AU595640B2 - Modified c-dna sequence for factor 8c - Google Patents

Description

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S- S -j 111 C.L IL AU-AI-78040/ 87 PfT WORLD INTELLECTUAL PROPERTY ORGANIZATIOA SInternation 6ea INTERNATIONAL APPLICATION PUBLISHED UNfi e P NT OPRAT' NTREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 00831 A61K 35/14, C12P 21/00, 21/02 Al C12N 15/00 C12P 21/00 21/02 A (43) International Publication Date: 11 February 1988 (11.02.88) (21) International Application Number: PCT/US87/01814 (71)(72) Applicant and Inventor: PASEK, Mark, P. [US/US]; 177 Lexington Street, Belmont, MA 02178 (US).

(22) International Filing Date: 31 July 1987 (31.07.87) (74) Agents: HALEY, James, Jr. et al.; Fish Neave, 875 Third Avenue, New York, NY 10022-6250 (US).

(31) Priority Application Number: 893,375 (32) Priority Date: 1 August 1986 (01.08.86) (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (33) Priority Country: US pean patent), FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (European pa- Parent Application or Grant tent), US.

(63) Related by Continuation US 893,375 (CIP) Filed on 1 August 1986 (01.08.86) Published 2 t MAR 1988 With international search report.

(71) Applicant (for all designated States except US)J.-BIE) -E-N-NV--['NI-/NL-]-Pietermaai- 15 rWillemstad, Cu- ragao

AUSTRALIAN

7 2 4FE3 1 98 8 T PATENT OFFICE (54) Title: DNA SEQUENCES CODING FOR MODIFIED FACTOR VIII:C AND MODIFIED FACTOR VIII:C- LIKE POLYPEPTIDES AND PROCESSES FOR PRODUCING THESE POLYPEPTIDES IN HIGH

YIELDS

(57) Abstract DNA sequences coding on expression for modified factor VIII:C and modified factor VIII:C-like polypeptides and methods of making them in high yields in appropriate hosts transformed with those DNA sequences. DNA sequences containing internal deletions removing a major part of the sequence which codes on expression for the maturation polypeptide of factor VIII:C express modified factor VIII:C and modified factor VIII:C-like polypeptides 20 times more efficiently than DNA sequences coding for the factor VIII:C.

'fa lteaent aontaina t Sanaftk'neats mrad mat.r Sectea 49.

and Is oorr r filter I i i~ r I i 1 -1- DNA SEQUENCES CODING FOR MODIFIED FACTOR VIII:C AND MODIFIED FACTOR VIII:C-LIKE POLYPEPTIDES AND PROCESSES FOR PRODUCING THESE POLYPEPTIDES IN HIGH YIELDS TECHNICAL FIELD OF THE INVENTION This invention relates to DNA sequences coding for modified factor VIII:C-like polypeptides and processes for producing them using those DNA sequences. More particularly, this invention relates to the production of modified factor VIII:C and modified factor VIII:C-like polypeptides which display the biological activity of factor VIII:C.

In addition, the polypeptides of this invention are produced in higher yields than previously produced factor VIII:C-like polypeptides and are more easily purified into biochemically pure mature factor

VIII:C.

BACKGROUND OF THE INVENTION Factor VIII:C, a large plasma glycoprotein, functions as the procoagulant component of factor VIII, which plays an integral role in the cascade mechanism of blood coagulation [see generally, W. J. Williams et al., Hematology, pp. 1085-90, McGraw-Hill, New York (1972)]. Factor VIII:C circulates in the blood as a complex with factor VIIIR:Ag (also known as von Willebrand factor protein) which is a large WO 8/00831 PCT/US87/018 14 -2protein associated with platelet aggregation and adhesive properties.

Factor VIII:C is synthesized as a single chain macromolecular precursor, which is later cleaved to yield the fragments which constitute "mature" factor VIII:C. Mature factor VIII:C is composed of two chains bridged by a calcium ion; an amino-terminal heavy chain of 740 amino acids, and a carboxy-terminal light chain of 684 amino acids. The primary translation product of factor VIII:C is a single chain in which the heavy chain of mature factor VIII:C is separated from the light chain by a "maturation polypeptide" of 908 amino acids. The excision of this maturation polypeptide is initiated by proteolytic cleavage of the primary translation product by an unknown or yet unidentified protease at the Arg 1648 Glu 1649 peptide bond. The initial nick event begins a series of successive proteolytic cleavages which shorten the nascent heavy chain from its carboxy terminus. Eventually the mature heavy chain of 740 amino acids results and in combination with the light chain of 684 amino acids, comprises mature factor VIII:C [see Andersson et al.

"Isolation and Characterization of Human Factor VIII: Molecular Forms In Commercial Factor VIII Concentrate, Cryoprecipitate, and Plasma," PNAS(USA), 83, pp. 2979-83 (1986)]. This complex is then activated by thrombin by cleavage at the Arg 1689-Ser 1690 bond Eaton et al., Biochemistry, 25, pp. 505-12 (1986)].

7Haemophilia A is a sex-linked hemorrhagic disease which is caused by a deficiency, either in amount or in biological activity, of factor VIII:C.

The symptoms of acutely bleeding haemophilia patients are treated with factor VIII traditionally purified from normal sera. Various methods of purification have been described in the literature [see, Zimmerman WO 88/00831 PCT/US87/01814 I -3et al., United States patent 4,361,509; Saundrey et al. United States patent- 4,578,218; E.G.D.

Tuddenhem et al., "The Properties of Factor VIII Coagulant Activity Prepared By Immunoadsorbent Chromatography, Journal of Laboratory Clinical Medicine, 93, pp. 40-53 (1979); D. E. G. Austen, "The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose," British Journal of Hematology, 43, pp. 669-74 (1979); M. Weinstein et al., "Analysis of Factor VIII Coagulant Antigen In Normal, Thrombintreated, and Hemophilic Plasma," PNAS (USA), 78, pp. 5137-41 (1981); P. J. Fay et al., "Purification And Characterization Of A Highly Purified Human Factor VIII Consisting Of A Single Type Of Polypeptide Chain," PNAS (USA), 79, pp. 7200-04 (1982); C. A.

Fulcher and T. S. Zimmerman, "Characterization Of The Human Factor VIII Procoagulant Protein With A Heterologous Precipitating Antibody," PNAS (USA), 79, pp. 1648-52 (1982); F. Rotblat et al., Thromb.

Haemostasis, 50, p. 108 (1983); C. A. Fulcher et al., Blood, 61, pp. 807-11 (1983)].

However, purification has proven to be difficult because of the relatively low concentration of factor VIII:C in serum, its tight association with the larger factor VIIIR:Ag and its sensitivity to degradation by serum proteases. Factor VIII:C when purified from plasma thus contains a heterogeneous mixture of heavy chains ranging in length from 1648 amino acids down to 740 amino acids which result from these numerous proteolytic events [Andersson et al., supra, p. 2983]. The heterogenous mixture of chains observed in plasma-purified factor VIII:C, has made recovery of a substantially pure mature factor VIII:C almost impossible. Furthermore, traditional treatment of haemophilia with factor VIII purified from plasma has serious drawbacks. Specifically, it can lead to the unintended transfer of the WO 88/00831 PCT/US87/01814 WO 88/00831 -4causative agents of hepatitis or the virus associated with Acquired Immune Deficiency Syndrome.

In view of its importance in the treatment of haemophilia, numerous attempts have been made to produce large quantities of factor VIII:C using recombinant DNA technology [See, for example, Genetics Institute, PCT application W085/01961; Genentech European Patent application 160,457; Chiron European Patent application 150,735; J. J. Toole et al., "Molecular Cloning Of a cDNA Encoding Human Antihaemophilic Factor" Nature, 312, pp. 342-47 (1984); and W. I. Wood et al., Nature, 312, pp. 330-37 (1984)].

However, such attempts have proven to be less successful than had been hoped. This is partially due to the fact that the recombinantly produced 2332 amino acid factor VIII:C chain is subject to proteolytic cleavage at many positions. It is also due to difficulties in producing recombinant factor VIII:C in sufficiently high yields.

SUMMARY OF THE INVENTION The present invention solves the problems referred to above by providing DNA sequences which encode modified factor VIII:C and modified factor VIII:C-like polypeptides. These DNA sequences code for polypeptides which are produced in approximately twenty-times higher yields than previous recombinantly produced factor VIII:C and are more easily purified into biochemically pure mature factor VIII:C.

