired deletion into an appropriate expression vector by preparing fragments with sticky, unique restriction enzyme ends (numbers refer to Figure according to the following steps: Step 1. For the expression of the mutant Factor VIII molecules a new plasmid pCLB211 was constructed. The SalI-HpaI fragment of 7440 bp (Figure 1) derived from pCLB89 was inserted into the expression vector pSVL (Pharmacia, No.

27-4509-01, Uppsala, Sweden) enabling transcription from a late SV40 promoter in mammalian cells. The plasmid pSVL was cleaved with XhoI and SmaI. The resulting plasmid pCLB211 contains an XhoI end linked to the SalI end, since both 5' protruding ends of these enzymes are identical, and the SmaI end linked to the HpaI end.

Step 2. Plasmid pCLB211 was digested with ApaI and Sail. The 1.4 kb ApaI(6200)-SalI(unique in pSVL-part of pCLB211) fragment was isolated.

Step 3. Plasmid pCLB100 was digested with NdeI, PstI and Apal. Two fragments were isolated: a PstI(5165)- NdeI(5527) fragment of 363 bp and second fragment, NdeI(5527)- ApaI(6200) of 674 bp.

Step 4. Plasmid pCLBlOwas digested with KpnI and SacI. A KpnI(1817)-SacI(19) of 1799 bp was isolated.

Step 5. Plasmid pCLB211 was digested with SalI and SacI. A 4.5 kb SalI(unique in the pSVL vector)-SacI(19) fragment was obtained.

Step 6. An M13-bacteriophage containing a HindII(1025)-PstI(5165) fragment with the desired deletion as verified with sequence analysis, was digested with KpnlU and PstI. A 584 bp PstI(5165)-KpnI(1819) fragment was isolated.

Step 7. The six isolated fragments from Steps 2 through 6 were mixed in equimolar amounts and ligated. A plasmid containing all six fragments was selected. The pas'id pCLB212 expressed the exemplary compound 3b (Table II). The ~fr 31 i i rPiY ii i I~ e ii a; s u ;i ii ji li i I i i 1 i i

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Example Construction of pCLB208, pCLJ3209 and pCLB2lO pCLB2O8, pCLB2O9 and pCLB2lO were contructed using the loop-out mutaigenesis technique as described in Kramer et al., Nucleic Acids Res. (1984) 12:9441-9456.

Loop out mutagenesis Deletion mutagenesis of the central region of Factor VIII cDNA was performed using the following oligonucleotides: Primer I: 3' TTA.CGG.TAA.CTT.GGT.TCT.CTT.TAT.TGA.GCA.TGA.TGA Primer II: 3' TTA.CGG.TAA.CTT.GGT.TCT.AGT.CAA.CTT.TAC.TTC.TTC Primer III: 3' TTA.CGG.TAA.CTT.GGTL.TCT.TCG.AAA.GTT.-LTC.TTTL.TGT to produce internal deletions in Factor VIII cDN$A of the sequences coding for amino acids S-741 through R-1648 (Primer S-741 through 1-1668 (Primer II) and S-741 tnrough R-16890 (Primer III). The corresponding proteins contain int-rnal deletions of 908 928 (11) and 949 (111) amino acid residues, respectively.

B. Preparation of Target Fragments To obtain a target fragment for loop-out mutagenesis a fragment of 0.8 kb derived from pCLBlO1 was

J-

Lecuyn.zes Tne wiia-type transcriptiona± ana trnsiaionai regulatory regions of Factor VIII, the entire gene with its wild-type and 3'-regulatory regions may be introduced into an appropriate expressionvetor arious expresi veto.Vaiusssion vectors exist employing replication systems from mammalian viruses, ~i WO 88/09813 PCT/NL88/00028 24 constructed as follows.

Plasmid pCLB101 was digested with EcoRI, the EcoRI sites were filled in and a further digestion with KpnI followed. A 479 bp Kpnl(1819)-EcoRI(2297) fragment (the numbers of nucleotide positions are as described in Figure 1) was isolated. Plasmid pCLB101 was digested with BamHI; the sticky ends were filled in and a further digestion with PstI followed. A 416 bp BamHI(4749)-PstI(5165) fragment was isolated. These fragments were ligated in equimolar amounts and subcloned in M13mpl9 amber. The M13 bacteriophage containing a KpnI-PstI fragment of 895 bp from 1819 upstream from the region to be modified to 5165 downstream from that region and with a large deletion in the Factor VIII coding sequence was selected for the loop out mutagenesis. After selecting the fragment with the precise deletion obtained with Primer I, II, or III in bacteriophage M13, the KpnI-PstI fragment containing the desired deletion was inserted into an appropriate expression vector derived from pCLB211 (see Example 4) using the following steps and preparing fragments with sticky, unique restriction enzyme ends (numbers refer to Figure 1): Plasmid pCLB211 was digested with Apal and SalI. The 1.4 kb ApaI(6200)-SalI (unique in pSVL-part of pCLB211) fragment was isolated.

From plasmid pCLBO00 digested with NdeI, PstI and Apal a 363 bp PstI(5165)-NdeI(5527) fragment and a 674 bp NdeI(5527)-ApaI(6200) fragment were isolated.

Plasmid pCLB00 was digested with KpnI and SacI and a 1799 bp KpnI(1817)-SacI(19) fragment isolated.

Plasmid pCLB211 was digested with SalI and SacI. The 4.5 kb SalI(unique in the pSVL vector)-SacI(19) fragment was isolated.

The M13-bacteriophage containing the fragment with the desired mutation was digested with KpnI and PstI. The KpnI-PstI fragments containing the desired mutation were isolated for all three mutageneses.

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iP j host cell, or heterologous to the host cell. The various DNA constructs (DNA sequences, vectors, plasmids, expression cassettes) of the invention are isolated and/or purified, or synthesized and thus are not "naturally occurring".

WO 88/09813 PCT/NL8800028 The six fragments from to above were isolated, mixed in equimolar amounts and ligated. Plasmids i containing all of the desired fragments were selected by hybridization and restriction enzyme digestion. Using Primers I, II or III shown above, three new expression vectors were constructed for the expression of the exemplary compound described in Table I: 1. pCLB208 for compound 7 with Primer I;s 2. pCLB209 for compound 8 with Primer II; and io 3. pCLB210 for compound 9 with Primer ITT, Example 6 Transient Expression of Recombinant Factor VIII DNA and Assay of Produced Proteins A. Transfection of COS-1 Cells and metabolic labeling The expression vectors obtained according to the previous examples were introduced into COS-1 cells using a DEAE transfection technique. Vector DNA was precipitated by incubating with DEAE-dextran for 2 hrs, followed by treatment with chloroquine shock for 2 hrs, as described by Lopata et al., Nucleic Acids Res. 12, 5707, (1984) and Luthman and Magnusson, Nucleic Acids Res. (1983) 11:1295. The medium uses for growth of the COS-1 cells and also for the conditioned medium was Iscove's DMEM (Flow) supplemented with 10% (v/v) heat inactivated fetal calf serum (Flow). The medium was changed 48 hrs after transfection. The conditioned medium was collected 48 hrs later.

Metabolic labeling of the proteins in the I transfected cells was carried out using serum free RPMI-medium (Gibco). Two days after transfection transfectants were incubated for 4 hrs with 50 uCi/ml L- 3 5 S-methionine (Amersham, 1985; 370 MBeq/330 ul; specific activity: over 100 Ci/mMole), followed by an overnight incubation with 1 mM L-methionine before harvesting the conditioned medium. In order to suppress protein degradation, protease inhibitors such as phenyl AA: L or interest for expression in mammaliani ceils such as COS cells are expression cassettes capable of (xpressing Factor VIII or congeners thereof which einploy a metallothionein promoter, or the SV40 early transcription unit transcription promoter (SVp), particularly the Rous Sarcoma Virus-Long Terminal Repeat (RSV-LTR) promoter in tandem witth t SVep WO 88/09813 .PCT/NL8800028, II 26methar.e sulphonyl fluoride (PMSF) were added to the conditioned medium after harvesting. To inhibit protein J glycosylation, tunicamycin can be added to the conditioned medium (final concentration 0.001 mg/ml). Conditioned media were harvested 4 days after transfection; produced proteins were assayed.