According the present invention, DNA sequences coding for modified factor VIII:C are produced and expressed in high yields. As will be apparent from the disclosure and examples to follow, the modified factor VIII:C and modified factor VIII:C-like polypeptides of this invention are characterized by deletions removing a major part of the maturation polypeptide of factor VIII:C. The DNA sequences in WO 88/00831 PCT/US87/01814 our preferred embodiment have a deleticn of substantially all of the nucleotides coding for the maturation polypeptide. Our most preferred embodiment contains a deletion of all the DNA sequence coding for the maturation polypeptide. On expression of our DNA sequences, the heavy chain of mature factor VIII:C is linked directly to the light chain. Following a one-nick proteolytic event, the mature form of factor VIII:C is generated.

Finally, the present invention provides various anti-haemophilic compositions containing modified factor VIII:C and modified factor VIII:Clike polypeptides produced by the DNA sequences of this invention, and various methods of using those compositions in haemophilia treatmenttherapy of acute or prolonged bleeding in haemophilia A.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a restriction map of the factor VIII:C cDNA.

Figure 2 is a schematic depiction of the construction of the recombinant DNA molecule with the QD deletion.

Figures 3A and 3B depict a schematic representation of the construction of the recombinant DNA molecule with the RE deletion.

Figure 4 depicts a restriction endonuclease map of the RE deletion inserted into the mammalian cell expression vector pBG312 indicating the positions of the SV40 origin of replication/enhancer, the adenovirus major late promoter, the factor VIII:C cDNA with the RE deletion, the 3' untranslated region of the factor VIII:C mRNA, and the polyadenylation site.

Figure 5 depicts the results of an Sl analysis of Factor VIIIC mRNA isolated from transfected BMT10 cells.

-6- Figure 6 depicts the results of a Southern analysis of plasmid DNA isolated from transfected cells.

Figure 7 depicts the published DNA and amino acid sequence of factor VIII:C (EPO application 160,457).

DETAILED DESCRIPTION OF THE INVENTION In order that the invention herein described may be more fully understood, the following detailed description is set forth.

In the description the following terms are employed: Nucleotide--A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine guanine cytosine and thymine The four RNA bases are A, G, C, and uracil DNA Sequence--A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

Codon--A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.

Amino Acid--A monomeric unit of a peptide, polypeptide or protein. The twenty amino acids are: phenylalanine ("Phe" or leucine r e~ WO 88/00831 PCT/US87/01814 1 -7isoleucine methionine valine serine proline threonine alanine tyrosine histidine glutamine asparagine lysine aspartic acid ("Asp", glutamic acid cysteine ("Cys", tryptophane arginine ("Arg", and glycine Reading Frame--The grouping of codons during the translation of mRNA into amino acid sequences.

During translation the proper reading frame must be maintained. For example, the DNA sequence GCTGGTTGTAAG may be expressed in three reading frames or phases, each of which affords a different amino acid sequence: GCT GGT TGT AAG--Ala-Gly-Cys-Lys G CTG GTT GTA AG--Leu-Val-Val GC TGG TTG TAA G--Trp-Leu-(STOP) Polypeptide--A linear array of amino acids connected one to the other by peptide bonds between the a-amino and carboxy groups of adjacent amino acids.

Genome--The entire DNA of a cell or a virus.

It includes inter alia the structural gene coding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine- Dalgarno sequences.

Gene--A DNA sequence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.

Transcription--The process of producing mRNA from a gene or DNA sequence.

Translation--The process of producing a polypeptide from mRNA.

S WO 88/00831 PCT/US87/01814 Expression--The process undergone by a gene or DNA sequence to produce a polypeptide. It is a combination of transcription and translation.

Plasmid--A nonchromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmiLd is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid.

For example, a plasmid carrying the gene for tetracycline resistance (TETR) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant".

Phage or Bacteriophage--Bacterial virus, many of which consist of DNA sequences encapsidated in a protein envelope or coat ("capsid").

Cloning Vehicle--A plasmid, phage DNA, cosmid or other DNA sequence which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.

Cloning--The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.

Recombinant DNA Molecule or Hybrid DNA--A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and able to be maintained in living cells.

WO 88/00831 PCT/US87/01814 -9- Expression Control Sequence--A sequence of nucleotides that controls and regulates expression of genes when operatively linked to those genes.

They include the lac system, the p-lactamase system, the trp system, the tac and trc systems, the major operator and promoter regions of phage X, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma virus and adenovirus, metallothionine promoters, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, Pho5, the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic microbial cells and their viruses or combinations thereof.

Factor VIII:C A polypeptide having the amino acid sequence of Figure 7, and upon maturation and activation, being capable of functioning as co-factor for the factor IXa-dependent maturation of factor X in the blood coagulation cascade. As used in this application, factor VIII:C includes the glycoproteins also known as factor VIII procoagulant activity protein, factor VIII-clotting activity, antihemophilic globulin (AHG), antihemophilic factor (AHF), and antihemophilic factor A [see W. J. Williams et al., Hematology, pp. 1056, 1074 and 1081].

Maturation Polypeptide The maturation polypeptide of factor VIII:C is made up of the 908 amino acids from amino acid Ser (741) to amino acid Arg (1648) (see Figure Maturation of factor VIII:C is initiated with a cleavage between amino acids 1648 and 1649 (which produces a C-terminal light chain) followed by a series of nicks which produce the mature N-terminal heavy chain.

I ii I I WO 88/00831 PCT/US87/01814 Mature Factor VIII:C As used in this application, mature factor VIII:C is composed of an N-terminal heavy chain (Ala 1- Arg 740) linked to a C-terminal light chain (Glu 1649-Tyr 2332) through an alkaline metal bridge, such as calcium (Figure 7).

Modified Factor VIII:C As used in this application, "modified factor VIII:C" refers to polypeptides characterized by a deletion of a major portion of the maturation polypeptide of factor VIII:C.

For example, where the entire maturation polypeptide has been deleted, "modified factor VIII:C" includes proteins that comprise the N-terminal mature heavy chain and the C-terminal mature light chain of factor VIII:C linked together as a single chain.

Modified Factor VIII:C-Like Polypeptide As used in this application, "modified factor VIII:Clike polypeptide" includes proteins having the biological activity of modified factor VIII:C. It also includes proteins having an amino terminal methionine, f-Met-factor VIII:C, and proteins that are characterized by other amino acid deletions, additions or substitutions so long as those proteins substantially retain the biological activity of modified factor VIII:C.

"Modified factor VIII:C-like polypeptides" within the above-definition also includes natural allelic variations that may exist and occur from individual to individual. Furthermore, it includes modified factor VIII:C-like polypeptides whose degree and location of glycosylation, or other post-translation modifications, may vary depending on the cellular environment of the producing host or tissue.

11 The present invention relates to processes for the production of modified factor VIII:C and modified factor VIII:C-like polypeptides. More particularly, it provides DNA sequences which permit the production of modified factor VIII:C and modified Sfactor VIII:C-like polypeptides in high yields, in appropriate hosts. Polypeptides produced by the DNA sequences of this invention are useful in the clinical treatment of haemophilia A.

As compared to factor VIII:C, the modified factor VIII:C produced by the DNA sequences of this invention lack a major portion of the maturation polypeptide of factor VIII:C. The DNA sequences of the present invention surprisingly express modified factor VIII:C in much higher yields than DNA sequences coding for factor VIII:C itself.

While not wishing to be bound by theory, we believe that the DNA sequences of the present invention produce modified factor VIII:C in high yields because of the absence of most or all of the maturation polypeptide. For example, the mRNA for the modified gene may be translated more efficiently, because the RNA coding for the long maturation polypeptide does not have to be translated. In addition, while factor VIII:C has many proteolytic targets which may be attacked while the polypeptide is in the cell, the modified factor VIII:C is less subject to such proteolytic attack because it lacks the proteolytic targets within the maturation polypeptide. Furthermore, when the maturation polypeptide is absent, 19 of the 25 N-linked glycosylation sites of native factor VIII:C are deleted, leaving only six N-linked glycosylation S e tide, comprising the N-terminal heavy chain of mature factor VIII:C linked directly to the C-terminal i.$hchain of mature factor VIII:C, said polypeptide bsrin WO 88/00831 PCT/US87/01814 -12sites on the modified polypeptide (three on the heavy chain and three on the light chain). Apparently, because there are fewer sites to be glycosylated, production and purification of the modified factor VIII:C is simplified.

In the processes of this invention, we modify the DNA sequence encoding factor VIII:C to delete from it 'a major portion of the DNA sequence encoding the maturation polypeptide. Having prepared a DNA sequence carrying the desired deletion we employ it in a variety of expression vectors and hosts to produce modified factor VIII:C encoded by it. For example, any of a wide variety of expression vectors are useful in expressing the modified factor VIII:C coding sequences of this invention. It also should be understood that DNA sequences encoding a modified factor VIII:C-like polypeptide can be similarly produced in accordance with this invention.

Useful expression vectors include, for example, vectors consisting of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of known bacterial plasmids, plasmids from E.coli including col El, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, the numerous derivatives of phage X, NM 989, and other DNA phages, e.g., M13 and Filamenteous single stranded DNA phages, yeast plasmids such as the 2p plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences. In the preferred embodiments of this invention, we employ pBG312, a pBR327-related vector Cate et al., Cell, 45, pp. 685-98 (1986)].

In addition, any of a wide variety of expression control sequences sequences that con- ~~h't 9 WO 88/00831 PCT/US87/01814 -13trol the expression of a DNA sequence when operatively linked to it may be used in these vectors to express the DNA sequence of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage X, the control regions of fd coat protein, the promoter for 3-phosphoglycerat3 kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. In the preferred embodiment of this invention, we employ adenovirus-2 major late promoter expression control sequences.

A wide variety of host cells are also useful in producing the modified factor VIII:C of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO cells, African green monkey cells, such as COS1, COS7, BSC1, and BMT10, and human cells and plant cells in tissue culture. In the preferred embodiments of this invention, we prefer BMT10 African green monkey cells.

It should of course be understood that not all vectors and expression control sequences will function equally well to express the modified DNA sequences of this invention and to produce our modified factor VIII:C. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation without departing from the scope of this invention. For A WO 88/00831 PCT/US87/01814 -14example in selecting a vector, the host must be considered because the vector must replicate in it.

The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the DNA sequence encoding the modified factor VIII:C of this invention, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of our modified factor VIII:C to them, their secretion characteristics, their ability to fold proteins correctly, their fermentation requirements, and the ease of the purification of our modified factor VIII:C from them and safety.

Within these parameters one of skill in the art may select various vector/expression control system/host combinations that will produce useful amounts of our modified factor VIII:C on fermentation. For example, in one preferred embodiment of this invention, we use an pBG312 vector, with an adenovirus 2 major late promoter expression system in BMT10 African green monkey cells.

The modified factor VIII:C and modified factor VIII-like polypeptides produced according to this invention may be purified by a variety of conventional steps and strategies. Useful purification steps include those used to purify natural and recombinant factor VIII:C [see, for example, Andersson et al., PNAS (USA), 83, pp. 2979-83 (1986)].

After purification the modified factor VIII:C and modified factor VIII:C-like polypeptides j 0 15 of this invention are useful in composition and methods for treatment of haemophilia A and in a variety of agents useful in treating uncontrolled bleeding.

While the modified factor VIII:C and modified factor VIII:C-like polypeptides of this invention may be administered in such compositions and methods in the Sform in which they are produced, as single chain polypeptides, it should also be understood that it is within the scope of this invention to administer the modified factor VIII:C after subjecting it to proteolytic cleavage. For example, modified factor VIII:C can be cleaved in vitro, into the heavy chain and light chain of mature factor VIII:C and linked with a calcium or other alkaline metal bridge, before, during or after purification.

The modified factor VIII:C and modified factor VIII:C-like polypeptides of this invention may be formulated using known methods to prepare pharmaceutically useful compositions. Such compositions also will preferably include conventional pharmaceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., human serum albumin or plasma preparations. See, Remington's Pharmaceutical Sciences W. Martin). The resulting formulations will contain an amount of modified factor VIII:C effective in the recipient to treat uncontrolled bleeding. Administration of these polypeptides, or 0. pharmaceutically acceptable derivatives thereof, may be via any of the conventional accepted modes of administration of factor VIII. These include parenteral, subcutaneous, or intravenous administration.

The compositions of this invention used in the therapy of haemophilia may also be in a variety of forms. The preferred form depends on the intended mode of administration and therapeutic application.

*US*

-j .i 0* L 6 6 66 6 666 6 6 66666 *6 6 6 666,* 6 6" 66 6 PCT/US87/01814 WO 88/00831 -16- The dosage and dose rate will depend on a variety of factors for example, whether the treatment is given to an acutely bleeding patient or as a prophylactic treatment. However, the factor VIII:C level should be high enough to prevent hemorrhage and promote epithelialization [see discussicn in Willias, Hematology, pp. 1335-43].

In order that this invention may be better understood, the following example is set forth.

This example is for purposes of illustration only and is not to be construed as limiting the scope of the invention.

EXAMPLE

We have constructed cDNA sequences which encode modified factor VIII:C molecules having a deletion of a major part of or all of the maturation polypeptide. To test the limits of our invention, we also constructed a cDNA sequence which encodes a polypeptide having a deletion of more than just the maturation polypeptide of factor VIII:C.

A. ASSEMBLY OF THE FULL-LENGTH FACTOR VIII:C cDNA Referring now to Figure 1, we have presented therein a restriction enzyme map of the factor VIII:C cDNA based upon the published sequence I. Wood et al., Nature, 312, pp. 330-37 (1984); (Figure The bar represents the coding sequence. Below the Srestriction enzyme map we have depicited the aminoterminal heavy chain of mature factor VIII:C attached by a calcium bridge to the carboxy-terminal light chain of mature factor VIII:C. Below the protein model on a bar congruent to the restriction enzyme map we have indicated the oligonucleotide probes (indicated with asterisks) which we used to screen human placenta, liver, and kidney cDNA libraries.

cally, it can lead to the unintended transfer of the WO 88/00831 PCT/US87/01814 -17- These libraries were made using oligo (dT) as firststrand primer and Xgtl0 as vector.

On this second bar are also located the oligonucleotide primers (left-arrows) which we used to initiate first-strand cDNA synthesis using human kidney mRNA as template. We made these singlestranded cDNA sequences double-stranded by the technique of Gubler and Hoffman Gubler, and B. J. Hoffman, Gene, 25, pp. 263-69 (1983)]. We cloned them at the dC-tailed EcoRV site in pBR322.

We then screened this plasmid-based kidney cDNA library with oligonucleotide probes located on the bar 5' to the oligonucleotide primers.

Below the primer/probe bar in Figure 1, we have displayed a collection of partial-length factor VIII:C cDNA and gbnomic subclones, which we isolated from these libraries. Together these encode the full-length cDNA gene. More information about these clones is presented below, in Table 1.

polypep.iLUe uoL a.C-LU vII..-

I

PCT/US87/01814 WO 88/00831 -18- TABLE 1 COMPENDIUM OF FACTOR VIII:C GENOMIC AND cDNA CLONES isolated from a genomic library constructed with cosmid pTCF [Grosveld, F.G. et. al., Nucleic Acids Research, 10, pp. 6715-6732 (1982)1 subclone pUC19.2874 length 2874 bp tissue 48,XXXX human lymphoblast isolated from oligo(dT)-primed Xgtl0 cDNA libraries clone length probe hybridization 1.7977 (placenta) 2.73 (liver) 4.73 (kidney) 1728 ~700 ~220 79+, 77+ 73+ 73+ isolated from a 85, 86-primed Gubler-Hoffman kidney cDNA library clone 1.82 2.82 3.7573 4.7573 6.7573 10.797783 11.797783 12.797783 13.797783 length -1200 -1200 -2700 -2700 ~2700 >1263 >1263 >1263 >1263 probe hybridization 82+, 79-, 82+, 79-, 74-, 75+, 74-, 75+, 74-, 75+, 82-, 79+, 82-, 79+, 82-, 79+, 82-, 79+, 77- 77- 73+ 73+ 73+ 77+, 83+ 77+, 83+ 77+, 83+ 77+, 83+ isolated from a 75, 77-primed Gubler-Hoffman kidney cDNA library clone 7.7475 length -2700 probe hybridization 74+, fected BMT10 cells.

19 Prior to assembling the full-length cDNA gene, we constructed two intermediate plasmids. This was necessary because of the excessive length of the factor VIII:C cDNA. For our first preliminary construction we isolated a fragment from clone 7.7475 extending from the PstI site at 5163 to the PstI site at 5755. We inserted this fragment into clone 4.7573 at the PstI site at 5755 thereby extending clone 4.7573. This PstI site is shared by the inserts of both clone 7.7475 and clone 4.7573. By extending clone 4.7573 in this manner, we provided a unique NdeI site at 5522 in the insert of this derivative of clone 4.7573. We needed to create this Nde site because we needed a unique site at which to extend the length of this insert at its 5' end.