B. Biological Activity of Recombinant Factor VIII The conditioned medium was assayed for Factor VIII activity using the standard coagulation or clotting assay and the chromogenic activity aF.say (Kabi Coatest).

The standard coagulation or clotting assay (socalled activated partial thromboplastin time) was performed using hemophilia plasma as described by Veldkamp et al., Thromb. Diath. Haemorrh. (1968) 19:279. The conditioned medium was citrated before examination.

The chromogenic activity or Kabi Coatest assay was performed according to procedures supplied by the manufacturer Kabi-Vitrum, except that all volumes prescribed were divided by four and 25 ul of conditioned medium was tested. The Factor VIII-like proteins were activated for 15 min, at which time the chromogenic substrate (S2222) was added.

Inhibition of Factor VIII activity was measured i according to the standard Bethesda protocol (Kasper et al., Thromb. Diath. Haemorrh. (1975) 34:869-871. The immunoglobulins used were purified by ion exchange and protein A Sepharose chromatography prior to use.

The standard for biological activity assays for Factor VIII was a pool of citrated plasma (0.5 mM final conc.) which was assumed to contain 1 U of Factor VIII activity or antigen per ml.

The biological'activity for various of the deletion proteins are shown in Table II.

Mutant proteins with deletions exhibited Factor VIII clotting activity.

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S WO 88/09813 PCT/NL88/00028.

27 In addition, the established activity of the recombinant protein solution appeared to be inhibited following addition of antibodies known to be inhibitors of plasma-derived Factor VIII activity and/or of so-called inhibitor-sera, obtained from patients with inhibitors, i.e., antibodies against Factor VIII, in their blood.

C. Immunologic Cross-reactivity with Factor VIII Preparation of monoclonal antibodies.

Balb/c mice were immunized with purified human Factor VIlI-von Willebrand factor complex obtained by agarose gel filtration of cryoprecipitate or a Factor VIII concentrate used for therapeutical purposes (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), Van Mourik and Mochtar, Biochim. Biophys. Acta 221:677-679 (1970). Lymphocyte hybridization was performed as described by Galfre et al., Nature (1977) 266:550-552.

Description of the techniques used for selection of clones producing monoclonal antibodies to Factor VIII has been provided elsewhere (Stel, 1984, Ph.D. thesis, University of Amsterdam, The Netherlands). Monoclonal antibodies to Factor VIII were identified according to their reactivity with Factor VIII polypeptides as described in European patent application EP 0253455.

Polyclonal antibodies to Factor VIII polypeptides.

Rabbits were immunized by standard procedures with a Factor VIII preparation which had been purified by 30 chromatography (Stel, supra). The antibodies thus obtained were identified by immunoblotting, as described in European patent application EP 0253455, using purified Factor VIII-von Willebrand factor or purified polypeptides. The antibodies were isolated by polyacrylamide gel electrophoresis then transferred to nitrocellulose sheets as target proteins. Three distinct polyclonal antisera were obtained: RH 63271, RH 63272 i:ii i i i: a

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ELISA

A newly developed ELISA (Enzyme Linked Immuno- Sorbent Assay) was used to detect Factor VIII antigen in the conditioned medium. The method is as follows. The ELISA plates were coated by adding 200 ul/well of an appropriate dilution of the Factor VIII specific monoclonal antibody CLB.CAgA (5 mg/1) in 0.05 M carbonate buffer, pH 9.8. The plates were sealed and incubated overnight at 4 0 C. Between all incubations the plates were washed three times with PBS containing 0.05% which was left on the plates for at least one minute.

A series of dilutions of test samples or normal plasma was pipetted into the wells (200 ul/well) in duplicate.

The plates were sealed and incubated at 37"C for 2 hrs without stirring, then washed as described above. For dilution of the antigen a buffer was used which contained 50 mM Tris-HCl buffer, 2% bovine serum albumin and 1.0 M sodium choride, pH 7.4.

CLB-CAgll7-horse radish peroxidase conjugates were diluted approximately 10,000-fold (depending on the sensitivity required) in a buffer containing 50 mM Tris-HCl buffer, 0.2% Tween-20 and 1.0 M sodium choride, pH 7.4. Of this dilution, 200 ul was added to each well, the plates were sealed and incubatedd for 2 hrs at 37 0 C in the dark.

After washing the plates as described above, 150 ul of a freshly prepared solution of tetramethylbenzidine (TMB) (0.1 g/l) and hydrogen peroxide (0.006%) in 0.1 M acetate/citric acid buffer, pH 5.5 was added to each well. The plates were incubated for 30 min in the dark at room temperature. The enzyme reaction was stopped by the addition of 150 ul 2N sulphuric acid. Adsorption was determii'ed at 450 nm with an ELISA microreader. As shown in Table II there was an increase in Factor VIII activity of successive i i;; i i jII j !I i tt ii i 1; ji .i i

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29 conditioned medium which was approximately proportional to the increase in the amount of Factor VIII proteins in the medium.

D. Size determination i The size of the produced Factor VIII proteins was established using gel electrophoresis as follows.

The monoclonal and polyclonal sera raised against plasma-derived Factor VIII were used for immunoprecipitation.

The antibodies were immobilized on protein A-Sepharose (15 serum per 10 mg protein A-Sepharose), then incubated with the metabolically labeled recombinant Factor VIII-like compounds.

The immobilized recombinant proteins were reduced using beta-mercaptoethanol, then separated on a 5% polyacrylamide SDS gel electrophoresis system (Laemmli, Nature (1972) 227:680-685). As shown in Figure 4 and Table III, the recombinant Factor VIII-like proteins have the size expected for glycosylated proteins. The smaller bands-were also present in the control lane.

Based upon the results presented in Table III it can also be concluded that the recombinant Factor VIII-like proteins of the subject invention are glycosylated, since there is a significant difference between the size of the proteins formed in medium with and without tunicamycin, a known inhibitor of asparagine-linked glycosylation.

Example 7 Construction of Stable Cell Lines Producing Proteins with Factor VIII Activity A. Expression in CHO Cells- Plasmids pCLB203 (10 ug) and pAdD26SV(A)-3 (1 ug, Kaufman and Sharp, Mol. Cell Biol. (1982) 2:1304-1319) were introduced into dhfr-deficient CHO cells (Chasin and Urlaub, Procq. Natl. Acad. Sci. USA (1980) 77:4216-4220) using the i with parenterally acceptable vehicles and other suitable excipients in accordance with procedures known in the art. The pharmaceutical preparations may conveniently be a sterile lyophilized preparation of the subject polypeptide which may 'I SWO 88/09813 PCT/NL88/00028' calcium phosphate precipitation technique (Graham and Van der Eb, Virology (1973) 52:456-467). Amplification of Factor VIII.

and dhfr coding sequences was achieved by stepwise administration of increasing concentrations of methotrexate (mtx) as described by Kaufman and Sharp, J. Mol. Eiol. (1982) 159:601-621. Independent transformants resistant to 200 nM mtx were picked and established as stable cell lines. Several of these cell lines produced about 75 mU of Factor VIII activity/ml of culture medium. The amount of Factor VIII activity secreted into the culture medium could be further raised by further amplification of the Factor VIII coding sequences, using mtx in increasing concentrations.

B. Expression in C127 Cells As described above, expression vectors may be introduced into eukaryotie cells, where they integrate into the genome of the host cell. Subsequently the expression cassettes may be amplified. An alternative to integration of the expression vector is its stable maintenance as an episome. An example of the latter is the expression of one of the "mutant" Factor VIII protein using the episomal BPV-system (Howley et al., Meth. Enzymol. (1983) 101:387-402). The BPV-1 lgenome (BamHI cleaved) was first introduced into a BamHI cleaved derivative of pTZl8R (Pharmacia) which contains Xhol sites on both sites of the BamHI site. Subsequently the resulting pTZX-BPV plasmid was cleaved using XhoI, yielding a 2.9 kb pTZ fragment and an 8 kb BPV-fragment with XhoI protruding ends. The latter fragment was ligated into plasmid pCLB212 which had been cleaved at its unique SalI site (at position 2040 in the original pSVL vector). The resulting expression vector pGB881 contains the SV40 late promoter, Factor VIII cDNA coding sequence lacking 2775 bp (mainly of the B-domain) and SV40 late polyadenylation signal. Due to the presence of the BPV-genome this vector can be maintained as an episome in the proper host cells, such as mouse C127 cells., L c c i :i X WO088/09813 PCTNL8/00028 31 Plasmid pGB881 (10 ug) was transfected into C127 cells using the calcium phosphate precipitation technique. (Graham and Van der Eb, supra). Foci were isolated 14 days after transfection and subsequently stable cell lines were established. Several cell lines produced 40 mU of Factor VIII activity/ml of culture medium.