As a second preliminary construction we introduced a polynucleotide linker in clone 2.82 at a location immediately 5' to the translation start codon of the signal sequence of factor VIII:C. The insert of clone 2.82 is at the EcoRI site of pBR322 and its orientation is opposite to that of tetracycline resistance. The 5' endpoint of the insert in clone 2.82 is at -133 in the 5' untranslated leader sequence. We cleaved clone 2.82 at the SalI site in tetracycline resistance and at the SacI site in the sequence encoding the signal peptide in the insert of clone 2.82 and inserted the synthetic duplex H

BH

SAI N N BSGNSS ACN R C APISAS LCC U O N1APCT 112 1 1 221211

GTCGACTCGCGACCATGGATGCAAATAGAGCTC

1 33

CAGCTGAGCGCTGGTACCTACGTTTATCTCGAG

MetGlnIleGluLeu This ligation resulted in the introduction of a SalI- NruI-NcoI polylinker immediately 5' to the start 0 1C *0 0 .0.

V: 0 1 I 11 WO 88/00831 PCT/US87/01814 codon which initiates translation of the signal sequence of factor VIII:C. These three restriction enzymes do not cleave the full-length factor VIII:C cDNA gene.

With these two intermediate constructions available, we assembled the full-length factor VIII:C cDNA in a six-fragment ligation reaction (bottom Figure It was necessary to create the full length DNA in this manner because we never isolated the full DNA in one single clone. We isolated fragment 1 from the above-described derivative of clone 2.82.

Fragment 1 extended from SalI in the polylinker to AvaI at 731. We isolated Fragment 2 from the insert in the AgtlO recombinant 1.7977. Fragment 2 extended from AvaI at 731 to EcoRI at 2289. Fragment 3 derived from the subclone pUC19.2874 of a genomic cosmid recombinant; it extended from EcoRI at 2289 to BamHI at 4743. Fragment 4 was isolated from clone 7.74575, starting from the BamHI site at 4743 and extending to the NdeI site at 5522. We isolated fragment from the above-described derivative of clone 4.7573.

Fragment 5 extended from Ndel at 5522 to NcoI at 7991. Fragment 6 is an assembly vector containing an E.coli replication origin and selectable marker for ampicillin resistance. We isolated Fragment 6 from pAT.SV2.tPA, a gift from Richard Fisher. This is a plasmid in which the transcription of the tPA gene is under the control of the SV40 early promoter.

We digested pAT.SV2.tPA with Sall which cleaves within the tetracycline resistance marker, and with NcoI which cleaves within the SV40 early region.

Of the 96 recombinants we analyzed, 32 contained all five factor VIII:C restriction fragments. We determined the DNA sequence of one of these clones, and we identified two changes with respect to the published sequence. One is a CTG to i^ II WO 88/00831 PCT/US87/01814 1 -21- CTA change at Leu 242 and the other is a TTC to CTC change at amino acid residue 1880 (Phe to Leu) (compare Figure 7).

B. INSERTION OF THE FULL-LENGTH cDNA INTO A MAMMALIAN CELL EXPRESSION

VECTOR

We excised the full-length factor VIII:C cDNA gene from the assembly vector by digestion with NcoI. We then treated the resultant NcoI restriction fragment with nuclease Sl to create a blunt end. We ligated this fragment to SmaI-digested pBG312. pBG312 is an animal cell expresion vector whose construction has been described elsewhere Cate et al., Cell, pp. 685-98 (1986)]. The sequence of BG312, from EcoRI to BamHI has (clockwise): a SV40 replication origin; an adenovirus-2 major late promoter and complete tripartite leader Zain et al., Cell, 16, pp. 851-61 (1979)]; a hybrid splice signal consisting of an adenovirus-5 splice donor and an immunoglobulin variable region gene splice acceptor J. Kaufman, and P. A. Sharp, J. Mol. Biol., 159, pp. 601-21 (1982)]; a polylinker containing sites for HindIII, XhoI, EcoRI, SmaI, NdeI, SstI, and BglII; the small t antigen intron flanked by its splice donor and acceptor; and the SV40 early polyadenylation site.

We verified the DNA sequence across the junction between the polylinker of pBG312 and the cDNA gene encoding factor VIII:C including the signal sequence for two independent clones: 8.1 and 8.2.

Clone 8.1 differs from 8.2 in the 3' untranslated region; T 7806 is fused to the Smal site of pBG312 in clone 8.1 instead of the C of the NcoI site at 7990 in clone 8.2. In addition, we isolated another clone, in which the fusion of the cDNA gene encoding factor VIII:C to pBG312 had occured within the sequence encoding the signal peptide of factor I i 22 VIII:C. This clone, which we named signal-minus, provided a negative control for our transient expression assays, described below.

C. CONSTRUCTION OF GLN 744 ASP 1563 (ABBREVIATED QD) DELETION In this section we demonstrate how we created the QD deletion which removes a portion of DNA sequence coding on expression for the maturation polypeptide (amino acids 741-1648). The QD deletion retains approximately 90 amino acids of the maturation polypeptide (four amino acids at the N-terminal end of the maturation polypeptide and 86 amino acids at its carboxy terminal end).

Referring now to Figure 2, we depict therein the construction of the QD deletion. We partially digested oie aliquot of the expression plasmid for the full-length factor VIII:C gene with EcoRI. This endonuclease cleaves between the codons for Gln 744 and Asn 745. We removed the 5'AATT overhang with nuclease Sl, and then subjected the plasmid to complete digestion with PvuI within the ampicillin resistance gene. We partially digested another aliquot with BamHl, which cleaves between the codons for Leu 1562 and Asp 1563 (see Figure We filled out the 5'GATC overhang with the Klenow fragment, and again digested the plasmid with PvuI within amp. We then combined the two mixtures of fragments and ligated them with T4 DNA ligase. A BamHl site between the codons for Gln 744 and Asp 1563 was created in this fusion.

The modified polypeptide produced on expression as a result of the QD deletion lacks 818 amino acids from within the 908 amino-acid maturation polypeptide, leaving 4 amino acids C-terminal to the carboxy terminus of the mature heavy chain, Arg 740, and leaving 86 amino acids N-terminal to the amino Op

OFFC

4 a a a 23 terminus of the light chain, Glu 1649 (Figure The 908 amino-acid maturation polypeptide is thus replaced by a 90 amino-acid maturation polypeptide, with the protease substance for both initial maturation of the primary translation polypeptide and subsequent maturation of the heavy chain remaining intact.

D. CONSTRUCTION OF THE ARG 740 GLU 1649 (ABBREVIATED RE) DELETION We demonstrate in this section how we created the RE deletion, which removes the entire DNA sequence coding for the maturation polypeptide.

Referring now to Figures 3A and B, we show how we obtained this RE deletion fusion in two steps.

In the first step we ligated four fragments which resulted in an intermediate plasmid. These four fragments were: the 462 bp fragment, obtained by digesting the expression plasmid for the full-length gene with HindIII between the codons for Arg 740 and Ser 741, removing the 5' AGCT with nuclease Sl, and subsequently digesting with KpnI which cleaves uniquely between the codons for Tyr 586 and Leu 587.

the synthetic oligonucleotide duplex fragment 5'pGAA ATA ACT CGT ACT ACT CTT CAG TCA CTT TAT TGA GCA TGA TGA GAA GTC AGT CTA Gp Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp 1649 1657 the 135 bp fragment obtained by digesting the expression plasmid for the full-length gene first with Sau3A; we isolated the 411 bp fragment which resulted from Sau3A digestion between the codons for Ser 1657 and Asp 1658 and between the codons for Glu 1794 and Asp 1795. Then, we digested the 411 bp fragment with PstI which cleaves between the codons :0 S: n• 0 0e 0 :0 WO 88/00831 PCT/US87/01814 -24for Ala 1702 and Val 1703, to obtain the 135 bp fragment.

pUC18 digested with KpnI and PstI.

We then isolated a fragment encoding the RE fusion from this intermediate plasmid. To do this, we digested the intermediate plasmid generated in the four-fragment ligation with Asp718 and PstI.

The fragment encoding the RE fusion was used to replace the corresponding fragment in the expression plasmid for the QD fusion. We ligated the resultant 624 bp fragment encoding the RE fusion to the mixture of fragments which we obtained by first completely digesting the expression plasmid for the QD internal deletion at the unique Asp718 site, next dephosphorylating the 5' GTAC overhang with calf intestinal phosphatase, and then partially digesting the plasmid with PstI.

Referring now to Figure 4, we depict therein a map of the RE deletion inserted into pBG312. In the modified polypeptide produced on expression the 908 amino-acid maturation polypeptide is entirely removed. The novel polypeptide produced iby this recombinant molecule cell is secreted, and may be purified as a single chain, the heavy chain is linked directly to the light chain. Because the Arg 1648 Glu 1649 peptide bond which is normally cleaved during the initial nicking of the full-length primary translation product is preserved in this deletion, the primary translation product for this internal deletion is nicked by the same protease that initiates nicking of the full-length primary translation product, thus producing directly the mature form of the heavy chain of factor VIII:C. Our Western blot analysis (data not shown) confirms that the RE modified factor VIII:C encodes a single chain molecule which is then processed into a 90K heavy chain and an 80K light j $c2 :y S WO 88/00831 PCT/US87/01814 chain in the culture medium. The resultant light chain possesses the peptide from Glu 1649 to Arg 1689 that binds the two-chain complex to von Willebrand protein. For this reason, this recombinant product, when secreted from a mammalian cell, will bind to the von Willebrand protein present in cell culture fluid. Similarly, when injected, it will complex to and circulate with plasma von Willebrand protein. Upon thrombin cleavage at Arg 1689 Ser 1690, the two-chain mature factor VIII:C will be activated and will dissociate from von Willebrand protein and assemble into its ternary complex with factor IXa and factor X on a platelet surface.

E. CONSTRUCTION OF THE ARG 740 SER 1690 (ABBREVIATED RS) DELETION In order to test the outer limits.of these deletions, we constructed a plasmid which codes for a polypeptide with a deletion of more than the maturation polypeptide alone we deleted the DNA sequence which codes on expression for the fortyone amino acids at the N-terminal end of the light chain of mature factor VIII:C).

We constructed this RS fusion with the two-step strategy described above for the RE fusion.

Our first step was a three-fragment ligation resulting in an intermediate plasmid. The three fragments which we ligated were: the 462 bp fragment, obtained by digesting the expression plasmid for the full-length gene with HindIII between the codons for Arg 740 and Ser 741, removing the 5' AGCT with nuclease Sl, and subsequently digesting with KpnI which cleaves uniquely between the codons for Tyr 586 and Leu 587.

the synthetic oligonucleotide duplex fragment: i j I I l riTlr_ ~r ;rr WO 88/00831 PCT/US87/01814 -26pAGC TTT CAA AAG AAA ACA CGA CAC TAT TTT ATT GCT GCA TCG AAA GTT TTC TTT TGT GCT GTG ATA AAA TAA CGp Ser Phe Gin Lys Lys Thr Arg His Tyr Phe Ile Ala Ala 1690 1702 pUC8 digested with KpnI and PstI.

In this fusion, we recreated the HindIII site between the codons for Arg 74J and Ser 741 (now Ser 1690).

We isolated a fragment encoding the RS fusion from this intermediate plasmid and used this fragment in our second step to replace the corresponding fragment in the expression plasmid for the QD fusion. In this second step, we isolated a 501 bp fragment encoding the RS fusion. We digested the intermediate plasmid with Asp718 and PstI and isolated the fragment encoding the RS fusion. We then used the strategy described above for the RE fusion to replace the related fragment in the expression plasmid for the QD fusion with the 501 bp fragment.

In addition to removing'the entire maturation polypeptide, the RS deletion removes DNA coding for the Glu 1649 Arg 1689 peptide, the putative von Willebrand binding domain. For this reason this recombinant molecule will not attach to circulating von Willebrand protein when it is secreted from an animal cell into culture fluid or when it is injected into a recipient.

F. TRANSFECTION OF AFRICAN GREEN MONKEY KIDNEY CELLS We transfected BMT10 cells D. Gerard and Y. Gluzman, Mol. Cell. Biol., 5, pp. 3231-40 (1985)] with the supercoiled expression plasmid. We used the DEAE-dextran technique M. Sompayvac and K. J. Danna, PNAS, 78, pp. 7575-78 (1981)] and chloroquine Luthman and G. Magnusson, Nucleic Acids Research, 11, pp. 1295-1308 (1983)] to trans- WO 88/00831 PCT/US87/01814 -27fect the cells. Transfectants are known to replicate the input expression plasmid to high copy number because SV40 T antigen is inducibly supplied in trans by BMT10 cells and binds to the SV40 origin of replication linked to the modified factor VIII:C gene in the expression plasmids. However, this technique is inefficient because, typically, only several percent of the transfected cells will actually incorporate DNA.

The transfectants will secrete modified factor VIII:C for up to 120 hours. For most experiments, the cm 2 /ml ratio is approximately 5.5; that is, a confluent monolayer of BMT10 transfectants in a 100 mm Petri dish (55 cm 2 is covered with 10 ml culture fluid.

G. FACTOR VIII:C ACTIVITY ASSAY We assayed the signal-minus, 8.1, QD, RE and RS expression constructs for factor VIII:C production after transfection in duplicate .nto BMT10 cells.

We used a 96-well plate adaptation of KabiVitrum's Coatest@ Factor VIII:C. One petri dish was used to prepare RNA for Sl analysis and the other petri dish was used to prepare Hirt DNA used in our Southern analysis. After 120 hours of incubation we assayed the cell culture fluids for factor VIII:C activity.

We expressed our results in terms of plasma level, where plasma factor VIII:C concentration is approximately 200 ng/ml.

In repeated transfections, both the signalminus construct (negative control) and the RS deletion have shown no detectable factor VIII:C activity.

This may be explained by the deletion of the von Willebrand protein binding domain in the RS deletion.

In the 120 hour experiment analyzed below, cells transfected with the full-length gene produced approximately 5% of the activity observed with both the QD and the RE deletions. The activity observed 1- WO 88/00831 PCT/US87/01814 -28with the QD deletion was 1.46% plasma level and that for the RE deletion was 1.30% plasma level.

Thus, we observed that BMTIO cells transfected with the QD and RE deletions produce at least 20 times more factor VIII:C than cells transfected with the full-length gene.

H. NUCLEASE Sl ANALYSIS OF FACTOR VIII:C mRNA In order to determine the levels of mRNA in each construction, we conducted a nuclease Sl analysis. This assay assists in the determination of the reason for the increased level of expression in our QD and RE deletions.

We isolated RNA from 100 mm Petri dish cultures of BMT10 cells 120 hours after transfection, using the unpublished method of W. Schleuning and J. Bertonis. Briefly, according to this method, we lysed BMT10 cells with 3 ml of 50 mM Tris-HCl (pH 5 mM EDTA 1% SDS containing 100 pg/ml proteinase K for 20 minutes at 37 0 C. We transferred the lysate to a 50 ml conical tube containing 3 ml of phenol and then mechanically sheared the DNA for seconds at high speed in a Polytron (Brinkmann Instruments). We extracted the aqueous phase with ether and adjusted it to 0.25 NaCl. We precipitated the nucleic acid fraction at 4 0 C, by the addition of an equal volume of isopropanol, collected it by centrifugation and redissolved it in 3 ml of water.

We selectively precipitated RNA overnight at 4 0 C, by adjusting the solution to 2.8 M LiCl.

We determined the amount of modified factor VIII:C mRNA for each construction. We isolated probes for the Sl analysis by digesting the QD expression plasmid with EspI. We labelled the 5' ends of the EspI fragments with [y-32P]ATP and T4 polynucleotide kinase, and annealed 10 pg RNA to 5000 cpm of the 32 P-antisense strand of the 477 nucleotide EspI fragi 0 0 A S h I 0 0 0 0 *0 00 0 0 0 WO 88/00831 PCT/US87/01814 -29ment isolated on a 5% strand separation gel M.

Maxam and W. Gilbert, Methods In Enzymology, pp. 499-560 (1980)]. We incubated the RNA overnight at 48 0 C in 10 -1 80% deionized formamide 400 mM NaCl 40 mM PIPES (pH 6.4) 1 mM EDTA. The hybrid molecules were then digested for 60 minutes at 370C by adding 190 p1 nuclease S1 at a concentration of 100 units/ml in 0.28 M NaC1 50 mM NaOAc (pH 4.6) mM ZnSO4. We terminated the digestion by adding EDTA to 10 mM and extracting with phenol. We denatured the protected fragments and subjected them to electrophoresis on a 5% strand separation gel.

We exposed the dried gal to Kodak XAR-5 X-ray film backed by a Lightning-Plus intensifying screen (Dupont) overnight at -700C. The 477 nucleotide EspI fragment has one end within the hybrid intron spliced out from the 5' untranslated region of the factor VIII:C mRNA J. Kaufman and P. A. Sharp, J. Mol. Biol., 159, pp. 601-21 (1981)] and the other end within the codon for Ala 62 (Figure 4).

We detected modified factor VIII:C mRNA by protecting a single-stranded 300 nucleotide DNA fragment from digestion. The experiment was repeated with 1 pg RNA in order to verify that the singlestranded probe was in excess.