Example 8 CONSTRUCTION OF pCLB204.

pCLB204 was constructed using the loop-out mutagenesis technique as described in Kramer et al. (1984) (see General Methods, The deletion mutagenesis of the central region of factor VIII cDNA was performed using the following oligonucleotide: primer V: 3' TAG.GTT.TAA.GCG.AGT.CAA.GTT.TTG.GGT.GGT.CAG.AAC 5' to produce in fac.tor VIII cDNA internal deletions of the sequences coding for amino acids Ala-375 through Ser-1637 (primer V).

The corresponding protein contains internal deletions of 12G3 amino acids.

To obtain a target fragment for loop-out mutagenesis a HindIII-PstI fragment of 1.4 kb derived from pCL203 (Example 3) was selected. The nucleotide position (Figure 1) of the Hind III-site is at 1025 in the full length factor VIII sequence upstream of the region to be modified; the PstI site is at position 5165 downstream of the region.

These sites are indicated in Fig. 2. The 1.4 kb fragment was subcloned in M13mp9 followed by the loop-out mutagenesis.

After selecting the fragment with the precise deletion obtained with primer- V in bacteriophage M13, the HindIII-PstI fragment containing the des-ed deletion was inserted into an i!

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t ii a i, i j i L C tli The unique HindIII site of the plasmid pSV2.tPA (Van Zonneveld et al., Proc. Natl. Acad. Sci. USA (1986) 83:4670-4674) was modified into a Sall site by making blunt the HindIII sticky-ends and ligating Sall-linkers WO 88/09813 -PCT/NL88/0028 32from pCLB201, using the following steps and preparing fragments with sticky, unique restriction enzyme ends (numbers refer to Figure 1): step 1. Plasmid pCLB201 was digested with Aval and XbaI, generating a vector fragment Aval (737) till XbaI (6951) of about 6 kb.

step 2. Plasmid pCLB201 digested with Aval and HindIII gave a 289 bp long fragment: Aval (737) till HindIII (1025).

step 3. Plasmid pCLB201 digested with PstI and NdeI gave a fragment PstI (5165) till NdeI (5527) of 363 bp.

step 4. Plasmid pCLB201 digested with Ndel and XbaI gave a fragment NdeI (5527) till XbaI (6951) of 1425 bp.

step 5. The M413 bacteriophage containing the fragment with the desired mutation was digested with HindIII and PstI.

step 6. The five fragments were isolated, mixed in equimolar amounts and ligated. Plasmids containing all fragments were selected. Using primer V, shown above, a new I expression vector was constructed for the expression of the examplary compound described in Table I: pCLB204 for compound 4 with primer V The DNA constructs and methods of the presentinvention provide a means for preparing polypeptides having Factor VIII activity by introducing a deletion mutant gene encoding a Factor VIII congener into a host cell. The subject compositions find use for treatment of the symptoms of Hemophilia A.

WX i"i- o i~eaar:-: _-r NVW, 088/098,13 PC~r/L0--00002 33 All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

j L_ i ligated, giving rise to plasmid pCLB1O1. Plasinid pCLB1Ol contained a MiuI-linker between the MaeIII and HgiAI sites (see Figure 3b) in addition to KpnI, BanI and ApaI sites. Four WO 88/09813 PCTN L88/0008 34

REFERENCES

Brinkhous, K.M. et (1985) Proc. Natl. Acad. Sci. USA 82, 8752-8756 Burke, R.L. et al., (1986) J. IDiol. Chem. 261, 12574-12578.

Carter, P. et al., (1985) Nucleic Acids Res. 13, 4431 Chasin, and tjrlaub, G. (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220 Eaton, D.L. et al., (1986a) Biochemistry 25, 505-512 Eaton, D.L. et al., (1986b) Biochemistry 25, 8343-8347 Fay, J.F. et al., (1986) Biochim. Biophys. Acta 871, 268-278 Galfr6, G. et al., (1977) Nature 266, 550-552 Gluzman, (1981) Cell 23, 175-182 G-orman, (1985) DNA cloning D. Glover), IRL-press, Washington, oxford, pp. 143-169 Graham, F. and Van der Eb, A. (1973) Virology 52, 456-467 k- Wormi-Mil

A-

WO W88/09813 PCT/NL88/00028 35 Hanahan, (1983) J. Mol. Biol. 166, 557-580 Howley, Sarver, N. and Law, M.F. (1983) Meth. Enzymol. 101, 387-402 Hurwitz, Hodges, Drohan, W. and Sarver, H. (.1987) Nuci. Acids Res. 15, 7137-7153 Kasper, C.K. et al., (1975) Thrombs. Diath. flaemorrh. 34, 869-871 Kaufman, R.J. and Sharp, P.A. (1982) Mol. Cell. Biol. 2, 1304-1319 Kaufman, R.J. and Sharp, P.A. (1982a) J. Mol. Biol. 159, 601-621 Kozak, M. (1983) Microbial. Rev. 47, 1-45 Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456 Laemmli, (1970) Nature 227, 680-685 4 7;;

I

I

p 11 Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res. 11, 1295 Maniatis, T. et al., (1982) Molecular Cloning. Cold Spring Harbor Laboratory WO 8809813PCT/NL88/000;8 36 -r Maxam, A.M. amd Gilbert, W. (1980) Methods Enzymol. 65, 499-5601 Melton-, D.A. et al., (1984) Nucleic Acids Res. 12, 7035-7056 Mulligan, R. and Berg, P. 1981) Proc. Natl. Acad. Sci. USA, 78, 2072 Sanger, F. et (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467 Stel, (1984) Ph.D. thesis, University of Amsterdam, The Netherlands Toole, et al., (1986) Proc, Natl. Acad. Sci. USA 83, 5939-5942 Van Mourik, J.A. and Mochtar, (1970) Biochim. Biophys. Acta 221, 677-679 Van Zonneveld, et al., (1986) Proc. Nlatl. Acad. Sci USA 83, 4670-4674 Vehar, et al., (1984)3 Nature 312, 337-3423 Veldkamp, J.J. et al., (1968) Thromb. Diathes Haemorrh. 19, 279 Wood, W.I. et al., (1984) Nature 312, 330-337 Yanisch-Perron, C. et al., (1985) Gene 33, 103-1191

L

Table I Deletion Mutant Factor VIII Proteins NR LR CR Expression Compound NR *Del. LR *Del. CR *Del. Vector 1. A-14R-7'4o 0 S-741lss-i,637 0 Q-1638.,Y-2332 0 pCLB2Ol 2. A-14#R-740 0 s-741l.A-867+ D-1563.*Sl637 695 Q-16384aY-2332 0 pCLB2O2 3. A-l-,D-712 28 P-R-V-A 897 Q-l638-),Y-2332 0 pCLB203 3b. A-lD-712 28 peptidebond 897 Q-1638-o-Y-2332 0 pCLB2l2 4I. A-14V-371 366 peptidebond 897 Q-1638+*Y-2332 0 pCLB2l4 7. A-l.R-740 0 peptidebond 897 E-16494Y-2332 11 pCLB2O8 8. A-l--R-710 0 peptidebond 897 S-16694oY-2332 31 pCLB209 9. A-1.jR-74I0 0 peptidebond 897 S-1690.iY-2332 52 pCLB2lO means an uninterrupted sequence of Factor VIII amino acids The number of deleted amino acids in each region compared with full-length Factor VIII are listed In the respective columns with the heading Del. The numbering of the amino acids is as used in Figure 1.

ii Table II: FACTOR VIII ACTIVITY AM6 PROTEIN AMOUNTS factor VIII activity (mU/mi) 7) chrom. ass stand. ass *3) rest activity 2 after addition of antibodies

CLB.