The results of nuclease S1 analysis of modified factor VIII:C mRNA for each construct are shown in Figure 5. Our results indicated that modified factor VIII:C mRNA levels are the same for all three deletions and the full-length factor VIII:C gene. Figure 5A is the analysis for 10 pg of input RNA, and Figure 5B is the analysis for 1 pg of input RNA. Lane 1 in both figures contains as marker 500 cpm of the labeled 477 nucleotide single-stranded DNA fragment used to protect modified factor VIII:C mRNA from Sl digestion; that is, 10% of the input to each hybridization reaction. Lane 2 contained RNA WO 88/00831 PCT/US87/01814 isolated from BMT10 cells transfected with the signalminus construct; lane 3, BMT10 cells transfected with the full-length factor VIII:C cDNA (construct lane 4: BMT10 cells transfected with modified factor VIII:C cDNA (QD deletion); lane 5: BMT10 cells transfected with modified factor VIII:C cDNA (RE deletion); lane 6: BMT10 cells transfected with the cDNA from the RS deletion; lane 7: marker fragments obtained by digesting pBR322 with HinfI and labeling their 3' ends with [a- 3 2 P]dATP and Klenow enzyme (a gift of Richard Tizard). Equal amounts of a protected fragment of the expected length of 300 bases are evident in both figures for the 8.1, QD, RE, and RS constructs. A protected fragment of approximately 220 bases in length for the signal-minus construct is evident in both figures, reflecting the absence of a portion of the DNA sequence encoding the signal peptide.

A comparison of Figures 5A and 5B demonstrates that the input 477 probe is in molar excess during the hybridizations for each construct.

Although the modified factor VIII:C activity levels are at least 20-fold higher for the QD and RE deletions compared to the RS and the full-length constructs, the amount of mRNA in all four constructs is very nearly the same. Therefore, the reason for the increase in expression for the QD and RE deletions is post-transcriptional in nature.

I. SOUTHERN ANALYSIS OF PLASMID DNA ISOLATED FROM TRANSFECTED

CELLS

We conducted this analysis to determine the DNA levels of newly-replicated modified factor VIII:C plasmids for our deletions, in comparison with the full-length gene. Again, this assay assisted in our determination of the reason for the high yields of modified factor VIII:C in our QD and RE deletions.

I i WO 88/00831 PCT/US87/01814 -31- In order to control for differences in DNA replication in BMT10 cells for the various constructs, we performed a Southern analysis of extrachromosomal DNA isolated from each transfection. We isolated DNA from 100 mm petri dish cultures of BMT10 cells 120 hours after transfection according to the method of Hirt Hirt, J. Mol. Biol., 26, pp. 365-69 (1967)].

For each construction, we digested 0.5 A260 units with DpnI to distinguish newly-replicated (DpnIresistant) DNA from input methylated bacterial DNA (Dpnl-sensitive). We electrophoresed the DNA fragments on a 0.7% agarose gel, and blotted them to GeneScreen Plus to analyze the DNA. The filter was hybridized at 65 0 C in 1 M NaCl 50 mM Tris-HCl (pH 7.5) 0.1% sodium pyrophosphate 0.2% polyvinylpyrrolidone 0.2% Ficoll 0.2% BSA 1% SDS using 105 cpm/ml denatured probe. We then washed the filter at 65 0 C with the same buffer and exposed it overnight at -70 0 C to Kodak XAR-5 X-ray film backed by a Lightning-Plus intensifying screen (Dupont).

The factor VIII:C probe was the 2924 bp EspI fragment isolated from the RE expression plasmid (see Figure 4) and 3 2 P-labeled to a specific activity of 109 cpm/pg by the random hexadeoxynucleotide primer method of Feinberg and Vogelstein P. Feinberg and B. Vogelstein, Anal. Biochem., 132, pp. 6-13 (1983)].

Our results, which are depicted in Figure 6, indicate that newly-replicated modified factor VIII:C plasmid DNA levels are the same for all three deletions and the full-length gene. Lane 1 contained the 1 kb ladder obtained from BRL and labeled with T4 DNA polymerase according to the manufacturer's protocol; lane 2: 1 ng supercoiled RE DNA; lane 3: 10 ng supercoiled RE DNA; lane 4: 10 ng RE DNA digested with DpnI; lane 5: DpnI digest of 0.5 A260 units Hirt fraction obtained from BMT10 cells transfected with the signal-minus construct; lane 6: transfected WO 88/00831 PCT/US87/01814 -32with the full-length factor VIII:C cDNA (construct lane 7: transfected with the QD deletion; lane 8: transfected with the RE deletion: lane 9: transfected with the RS deletion. Figure 6 shows nearly equal amounts of the supercoiled form of each construct after digestion with DpnI (lanes thus excluding the possibility that differences in DNA replication enhance the expression of the QD and RE deletions. Lane 2 contains 108 molecules of the RE construct and lane 3 contains 109 molecules, suggesting that the copy number is approximately 103 in the approximately 105 cells successfully transfected.

J. CONSTRUCTION OF ARG 740-ASP 1658 (ABBREVIATED RD) DELETION In this section, we demonstrate how we created the RD deletion which removes the DNA sequence coding on expression from Ser 741 to Ser 1657. We constructed this RD deletion fusion in three steps. In the first step, we digested plasmid QD (Figure 2) with Sau3A between the codons for Ser 1657 and Asp 1658 and between Glu 1794 and Asp 1795.

This produced a 411 bp fragment. We also linearized plasmid tsa pML Dailey et al., J. Virol. 54, pp. 739-49 (1985)] at the unique BclI site. We then ligated the 411 base pair fragment derived from plasmid QD with T4 DNA ligase (the ligase for this and the following examples) to the linearized tsa pML at the unique BclI site to generate plasmid 411.BclI, which contains the BclI site on the Asp 1658 side of the 411 bp insert 5' to the sequence encoding Asp 1658). Plasmid 411.BclI may be linearized uniquely with BclI, resulting in a GATC overhang which consists of the GAT codon for Asp 1658 and the first base of the CAA codon for Gln 1659.

I N.^l L- INrU.LINCLUJ pUIY.y!1MUeL ±ILuILt±UA.tLt=Uy ZJ- VL.j ULlL 9. sea 00 9 9i 9 WO 88/00831 PCT/US87/01814 -33- We also digested plasmid QD with HindIII to cleave the plasmid between Arg 740 and Ser 741 and within the codon for Glu 321 to generate a 1258 bp fragment. We then removed the 5' AGCT overhang with mung bean nuclease and ligated it to the BclIlinearized 411.BclI fragment which had previously been rendered flush by treatment with Klenow enzyme and all four deoxynucleoside triphosphates. This resulted in plasmid RD.411, which contains an Asp718 site 5' to the fusion site within the 1258 bp HindIII fragment. RD.411 contains a PstI site 3' to the fusion site within the 411 bp Sau3A fragment.

Subsequently, we digested plasmid RE (Figure 3B) with Asp718 to cleave within the codon for Trp 585.

We then dephosphorylated the 5' GTAC overhang with calf intestinal phosphatase and then partially digested with PstI. This partial digestion cleaved the linearized RE plasmid between the codons for Ala 1702 and Val 1703, thus removing a 628 bp fragment spanning the RE fusion.

We then cleaved plasmid RD.411 with Asp718 and PstI to generate a 601 bp fragment spanning the RD fusion. We then ligated this fragment to the Asp718-cleaved, PstI-partially cleaved RE plasmid DNA to generate plasmid RD. As demonstrated below, plasmid RD directed the expression of a factor VIII polypeptide with a fusion between Arg 740 and Asp 1658. Cleavage of the RD polypeptide after Arg 740 generates a twochain factor VIII molecule with a mature heavy chain calciumbridged to a 69 light chain, i.e. a light chain lacking the first 9 amino-terminal amino acids.

K. CONSTRUCTION OF ARG 740-SER 1657 (ABBREVIATED RSD DELETION) In this section, we demonstrate how we created the RSD deletion which removes the DNA I, WO 88/00831 PCT/US87/01814 -34sequence coding on expression for Ser 741 to Gin 1656 of the mature polypeptide. Initially, we constructed plasmid 411.BclI and linearized it with BclI as described in example Subsequently, we digested plasmid QD with HindIII, cleaving the plasmid between the codons for Arg 740 and Ser 741 and within the codon for Glu 321 to generate a 1258 bp fragment.

We preserved the AGC codon within the 5' AGCT overhang with Klenow enzyme and dATP, dGTP and dCTP and then removed the leftover 5' T overhang with mung bean nuclease. We then ligated this modified HindIII fragment to BclI-linearized 411.BclI, which had been previously treated with Klenow enzyme and all four deoxynucleoside triphosphates, to produce plasmid RSD.411, which contains an Asp718 site 5' to the fusion site within the 1258 bp HindIII fragment and a PstI site 3' to the fusion site within the 411 bp Sau3A fragment.

We then prepared Asp718-cleaved, PstItially cleaved RE plasmid DNA as described in example Subsequently, we cleaved plasmid RSD.411 with Asp718 and PstI and ligated the resulting 604 b7, fragment spanning the RSD fusion to the Asp718-cleaved, PstI-partially cleaved RE plasmid DNA to generate plasmid RSD. Upon expression, the RSD plasmid encoded a factor VIII polypeptide with a fusion between Arg 740 and Ser 1657. A cleavage of RSD polypeptide after Arg 740 generates a 2-chain factor VIII molecule with a mature heavy chain and a delta 8 light chain, i.e. a light chain lacking the first eight amino terminal amino acids. Furthermore, because in the primary translation product Ser is also at position 741, RSD may also be viewed as a fusion between Ser 741 and Asp 1658. A cleavage after Ser 741 may generate a 2-chain factor VIII molecule with a heavy chain terminating at Ser 741 and a 69 light chain.

I 77 7 WO 88/00831 PCT/US87/01814 L. TRANSFECTION OF AFRICAN GREEN MONKEY KIDNEY CELLS We first produced African green monkey kidney cell line 6L by cotransfecting cell line (BSC1 African green monkey kidney cells which have been adapted to grow at 40 0 Brackman and D. Nathan, Proc. Natl. Acad. Sci. USA, 71, pp. 942-46 (1974)] with pLTRtsA58 and with pY3, which has a transcription unit for hygromycin B phosphotranferase Blochlinger, and A. Diggelmann, Mol. Cell Biol.

4, p. 2929-31 (1984)]. Plasmid LTRtsA58 contains a transcription unit for a temperature sensitive T-antigen allele. A mutant tsA58 virus is a temperature-sensitive mutant of SV40 which does not produce progeny at 39 0 C. The large T-antigen protein specified by the tsA58 mutant is much more labile at the nonpermissive temperature than wild type large T-antigen protein Tegtmeyer et al., J. Virol 16, pp. 168-78 (1975). The resulting cell line 6L inducibly expresses SV40 T-antigen at 33 0

C.

We then transfected 6L cells with supercoiled expression plasmids RD or RSD. The transfection was carried out using the DEAE-dextran technique and chloroquine as described in Example We then incubated the transfected cells at 33 0 C. During incubation, the transfected cells synthesized and secreted modified factor VIII:C into the culture fluid. The transfectants will secrete modified factor VIII:C for up to 120 hours. For most assays, the cm 2 /ml ratio was approximately 5.5; that is, a confluent monolayer of 6L transfectants in a 100mm Petri dish (55cm 2 was covered with 10ml culture fluid.

M. FACTOR VIII:C ACTIVITY ASSAY We assayed the RE (Example RD and RSD expression constructs for factor VIII:C production after transfection and incubation at 33 0 C for three OF\C 0 0* -0 0 0 0 00 0 0 WO 88/00831 PCT/US87/01814 -36days using KabiVitrum's Coatest® factor VIII assay kit adapted to a 96 well plate. Cells transfected with plasmid RE produced culture fluid having a factor VIII concentration which was 0.48% plasma level [normal plasma factor VIII concentration is approximately 150 ng/ml]. Cells transfected with plasmid RD produced culture fluid having a factor VIII concentration which was 0.41% plasma level. Cells transfected with plasmid RSD produced culture fluid having a factor VIII concentration which was 0.71% plasma level.

In a similar assay, cells transfected with plasmids RE or RSD which had been incubated at 33 0

C

for three days and then for an additional two days, yielded the following factor VIII concentrations in the cell culture fluid: Factor VIII:C Concentration In Culture Fluid As Of Plasma Level 3 Days 5 Days RE Transfected Cells 0.30% 0.77% RSD Transfected Cells 1.50% 1.16% Microorganisms, recombinant DNA molecules and the modified factor VIII:C DNA coding sequences of this invention are exemplified by a culture deposited in the culture collection of the American Type Culture Collection, in Rockville, Maryland, on July 22, 1986, and identified there as: E.coli HB101 (RE) This culture was assigned ATCC accession number 53517.

Two additional cultures were deposited in the American ~iu~--n.rn S a* a S* Sa a* WO 88/00831 PCT/US87/01814 -37- Type Culture Collection, in Rockville, Maryland on July 27, 1987, and identified there as: Ad.RD.2 [E.coli HB101 having ATCC accession number 67475; and Ad.RSD.1.2 [E.coli HB1C1 having ATCC accession number 67476.

While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

1;

Claims (9)

1. A recombinant DNA molecule characterized by a DNA sequence coding on expression for a modified factor VIII:C polypeptide, which modified factL. VIII:C polypeptide has the maturation polypeptide, or ajor portion thereof, deleted, and displays procoagulant activity comparable to native factor VIII:C, said DNA sequence containing a deletion of a major part of the DNA sequence which codes on expression for the maturation polypeptide of factor VIII:C.

2. The recombinant DNA molecule according to claim 1, wherein the deletion is all of the DNA sequence which codes on expression for the maturation polypeptide of factor VIII:C.