CagA 4 pat.j 5 antigen determination 8 length (deletion) in amino acids compoundl) 1 2332 2 1637 3 1411 3b 1407 4 .1069 7 1424 8 1404 9 1383 mock (pSV2) (0) (695) (925) (925) (1263) (908) (928) (949) 1.0 6.7 17.3 15.0 0 15.0 15.0 0 0 6) 2 5

ND

9 0

ND

ND

ND

0 <5 mU I1% <1 7 mU .20 mU

ND

9

ND

9 20 mU 0 0 2OmJ ND ND ND ID ND ND ND ND. ND 0 1) see Table 1; 2) according to chromogenic (Kabi Coatest) assay 3) standard clotting assay; 4) CLB.CAgA, monoclonal antibody to facto: VIII (Stel, Ph.D. Thesis, University of Amsterdam, The Netherlands, 1984) inhibitor isolated from serum of patient J.

6) initial activity too low to distinguish possible inactivatio,.i 7)conditioned media af.-'er 48 h~ours incubation. One unit factor VIII C')r2Sr1r1s L100 ng factor VIII protei~n mzme1inc-C'immunosorbe-t :tssa'%, (ELISA); 9) NI) =-not detei '.ned Fp :t Table III. SIZE OF SECRETED FACTOR VIII PROTEINS compoundl) expression vector length 2 deletion 3 calculated established glycosylation size 4 size 5 inhibition 6 1 pCLB 201 2332 0 265 kDa 2 pCLB 202 1637 695 188 kDa 192 kDa 185 kDa 3 pCLB 203 1411 925 162 kDa 168 kDa 158 kDa 3b pCLB 212 1407 925 162 kDa 168 kDa 4 pCLB 204 1069 1263 123 kDa 135 kDa 1) see Table I 2) The length is the total number of amino acids in the factor VIII coding sequence of the expression vector.

3) deletion means the number of amino acids deleted from the coding sequence of the full length factor VIII in the corresponding expression vector.

4) The size in kilodalton determined on basis of the known amino acid composition.

The size in kilodalton determined by immunoprecipitation of labeled protein followed by electrophoresis.

6) The size in kilodalton of proteins from cells grown in a medium containing tunicamycin (0.001 mg/ml).

.4^

B'

I

-r

I

_i:i i;l 4i.

b 1

Claims (12)

1. A protein having biological activity of Factor VIII i and having an amino acid sequence essentially corresponding to the amino acid sequence of Factor VIII except for:- a deletion of the amino acids Ser-741 through Ser-1637 and a deletion of at least part of the amino acids Lys-713 through Arg-740; optionally a deletion of the amino acids Gln-1638 through Ile-1668, or part of them; and optionally an insertion of a linking group comprising at least one amino acid.

2. A protein according to claim 1 having an amino acid sequence essentially corresponding to the amino acid sequence of Factor VIII except for a deletion of the amino acids Lys-713 through Ser-1637 and optionally a deletion *of the amino acids Gln-1638 through Ile-1668, or part of them.

3. A protein according to claim 2 having an amino acid 20 sequence essentially corresponding to the amino acid sequence of Factor VIII except for a deletion of the amino acids Lys-713 through Ile-1668.

4. A protein according to claim 2 having an amino acid I sequence essentially corresponding to the amino acid i sequence of Factor VIII except for a deletion of the amino acids Lys-713 through Ser-1637.

5. A protein according to claim 1 containing a linking I group comprising the amino acids Pro-Arg-Val-Ala in the place of the deleted amino acids.

6. A protein according to claim 5 wherein the amino acids Pro-Arg-Val-Ala connect the amino acids 1-712 and

1638-2332.

7. A protein having biological activity of Factor VIII and having an amino acid sequence essentially corresponding to the amilio acid sequence of Factor VIII except for a deletion of the amino acids Ser-741 through Ile-1668.

8. A r-combinant DNA encoding a protein as defined in I any of the claims 1 to 7. temperature. The enzyme reaction was stopped by the addition of 150 ul 2N sulphuric acid. Adsorption was determined at 450 nm with an ELISA microreader. As shown in Table II there was an increase in Factor VIII activity of successive -41- II

9. An expression vector comprising, in the direction of transcription, a transcriptional regulatory region and a translational initiation region functional in a host cell, i a recombinant DNA encoding a protein as defined in any of the claims 1 to 7, and translational and transcriptional termination regions functional in said host cell, wherein said initiation and termination regions are capable of regulating expression of said recombinant DNA. A transformed host cell and progeny thereof, wherein said host cell comprises an expression vector comprising, in the direction of transcription, a transcriptional regulatory region and a translational initiation region functional in a host cell, a recombinant DNA encoding a protein as defined in any of the claims 1 to 7, and translational and transcriptional termination regions functional in said host cell, wherein said initiation and :termination regions are capable of regulating expression of said recombinant DNA. 11. A transformed host cell and progeny thereof S 20 according to claim 10, wherein said cell is a mammalian cell. 12. A transformed host cell and progeny thereof il according to claim 10, wherein said cell is a prokaryotic cell or a yeast cell. S 25 13. A method for preparing a protein as defined in any one of the claims 1 to 7, comprising growing, in a nutrient medium, a transformed host cell which comprises lan expression vector comprising, in the direction of transcription, a transcriptional regulatory region and a translational initiation region functional in said host cell, a recombinant DNA encoding said protein, and translational and transcriptional termination regions functional in said host cell, wherein said initiation and termination regions are capable of regulating expression of said r-combinant DNA. 14. A Factor VIII deficiency symptom-alleviating composition cofprising a symptom-alleviating amount of a protein as defined in any one of the claims 1 to 7 in a physiologically acceptable carrier. -n~lsea~sazm~Pne3--3-rr, i i I- Plasmids pCLB203 (10 ug) and pAdD26SV(A)-3 (1 ug, Kaufman and Sharp, Mol. Cell Biol. (1982) 2:1304-1319) were introduced into dhfr-deficient CHO cells (Chasin and Urlaub, Proc. Natl. Acad. Sci. USA (1980) 77:4216-4220) using the A protein according to claim 1 substantially as hereinbefore described with reference to any one of the examples. 16. A recombinant DNA according to claim 8 substantially as hereinbefore described with reference to any one of the examples. 17. An expression vector according to claim 9 substantially as hereinbefore described with reference to any one of the examples. 18. A transformed host cell and progeny thereof according to claim 10 substantially as hereinbefore described with reference to any one of the examples. 19. A method according to claim 13 substantially as hereinbefore described with reference to any one of the examples. DATED: 18 May, 1992. PHILLIPS ORMONDE FITZPATRICK 20 ATTORNEYS FOR:- °k< STICHTING CENTRAAL LABORATORIUM VAN DE BLOEDTRANSFUSIEDIENST VAN HET NEDERLANDSE RODE KRUIS 8502i S 25 i I i I I I ssl L I WO088/09813 3PCT/NL88/00028 Figure1 3 4 50 GTCGACATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTT 9 YM 0 1 E L S T C F F L C L L R F C F -2 80 90 100 110 120 AGTGCCACCAAGATACTACCTG6TGCAGTGGACTTCATGGGACTATAT6CAAAGT S( T R R Y Y L G A V ,E L S W D ,Y MO 0 19 13-0 140 150 160 170 180 GATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGALAGTGCCAAATCTTTTCCI D L G EL P V D A R F P FP R V F' K 9 F 190 200 210 2 TTCAACACCTCAGTCGTGTACAAAAAGACT'CTG3TTTGTAGAATTCACGGATCA--r*,TT: F N T S V V Y 1- VT L F V E F T D H L F 260 270 280 290 700 AACATCGCTAGCCAAGGCCACCCTGG~ATGGGTCTGrCTAGRi7TCCTACCATrCCAr-"'-r:-I- N~ I( PR P FP W M G L L G F'TP0AE 110 320 30340 35-60 GTT'T. YI"LTACAGTGGTCATTACAC'TTAAGAACATGGCTTCCCATCCTG'TCAGTCTTCAT' V Y D T V V I T L N NM A S H P' V S L H 99 37 0:9040410 420 GC TOT TOOTOT GATC CTACT GSAA GCTTC TGA GGGAoc TGAATAT GATG ATCAGACCAGT A V G V S Y W K< A S E S A E Y D D Q T S 430 440 450 460 470 400 (AA 6 OGA GAAA GAAOjA T OAT AAAG r'cTrcc CTGGTGG 6 AG BCCAT AC AT AT TCTG66CAGC- (.IR E K E D D VV F P' G GS H T Y V W 0 12; 490 500 510 520 53.0 540 (3CIGAGGAG'CAT(CTTAC~('GGC'TCTCCTTT V L E F N G P M A D FP L C L T Y S Y L )60 570 560 590 6 00 TCTCA 1'-.TGG-2ACCTGGTA )AAGA.CTTGAATTrCAGGCCTCA'.T''GGAGCCTACTA51(' TGT S, H VODL V 1)L N (S G L I GSA LL V C 610 620 630 640 650 660 r 1 36i(::c AAGi(AAGACACAGACCTTi13C OCAAAT'1TA(-TACTACTTTTT R E 6~ 1 L A E I I T 0 T L, H I F I L L F 670 600 690 700J 710 720 UC TOTAT'T T GAT GAAGGOi)AAAGT1 S GCACTCAGAAAC(AAPAACT'CCTTGATGCIACGAT A V F D E G SW H E T N S L MO0D 770 740 750 760 770 780 A (GGATGCI GCATCTSGCTCR-GGC'.CTGGCCTAA ATGCACACAG3TCAATGGTTATGTAAAC R D A A S A R A W P K M H T V N C-3 Y V N 790 600 810 a20 6350 810 AGO rFCCCCAGGTCTE3ATTOGATGCCACASGAAATCAGTC rATT'GGCATGTGATTGGA R S L FP G L 1: (3 C: H R k' S V Y W H V I r 259 850 F360 870 680 890 900 ATGGGCACCAC:TCCTGAAGTGCACTCAATATTCCTCGAAOGTCA2,ACATrTTCI-TGTGAGG M 6 T T F' E V H S I F I G; H T F L V R ericoniing ;a i-'actor VIIIL congener into a hiost cell. T~he subj)ect compositions find use for treatment of the symptoms of Hemophilia A. WO 88/09813 PCT/NL88/00028 910 920 930 940 950 960 ACCATCGCCAGGCTCCTTGAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTC N H R (2 A S L EI S P IT F L T A T L 299 970 980 990 1000 1010 1020 TTGATGGACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGC. *L M DL GO0 F L L F C H IS S H O H D G 1030 1040 1050 1060 1070 1080 ATGGAAOCTTATOTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATOAAAAAT M E A Y V K V D S C P E E FP 0 L R M K, 339 1.090 1100 1110 1120 1130 1140 A AT GAAGAAGCGGAAGAC TAT OAT GATGAT CTT ACT GA TTCTGAA ATGGAT GT OGTCAG 0 N E E A E D Y D D D L T D S E M D V V R 1150 1160 1170 1160 1190 1200 TTTG3ATGATGACAACTCTCCTTCCT*TTATCCAAATTCGCrCAA'TTGCCAAGAAGCATCCT F D D D N S FP S F I Q I R S V A K K H FP 379 1210 1220 1230 1240 1250 1260 AAAACTTGGGTACATTACATT3C'TGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTC K T W' V H Y I A A E E E D W D) Y A P L V 1270 1280 1290 1:700 13110 1320 C TCGCCC CC GATB(ACA GAAGTTAT AAAAG TCAA TATT TGAA CAATGGCCCTCAGCGGAT T LAP PD DR SY KS O 0VL N NGPO RI 419 1301:40 1:350 1360 1370 1380 GGTAG66AAGTACA AAAAAOTCCGATTrTATGC3CATACACCoGAT6A( ACCTTTAAGACTCGT G R Y~ V~ 13~ vi F MA Y T D E T F T: R 1390 1400. 1410 1420 1430 1440 GAAGCTATrTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTAT'GGGGAAGTTGGAGAC E A

1 0 H E S G I L G P L L Y G E V G D 459 J.450 1460 1470 1480 1490 1500 ACACTGTTOATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGA T L- L I I F PN (2 A SR F' P N I Y F' H G 1510 1,520 1530 154 0 1550 1560 ATCACT(3AT(3TCCGTCC'r TTGTATTC AAGGAO3AI'T('CCAoAAAGT6'TAAAACATTTGAAG, I T D V R F' L Y S R R L, F' kG V K H L K499 1570 1580 1590 1600 1610 1620 GA TTT TCCAA TT CTGCCAG66 GAAA TAT TCAAA TATAAAT GOAC AGTG ACTGTAGAA BAT D F F' I L F' G E I F K Y K 14 T V T V E D 1630 1640 1650 1660 1670 1660 006 CCAACTIAAATCAGA*r CC T C 10CC T6ACCC OCTATTACT CTAOITTICOTTAATA T S G P T K S D FP R C L T R Y Y S S F V N M 5 9 1690 1700 171.0 1720 1730 1740 GAOAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCT OTA E R D L A 9 6 L I C? P L L I C Y K E S V 1750 1760 1770 1760 1790 1600 rOATCAAAGAGGAAAC('CGATAAI1GY'CAG)ACAAGAG6AA(:.TCACCT*GTTTTCTGTATTT L D 0 RG NO I MS D k. R N V I L F S V F 570. "WO 88/09813 PCT/NL88/0002.8 3/13 1810 1820o 1830 16E40 1850 1860 G.ATGAGAACCGAAGC TOGTA CtTCACAG3AGAA)T ATACAACGC TTTC -TCCC:CAA TC CAGCT,, DE N RS WY L T EN I R F L FN PA 1670 18130 1890 1900 1910 1920 GGAGTOC AGC T TAGGA TCC AG AG"T TC CAAGC CT CCAACATCA TOCAC AG C ATCAAT GGC G3 V 0 L E D FP E F Q2 A S N I M H S I N4 G 619 19:30 1940 1950 1960 1970' 1980 TATGrGTTTTTGATAO-T'TTOCAGTTGTCAGTTTGTTTGCATC GGGTGGjCATACTGGTr-APT Yv V F D S L Qi L S V C L H E V A Y W T 199 0 2000 2010 2020 20302r.4 CTAGCATTGGAGCACAGACTGACTTCCTTTrCTGTCTTCTTCTCTGGATATACCTrTCAAA L S 1 63 A Li T 1) F L S V F F S G Y T F fK 659 2050 2060 2070 20830 2090 CACAAAA TGGTCTATGAA GAGACAC TCACCCTA TTC CCATTCTCAGGAGAAACTGTCTT C H M V Y E D T L T L F P F S 6 E T V F 2110 2120 21.30 2140 2150 2160 ATGTCGATGGACCCAGG-rCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAAC M S M E N FP G L. W I L G C H N S D F R N 699 2170 21e0 2190 2200 2210 2220 AGgIGGC ATGACCGC CTTACTGAAGG TTTCTAGT TOTGACAAGAAC AC TGOTGATTATTA C R G M T A L L KV S S C K N T G D Y Y 227-0 2'240 2250 2260 2270 2290 GAG GA CAG TTAT GAAG ATAT T TCAG CATAC TTOC TGAGT AAA AAC AATBC CAT TGAACC A E D S Y E D 1 S A Y L L S K N N A I E P 739 2290 2300 2: 310 2:320 2330 23740 AGAAGCT["rCTCCCA(3AATTCAAOACACCCTAGCACTAGGCAAAAG7CAATTTAATGCCACC R S F 3) 0 N S ,R Hi P S T R QK k'0F N AT 23 50 2360 2370 2380 2:390 2400 ACA AT TCC AGAA AAT GAGAT ABAGAA GACT GACCC TTO GT TTGC ACACAGAAC C CT ATE- T I P E N D I E T D FP W F A H R T P M 779 2410 2420 2430 2440 2450 2460 CC TAA AAI'ACAAAA TOTCT( CC TC TAB T OAT rT 6 TiGA TGCT'CTTGCGA)CAGAGTC C'ACT P K 1 0. N~ V S S S D L L M L L, R 0 S FP T 2470 2480 24.90 2500 2510 2520 C GAGATGGBC TAT CC TTAT CTGATCT CCAA GA AGCC AAATAT GA GA CTTTT TCTGATGAT P H G L S L S D) L (2 E A V' Y E T F S D D 810 25370 2540 2550 2560 2570 2580 C CAT CAC CTGGAGCAATAGACABT AATAACAGC C TGTCTGAAA T"GACACACTTCAG C CA P' S 15 B A I D S N N S L. 5 E M T H F R P i Iv I' 2590 2600 2610 >26' I 2630 2640 CAGCTCCATCACAGTGGGGACATGGTATTTACCC C\ GATCAGGCCTCCAATTAAGATTA 0L H H S G D M V F 8P B LQLRLS9 2650 2602670 ~'~2680 2690 2700 A AT GAGAAACT GGGG'AACTBCAGC-ACAAGT'flAAGAAACTTGATTTCAAAGI*TTC'T N E K L GV TA A TE L. K K L D F K V S 'WO 88/09813/3 PCT/N L88/00028 2710 2720 27370 2740 2750 2760 4 S T S N N L IS T I FP S DI) N L A, A G T D 899 2770 2780 2790 2600 261 0 2820 ATACAAGTTCCTTAGG()CCCCCAGTAT6CCAGTI-fCAT1TATAGTCAATTAGATACC N T S S L. G3FF' S M( P V H Y D S 0L. D T 26370 2640 2650 2860 2870 2680 4ACTCTATTTGGCAAA(3TCATCTCCCCTTACTGAGTCTG6TGGACCTCTGAGCTTGAGT T L F 6G K 6k:S F' L T E S 66G P L S L S 93 2690 2900 2910 2920 2930 294-0 GAAGAATAATGATTCAAGTTGTTAAATCAGTTTT6TAGCCAGAAGTTCA E E N N D S K L L ES 6 L M N S 0 E S S 2950 2960 2970 2960 2990 3000 TGGGGAAATGTATCTCAACAGAGA6TGGT(AGTTATTCAGGAAAGAGCTCAT 14 6G N V S S T E S 6 R L F iK G K R A H 979 37.010 3020 3030 3040 3050 3060 GGACCTGCTTTGTTGACTAAAGATATCCTTATTCA6TTA6CATCTCTTTGTTAAAG G P A L L T K D N A L F KV (3 1 S L L f,, 3070 3080 3090 3100 3110 3120 T N iK T S N N S A T N R T H I D 6 F' S 101Y .3130 3140 3150 3Y16031016 T TArTAA T TGAGA A TAOTC CATC AGTC TGGCAAAA T ATA TTA LAOAG TGACAC TGA 6TT T L L IE NS P S V WO N I LE S D T E F 3190 3200 3210 31220 3270 3240 AAAAAAGTGACACCTTTGAT'T'CATGACAGAATCTTAT66ACAAAAMTGCTACAGCTTTGz 1" K VT FP L I H D R M L M D K NA TA L 1059 3250 :3260 3270 3260 3290 3300V AGGCTAAATCATAT6TCAAATAAAACTACTTCATCAAAACAT6AAATGTCCAACAG R L N H M S N K T T SB S N M E M V Q 0 :310 33.40 34530 340 3470 34360 T C 66OAAA66CCCCAGTTCCAA AGACCAAAA TCCATTCTCTTAGGCCGAA CGTG P' K E 6 P F' 0 P D L 6 F E K M 1099

3497.0 3500 13910 3400 .3410 3420 FGTCGOAATTTCTTGTCGAAAAACAA6TAGCTA6GAMGGGAAATTTCAA 6 Q N F L S E le 19K V V V G IC 6 E F T K 35.F50 37560 3.570 '3180 3590 3600 GACGTA6L3AC TCAAAGA GATL3GTTT TTCCAAGCAGCAGAAACC TAT TTCTTAC TAACTT(3 D V 6 L K" E M V F r' S S R N L F L T N L 1179 'wb088/09813 PCT/NL88/00028 5/13 37610 -Z620O 36:30 37640 31650 .3660 D) N L H E N N T H NO0 E K I1Q E E I E :3670 60 6 3700 3710 .372 0 37730 3740 3750 37760 37770 :7B6 T KN F M KN L. FL LBS T RO0N V E G~ 3790 :3600 36310 3820 .3630 Y D 6 A Y A P' V L 0 D F R S L N D S T N 1259 3903660 :3870 38380 3639L7 R T I H T A H F S fl' f" G E E E N L E 0 3910 7"920 3930 3940I 3950 3960 TTGAACACAGAATTGGAAAGAGACCAGTTTC L G N\ 0 T K0 1 Y E K Y A C T T R 1 13 P 1299 3970 3990 .3990 4000 4010 4020 N T E 0 0 N F V T 0 R S 1K*' R A L K 0 F R 4030 4040 4050 4060 4070 4080 L. F' L.E E TE L E f-K R I I V D D T S T 01339 4090 4130 4110 4120 413>0 4140 T GGT CCAAAAACAT GA('C T T T GACCCCG4GCACCC TCACA CAGATAGACTA~C r T GAG W S k" N M H L T F' S T L TO I,1D Y rqE 4150 4160 4170 4180 4190 420 4- AAG 6GGAAGGGGC CA~T T (CT C(CT C CC CTT~T CA6 T T 6CC TT AC GAGGAGTCA1Y' CC 1, E K G A IT GI S F' L S D CL T RBS H S 1379 4210 4220 42:30 4240 4250 4260 A T CC CTC A 6CAA TAAT CT C CATT ACCCAT TGC/ AG66T 7CATCA'TT TCCATCT T T 0 N R S F' L F' I A k' V S S F FP S I 4270 42B0 4290 4300 4310 4320 4 A GAC CT~Th T ATC TGAC CA 6667CCTA~T T ;CAAGACACTCTT C TCATC T CC AGCCAC R P' I Y L T R V L F 0 D N S S H L P A~ 1419 433.0 4340 4350 4360 4370 4380 TCT7 AAGAAAGTTC TGGGG CCAAA GC(ATT77CT TACAPGGAGCCAAA S Y R K" K D S 6 V Q0 E S S H F L M 6 A K 4- 1)9 0 44-00 4410 A420 44.30 4440 /W'AAATAACCT777CT T TGC CATTCTAACCTT6GAGATGAC T 667 CAAGGAGTT K N N L S L A 1: L T L E M T B3D 0.R E V 1459 4450 41460 4470 4460 4490 4500 GG~c'SGAAGGCCATCSCCTCAAATGGAATT C SL 6 T S 7N SV 7VK K V ENT7V Yanisch-Perron, C. et al., (1985) Gene 33, 103-119 I A WO088/09813 PCT/NL88/00028 61 -13 4510 4520 457.0 4540 4550 4560 CTCCCGAe)CCAGACTI*GCCAAACATCTG(CAAATTAATGCTTCCAAAATTCAC L P P D L PF' T S G6:K.V E L L P K V H 149 4570 4580 4590 4600 4610 A4620 ATTTATCAGAAGACCTATTCCCTACGGAACTACAATGGTCTCCTGGCCATCTGI3AT I Y, 0 K D L F P T E T S N 6 S P 6 H L-D 4630 4640 4650 4660 4670 4680 CTCGTGGAAGGGAGCCTTCTTCAG6AACAGAGG6CGATTAAGTGGATGAAGCAAAC L V E 6 S L L 0 6 T E 6 A I KW N E A N 1539 4690 4700 4710 4720 4730 4740 AGACCTGGGTTCCCTTTCTGAGAGTAGCAACA6AAGCTCTGCAAAGACTCCCTCC R P 6 K V F' F L R V A T E S S A K T P S 4750 4760 4770 4780 4790 4800 AAGCTATTG GATCCTCTTGCT'T6GGATAACCACT ATGGTAC TCAGATACCAAAAGAA GAG L /L D P L A w D) N H Y G T 0 1 P K E E 4810 4G20 4830 4040 4850 4860 T 60 AAArCCC AAGA6AAGTC AC CAGAAAAAACAGC TTT T AGAAAGGATACCATT TTG W Ki S E S FP E K T A F k' k D T I L 4870 4820 4890 4900 4910 4920 TCCCTGAACGCTTTGAGCAATCATGCAATAGCAGCAATATGAGGGACAAAATAAG S L N A C E S N H A I A A I N E 6 Q. N K 1579 1.619 2 4930 4940 4950 4960. 4970 4980 CCC 0thAATA GAAGT CAC CTG66GCAAAGC AA 66TA 06AC TGAAA GOC TO TCTCT CAAA AC F' E I E V T W A 1" Q. 6 R T E R L C S C N 397 38 4990 5000 5010 5020 50:30 5040 CCACCAGTCTTGAAACGCCATCAACGGGAAATAACTCGTACTACTCTTCAGTCAGATCAA P P V L Ke R H 0 R 4 I T R T T L Q S D Q 1659 5050 5060 5070 5005090 5100 OAGGAAATTGACTATGATGATACC'ATATCAGTTGAAAT6AAGAAGGAAGATTTTGACATT IE E I D Y D D T I S VC M~HKI E DF D I 5110 5120 51:30 5140 5150 5160 r A TGA TGAG OAT GAAAA TCAGAGCC CCC C AGCTTTC AAAA 6AAAACACGACACTATTTTT Y D E D E N c0 S P F Q K- W T R H Y F 1699 5170 5180 5190 5200 5210 5220 ATTGCT6CAOTGGAGAGGCTCTGGEATTATGGGATGA6TA6CTCCCCACATGTTCTAAGA I A A V E R L W D Y 6 M S S S P H V L R 5230 5240 5250 5260 5270 5280 A ACrA GGGCT CAGAG TG6CAOT 6TCCC TCAGTrTCAAGAAAGTTG6TTTTCCA6GAATTrTACT N R A Q S 6 S V F' 0 F K V V F Q. rz F T 17139 5290 53LIO S53.10 5320 533-:--0 53-40 GATGGtT CCTT TA CTCAOCCC TTATACC 6T(3GA6AAC TAA ATGAACA TT TGGGACTCC TG D G F T 0 P L. Y R G E L N E H L 6 L L 5:350 53Z60 5'370 5:3630 5390 S400 GGOCCATrAT ATAAGAGCAGAAGTTGAAGATrAAT ATC ATGGTAACT TTCAGAAATCAG6C C G FP Y I R A 5 V 5~ b N 1 M V T F R N Q A 1779 WO 88/09813 7/1PT/NL88/00028 5410 5420 5430 5440 5450 5460 TCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGG(3OATCAGAGCAAGGA SBR P Y S F Y SBS L I BYE E D Q RD 6 5470 5480 5490 5500 5510 5520 GCAGAACCTAGAAAAACTTTGTCAAGCCTAATACCCTTACTTTTGGAAAGTG *A E P' R K N F V KP N E T T Y F L4 K V 1819 *5530 5540 5550 5560 5570 5560 *(:ACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAACCTGGGCTTATTTCTCT Q OH H M A F T KD E F D C K A W AY F 3 5590 5600 5610 5620 5630 54 GATGTTGACCTGGAAAAAGATGTGCACTCAOGCCTGATTGGACCCCTTCTGC T- icCCAC D V D L E K D V H 66G L I G F L L V C H 159 5650 5660 5670 5660 5690 5700 ACTAACACACTBAACCCTGCTCAGGA(ACAAGTGACATACAGGTTTI3-.- (3,TT T N T L N P A Hl 1- R Q V T V Q E F A L F 5,710 5720 5730 5740 5750 5760 TTCACCATCTTT(ATAGACCAAAAGCTGGTACTTCACTGAAAA;TATGGAAAGAAACTOC F T I F D E T K S W Y F T E N M E R N C 1899 5770 5760 5790 5800 5810 5620 AGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAATTATCGCTTCCAT R~ A, P C N I 0 M E D P T F R' E N Y R F H 5630 58)40 5850 56360 5670 5680 OC AAT CAAT GGC TACAT AAT OGA TACACT AC C TOCT TAG TAA TOC T CAGG ATCAAAGG A I N 6 Y I M D T L P LV M A 0 D 0 R 1939 5690 5900 5910 5920 5930 5940 ATTCGATGGTATCTGCTCAbCATG(3OCAGCAAT6AAAACATCCATTCTATTCATTTCAGT I R W Y L LBS MOB N E N I H S I H F S 3950 5960 5970 5980 5990 6000 GGACAT(TOTTCACTOTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTAT G H V F T V R k E E Y K M A L Y N L Y 1979 6010 6020 60:30 604,0 6050 6060 K C ~1CAGG0TOIT TT TOAACAOTGGAAA T 6TTAC CA TCCAAAGC TGGAATTTGGGGTOGAA P G V F E T V E M L F'S K A G I W R V E 6070 6080 6090 6100 6110 6120 TO C CTTA TTGOCO A CAT C TACATGC TG 6ATGAOCACACTTTTTCTGo T TACAOCAAT C L I 0 E H1 L H A 0 M S3 T L F L V Y S N 2 1 *6130 6140 6'IZO 6160 6170 6180 AAGTGTCAGACTCCCCT6(3GAATO(3CTTCTO6ACACATTA(3AGATTTTCAGATTACAGCT KC0 T P L 0 M A S G H1 1 R D F 0 I T A 6190 6-00 621.0 6220o 62:30 6240 TCAOOACAATATGGACAGI'G6OCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATC co GD V (3DG W A P K L A RI.L H YB S(GS1 2050 6250 6260 6270 6200 6290 6300 AAT OCCTOOA(3CACCAAGCAOCC CTT TTCT TO OATCAAOOGTGGATCT13TT(GCACCAA TO N A W S T K E P F S W 1 V D L L A F' m f WO 88/09813 PCT/NL88/00028 6310 6320 6330 63740 6350 6360 I I H G I tKl T~ t! A R Q 14 F S S L Y I S 2099 6370 63180 6390 6400 6410 6420 CAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAATTCC 0 F I I M Y S L D 6 K W 0 T Y R G N S 6430 6440 6450 6460 6470 6480 ACT86 ACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGATAAACACAATATT TOGT L MV F F6 N YEV) s sG IK H N 1 2139 6490 6500 6510 6520 653.0 6540 TTT *AAC CCT CCAAT TAT TOCT COAT ACATCCGTTTGCA CC CAAC TCATTATAGCA TTCG C F N P FP I I A R Y I R L H P' T H Y S I R 6550 6560 6570 6580 6590 6600 AGCACTCTTCGCATGGAGTGGATGGCTGTGiATTTAAATAGTTGCAGCATGCCATTGGOA T I. R mE M G C DL N o C S M P L G 2179 6610 6620 6630 664-L 6650 6660 ATGGAGAGTAAAGCAATATCAGAT6CACAGATTACTGCTTrCATCCTACTTTA :CAATATO M1 E S K A I S D A 0 I T A S S Y F T N M 6670 6680 6690 6700 6710 6720 T TTG6C CACCT GOTCTCC TTC AAAAGC TCGAC TT CACCT CCAAGOGAGGAGTAA TGCCTGG F A T W S P S K A R L H L Q G R S N A W 2219 6730 6740 6750 6760 6770 6760 AGACCTCAGGTGAATAATCC'AAAAGAGTGBGCTG3CAAGTOGACTTCCAGAAGACAATGAP.A R FP 0 V N N P' K E W L Q V D F Q k T M K" 6790 6600 6810 6820 6630 6840 CTCACAGGAOTAACTACTCAOGGA6TAAAATCTCTGCTTACCAGCATGTATGTGAAGGAG V T 0 V T T QO V K S L L T S M Y V K" E 22bv 6850 6B60 6670 6880 6690 6900 Tt'CTCATCTCCA(CATCAAI-ATGCCATCAGTGACTCTCTTTTTTCAGAATGGCAAA L I S S SO O GH Q. WT L. F F0Q NOG K 6910 6920 6930 6940 6950 6960 GTAAAGGTI TTTCAGOOAAATCAAGACTCCTTCACACCT6G6TGAACTCTCTA6ACCCA V f, V F 0 G N 0 D S F T P V V N S L D P 2299 6970 6960 6990 7000 7010 7020 CCOTTACTGACTCGCTACCTTCGAATTCACCCCCAGAOTTGGGTGCACCAGATTGCCCTG P L L T RY L R I H P 0,S W V HO I1A L 7030 7040 7050 7060 7070 7080 AGGATGGAGGTTCTGOCTGCGAOOCACAGGACCTCTACTGAGGGTGGCCACT6CAOCAC R m E V L 0 C E A 0 DLVY 2332 stop 7090 7100 7110 7120 7130 7140 CTOCCACTGCCGTCACCTCTCCCTCCTCAGCTCCAGGGCAGTGTCCCTCCCTGGCTTGCC 7150 7160 7170 7160 7190 7200 TTCTACCTTTGTGCTAAATCCTA6CAGACACTGCCTTGAAGCCTCCTGAATTAACTATCA -i m mv. Q) 4 J4. 0 rc 04 w 10 41 E U U) E4J WO 88/09813- PCT/NL88/00028 .~9.L13 7210 7220 17230 7240 7250 7260 I'ATCGATCTGlG3(GCAGA7GGACATTATACCT 7270 7280 7290 7300 7:310 7320 SC CTT T T TC TEC AGCTGCrC C .Ci ATTACTcc-TTCC TTC CAATAT TAGAAA 7330 7340 7350 7360 7370 7360 AATAGGACTCTAACTCTCCGAATASCCCCGC 7390 7400 7410 7420 7430 7440 CCCTC*TTGAAAATTIGAGACTGAAGTTTTAG 7450 7460 7470 7480 7490 -7500 -rAATCGTAGCCTC'TrAAAACCCGTTTTACT 7510 7520 7530 7540 7550 7560 A TC A(GCAT GGACAA~O CA'TGT TTCC GAGA T ATAAC TT 66A6TCAG 6 7570 7560 7590 7600 7610 7620 C AAT CAT TTG6AC AArCTGCAAGAAT6AAACTAC TACAGTAAGT CT 6 76:30 764.0 7650 7660 7670 7680 7690 7700 7710 7720 7730 7740 7750 7760 7770 7780 7790 78300 0 TCC CC TGAi TTAI'A~~t TA -T C TGI'TAAATGCAAATGT (CATTTTTCTGAC6G OT 7610 7820 7830 7840 7850 7660 6 TCCA TA 3A i AACCATT'TGOT CTTAATTC TGACCAA AT iAAATAGT CAGGAGG 7870 780 7890 7900 7910 !7920 ATCA~GTAACTGATAAACAGCTTGATTTAGC 7930 7940 7950 7960 7970 7980 AAAAAATAGTA()TG3TC'PGA.TCTTAATCGG 7990 a U. 0 6010 8020 6030 6040 AATTAGAACAC" T'TCGGTi(AATCACTSATCAT 81050 83060 8070 83080 83090 6100 A CCTA TTCT C CCCTcrATCTG tAA~ACAACAAAA WO VO88/09813 .10/13 PCT/N L88/00028 811o 8120 61E130 8140 8150 8160 GTAAGGATAAACTATAAATGOCTCTCT~TAA 9170W 18 8190 6200 6210 6220 ATAGAGCGAAATGATGTAACGAAAATCGCGC 8230 8240 T iAT C(CCAC ACATAGGATC C 6- Ij I U I I I- I- I- A Figire 2 Ec oR1 (HI (S it) NdeI H KpnI Psi I XboI EcoR i pSV2 hRe LT actor M cDNA pSV2 H (Sol) (Hpal) (BgUI) I II II I L Figure 3 mRNA IOW-LTR c DNA insert 3 -pc4A intro.n start reading frame ib 711-""4pI a0i base pairs AM TC Id Al A3 Cl C2 so8 2020 2172233 2332 aino acids APC la so~SJ d ~4 2332 I rr- C1TCA :GATC" 1637 amfino acids I GACCCACGCG~rG iTA 1A 2332 UlII amino acids 00 00 Pwb 88/09813 it P CT/N LF,/0O28 13/13 I I Figure 4 12 Mr -x i-3 -200 -192 1w 68 -97 -68 "r M S A R A W P K M H T V N G3 Y V N 790 800 810 820 830 810 AGOTCT-rCTCCAGGTCTGATTGGATGCCACAGG AATCAGTCTATTGGCATGTGATTGGA R L F G L I G C H R K S -V Y W H V I G 259 850 860 870 880 890 900 ATGGGCACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTC:AICAATTTCTTGTGAGG M G T T P E V H S I F L E Vi H T F L V R 'N^ i, I w rl INTERNATIONAL SEARCH REPORT International Application No PCT/NL 88/00028 i' t i :f i i ii r, i%- i ~E i i ir 1. CLASSIFICATION OF SUBJECT MATTER (it several classification symbol apply, Indicate ill) According to international Patent Classification (IPC) or to both National Claasificaion and IPC 4 C 12 N 15/00; A 61 K 37/02; C 07 K 13/00; C 12 P 21/02; I PC C 12 N 1/00 II. FIELDS SIARCHED Minimum Documentation Saarched 7 Classification System Classification Symbols IPC 4 C 12 N; C 12 P; A 61 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched a III. DOCUMENTS CONSIDERED TO *E RELEVANT* Category Citation of Document, i" with Indication, where appropriate, of the relevant passages I Relevant to Claim No. X WO, A, 86/06101 (GENETICS INST.) 1-7,11-21 23 October 1986 see the whole document cited in the application X Biochemistry, volume 25, no. 26, 1-7,11-21 December 1986, American Chemical Society, (Washington, DC, US), D.L. Eaton et al.: "Construction and characterization .of an active factor VIII variant lacking the central one- third of the molecule", pages 8343- 8347 see the whole document cited in the application X EP, A, 0160457 (GENENTECH) 1-7,11-21 6 November 1985 see the whole document cited in the application P,X WO, A, 87/07144 (GENETICS INST) 1-7,11-21 SSpecial categories of cited documents: is later document publishe, s;aler the International filing date A" doeument defining the anerl state of the art which i notor priority date and not in conflict with the application but do nt defnng the gner tat o th art which no cd to understand the prnciple or theory underlying the considered to be of particular relevance invention earlier document but published on or after the International document of particular relevance: the claimed Invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an Inventive step which is cited to establish the publication date of another document of particular relevance" the claimed invention citation or other special reason (as apecified) cannot be considered to Involve an Inventive step when the document referring to an oral disclosure, use, eshlbltion or document is combined with one or more other such docu. other means ments, sucn combination being obvious to a person skilled document published prior to th.~ international filing date but In the art. later than the priority date claimed document member of the ame patent family IV. CERTIFICATION Date of the Actual Completion of the International Search September 1988 international Searching Authority EUROPEAN PATENT OFFICE Form PC'FIISA/210 (second shelt) (January t195) of this International Search Report fR DER PUTTEN International Aplication INo. PCT/NL 88/00028 ~WK~* Ill. DOCUMENTS CONSIDERED TO SE RELEVANT (CONTINUED FROM 1THE SECOND SWEET) Category *j Citation of DOU t with Iniain wh~ie &PWOPrtat@, Of the felvent passages Relevant to Claim No S P,x P,x P, X A A 3 December 1987 see page 14, paragraph 3 page 16, end; page 12, penultimate paragraph; claims 10-14 WO, A, 88/00831 (BIOGEN)

11 February 1988 see the whole document EP, A, 0265778 (ROHRER INT.) 4 May 1988 see the whole document EP, A, 02,53455 (GIST-BROCADES) January 1988 see page '17, line 29 page 19, line 34; page 53, lines 1-20; examples 9, 1-2j14; table 1; figures 11,17; claims 3,9,21,32 cited in the application EP, A, 0232112 (CHIRON CORP.) 12 August 1987 The Journal of Biological Chemistry, volume 261, no. 27, 25 September 1986,'The American Society of Biological Chemists, Inc., (US), R.L. Burke et al.: "tThe functional domains of coagulation factor VIII:C"1, pages 12574-12578 1-7,11-21 1-7,11-21 1-7,11-21 I K! .1 Form PCT ISA.;21Q (extra iiheet) (Jai~uary 1945) A .iii iliiii-il~ilC:: .i ii J jH i!;j j i:i- :i :f ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. NL 8800028 SA 22608 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 20/09/88 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date WO-A- 8606101 23-10-86 AU-A- 5772886 05-11-86 EP-A- 0218712 22-04-87 JP-T- 62502941 26-11-87 EP-A- 0160457 06-11-85 JP-A- 60243023 03-12-85 AU-A- 4134585 24-10-85 WO-A- 8707144 03-12-87 AU-A- 7486887 22-12-87 EP-A- 0270618 15-06-88 WO-A- 8800831 11-02-88 AU-A- 7804087 24-02-88 EP-A- 0275305 27-07-88 EP-A- 0265778 04-05-88 AU-A- 7981687 21-04-88 EP-A- 0253455 20-01-88 AU-A- 7575487 21-01-88 EP-A- 0232112 12-08-87 AU-A- 6801487 30-07-87 JP-A- 62282594 08-12-87 3 For more details about this annex see Official Journal cf the European Patent Office, No.

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