3. The recombinant DNA molecule according to claim 1, wherein the DNA sequence coding on expression for the modified factor VIII:C-like polypeptide is selected from the group consisting of: ATG GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTG TCA TGG GAC TAT ATG CAA AGT GAT CTC GGT GAG CTG CCT GTG GAC GCA AGA TTT CCT CCT AGA GTG CCA AAA TCT TTT CCA TTC AAC ACC TCA GTC GTG TAC AAA AAG ACT CTG TTT GTA GAA TTC ACG GAT CAC CTT TTC AAC ATC GCT AAG CCA AGG CCA CCC TGG ATG GGT CTG CTA GGT CCT ACC ATC CAG GCT GAG GTT TAT GAT ACA GTG GTC ATT ACA CTT AAG AAC ATG GCT TCC CAT CCT GTC AGT CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA GCT TCT GAG GGA GCT GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT GAT AAA GTC TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG AAT GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT CAT GTG GAC CTG GTA AAA GAC at t. seuc wc e o 0 t plei of VI 2. Th ooominn DN oecl acodigt i S~XI~-l i TTC GGG ATA TCA TCT GTA AAA CAA AGG TTC CTA GAA CTA CAT GAC AAG GAG AGA ATT GAT GGA CTG ATC AGG ATT GTA CGC TCA CTA CTC CTC GTG CAC GTT GGA AAT AGT CTA CAA CCT AAC TCA GTG AAC CTT CTC CCT CGA CTT AAC CAT CAC AGT GGT CAA ATC TTG TAC AGA CTC GAA TAT GGA CAT ATC ACA CAC AGC TGT CCA TCA CTC CTT ACA CGG AGG CTC CAC CAT ACT TTT TAT ATG ACT TCT CCT TGG TAT AGG ACC TTG ATT CCT TTA CCA CAT TAC CTC CAA CTG GAG CTT ATC TTC CAC GGC GCC TTT AAG GCC TCT TAT TCA CGC CCT TGT GTC AAA CAT CCT AAA CAC AAA AAC TTT GGA ATA CAC CCA GGA GGG TCT ATT AGA TTT AAT GAG AAT CAT ACT CTC ATT AAG GAA CCT GTA AAC TCC TGG CCT CTC (CTA) TGG CAT ATA TTC CAG GCG CAA ACA CAT ATC AAA CTA AAT AAT TCT GAA TCC TTT ACT TGG TAT CCT AGT CAA TAC AAA AAC ACT CCT TTA TTT AAC GGA ATC AAA GGT CAA ATA CCA ACT AGT TTC GGC CCT GGA AAC TCT CTA ATA CAA CAT CCA GGC TAT GAG GTG CAC TTC 39 GGA CCC CTA CTA CTA TCT ACA CAA AAC ACA CAC ACC TTC CAC AAA TTT TTT CAT CAA GGG AAA AGT TGC CAC TTC ATC CAG GAT AGG GAT CCT GCA AAA ATC CAC ACA CTC AAT GGT TAT CCA GGT CTC ATT GGA TCC CAC AGC GTG ATT GGA ATG GGC ACC ACT CCT CTC CAA GGT CAC ACA TTT CTT GTG TCC TTG GAA ATC TCG CCA ATA ACT CTC TTC ATG GAC CTT GGA CAC TTT TCT TCC CAC CAA CAT CAT GGC ATG CAC AGC TCT CCA GAG CAA CCC CAA GAA GAA GCG GAA CAC TAT CAT CAT ATG GAT GTG GTC AGG TTT CAT CAT ATC CAA ATT CCC TCA CTT CCC AAC CTA CAT TAC ATT CCT CCT CAA GAG CCC TTA CTC CTC CCC CCC CAT GAC TAT TTC AAC AAT GGC CCT CAC CGG AAA CTC CCA TTT ATG GCA TAC ACA CGT CAA CCT ATT CAC CAT CAA TCA CTT TAT GGG GAA CTT GGA, GAC ACA AAT CAA CCA ACC ACA CCA TAT AAC ACT CAT CTC CCT CCT TTC TAT TCA CTA AAA CAT TTC AAG GAT TTT CCA TTC AAA TAT AAA TGG ACA GTG ACT AAA TCA CAT CCT CGG TCC CTC ACC CTT AAT ATG GAG ACA CAT CTA CCT CTC CTC ATC TCC TAC AAA CAA TCT CAC ATA ATC TCA CAC AAC AGG AAT TTT CAT GAG AAC CCA ACC TGG TAC CGC TTT CTC CCC AAT CCA CCT GGA GAG TTC CAA CCC TCC AAC ATC ATC CTT TTT CAT ACT TTC CAC TTC TCA CCA TAC TGG TAC ATT CTM ACC ATT CTT TCT CTC TTC TTC TCT GGA TAT S I S 5 r S I S 5 5 5 0 5 50 5 a. S S S S S C** A l.;i;I i; i ACC TTC AAA CAC AAA ATG GTC TAT GAA GAC ACA GTC ACC GTA TTC CCA TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA AAG CCA GGT CTA TGG ATT CTG GGG TGC CAC AAC TCA GAG TTT CGG AAC AGA GGC ATG ACC GCC TTA CTG AAG GTT TCT AGT TGT GAG AAG AAC ACT GGT GAT TAT TAG GAG GAG AGT TAT GAA GAT ATT TCA GCA TAG TTG CTG AGT AAA AAC AAT GCC ATT GAA CGA AGA AGC TTC TCC GAG CAT CGT CTT CCT TGG GAT AAC CAC TAT GGT ACT GAG ATA CCA AAA GAA GAG TGG AAA TCC CAA GAG AAC TCA CC7L CAA AAA ACA GCT TTT AAG AAA AAC GAT ACG ATT TTC TCC CTG AAG GCT TCT GAA AGG AAT CAT GCA ATA CCA GCA ATA AAT GAG GCA CAA AAT AAC CCC GAA ATA GAA CTC ACC TGG GCA AAC GAA GGT AGC ACT GAA AGC CTG TGC TCT CAA AAC CCA CGA GTC TTC AAA CCC CAT CAA CGG GAA ATA ACT CGT ACT ACT GTT GAG TCA GAT CAA GAG GAA ATT GAG TAT CAT CAT ACC ATA TGA CTT CAA ATG AAG AAG GAA CAT TTT GAG ATT TAT CAT GAG CAT CAA AAT GAG AGG CCC CCC ACC TTT CAA AAC AAA ACA CGA CAC TAT TTT ATT CCT CCA GTC GAG ACG CTC TGG GAT TAT GGG ATG AGT AGC TCC CCA CAT CTT CTA ACA AAC AGG GCT GAG ACT GGC AGT CTC CCT GAG TTC AAG AAA CTT CTT TTC GAG GAA TTT ACT CAT GGC TCC TTT ACT GAG CCC TTA TAC CGT GGA GAA CTA AAT CAA CAT TTG GCA CTC CTG GGG CCA TAT ATA AGA CCA CAA CTT GAA CAT AAT ATC ATG GTA ACT TTC AGA AAT GAG CCC TCT CCT CCC TAT TCC TTC TAT TCT AGC CTT ATT TCT TAT GAG GAA CAT GAG AGG CAA GCA CCA GAA CCT AGA AAA AAC TTT CTC AAG CCT AAT CAA ACC AAA ACT TAG TTT TGG AAA GTC CAA CAT CAT ATG GCA CCC ACT AAA CAT GAG TTT GAG TCC AAA CCC TGG GCT TAT TTC TCT CAT CTT GAG CTC GAA AAA CAT CTG CAC TCA GGC CTG ATT GGA CCC CTT CTC GTC TGC CAC ACT AAC ACA CTC AAC CGT GCT CAT GGG AGA CAA GTG ACA CTA GAG CAA TTT CCT CTC TTT TTC (CTC) ACC ATC TTT CAT GAG ACC AAA ACC TGG TAG TTC ACT A-AJ AAT ATG CAA AGA AAC TGC AGG GCT CCC TCC AAT ATC GAG ATC CAA CAT CCC ACT TTT AAA GAG AAT TAT CCC TTC CAT GCA ATC AAT GGC TAG ATA ATG GAT ACA CTA CCT GGC TTA CTA ATC GCT GAG CAT CAA AGG ATT CCA TGC TAT CTC CTC ACC ATG GGC AGG AAT CAA AAC ATC CAT TCT ATT CAT TTC ACT GGA CAT GTG TTG S .1 .55 5~ 41 ACT CTC AAA CTA AAC CAT CCA TGC TTG CGT ATG AAT TCA GCT AGC TGC GCA ACC ACT CTC ACT AAC ACT AAT CCC GTC GCA CTG CTC TTT CTA COA GTT CAT CT CTA TAT CT CAT TCT TTT AAC AGO CA CAC TAT TC TOT CA ACT AC OAG TCC AAT CAA ACT CAC OTO CAA TTA CAC CAC CAA OCT Ch CAA 000 TAT OCT TOT CCA CCA CCA CT CAC CAC OTC ACC OCA AAC ACT ACT CCC TAO OTT ATC ATT TOT GC CTC CAC TTO TTT GAO OTG CAC GAO OTC CTC TTO TTO TCC CAT GTO GAG AAA CCT ATT CCC ACT ATT GC AAC ATC TTO OTT CCA ATA ATO CC CCA ACT COT TGC CAC CCA OTO TTT TOO ACT ATT OTO TOA GAO AAC AOG ATG ACA ACT CCA AAA CTT TCC ATC COO ACA AGA CAG ATT TOO CAT ACC AAA CT ATC TTC CT TCA ACA TTO CTA ATO CAC TTO CGC CO TAO TCC CCA ACC GAT GGT GTG OTT CT CAC CAC TAT TTT CAC ACA CCC CTC GAA AC ACA OTT CTC CCA ATC CT TCA CCA OTT OAT TAT COO TTT TOT ATT CAC GC AC OTO TAO CCC AAC AAC TTA ATC GTO CAC AAT ATT TTC CAC OCA CAC TTC ATC CCA ATC CAG TOA TOO TAO AAA CT CA COT CAC CTC CAC AAC ACA AAA TOT OTG TOO AC ACT AAT CCC AAA ACA OCT GTG TAO OTT OGA OTC ACC ATG CCC ACC ACG CAC TAT ATC ACA TTT COT TOA GTO GTG CAC OTT TTO OTG OTA GGT OTO ATT ACA OAT CT CTT CAA TAT CAT AAA ATC CCA CTC 9] CTC CAA ATC TTA TC OTT ATT CCC C TTT OTC CTC TAO CT TOT CCA CAC I CAA TAT CCA CAC 9J TOO CCA TCA ATO TCC ATO AAC CTC ATO AAC ACC CAC ATO TOT CAC TTT2 TCC CAC ACT TAT TTO TTT CCC A.AT TTT AAO OCT OCA ACT OAT TAT AC GC TCT CAT TTA2 ACT AAA CCA ATA TTT ACC AAT ATC OTT CAC OTO CAA AAT AAT OCA AAM. ATC AAA GC ACA OTT ACC AC ATC CAA CAT CCC OAT CTA AAC CTT TTT GTC AAC TOT OTA ATT CAC 000 CAG CAC CTT CTG GC k ACA TAO TAO CTC CAA ACT CAT OTO OCT ACA CTGCOCA TAO AAA AAC ACT AAO ATO CT AAC OCT AC ATO OAC OTT AAC AAO ATC CCT GTA TOO TAO CAT CAC ACC ACT ACAAT ~CA TOO AC CAT WGC AAT .TT ACA G'C CO AT CO 7AT CTC CGT GCC kTC ATO CA CCA 3TC CAT MT ATT MT CC \AT ACT ['CA CAT L'TT CO 3GG AGC 3AC TCC 3CA CTA PAT CTC CAC TCC CAG CCA GAC OCA kCT TCC FCC CAC CCT CCA GCT CAC AAA TOT CTC TTT COA ACC GCT CAC GOT TOO FCC AAA CAA ACC 0 0 0 0 0 0 0 0 0 000 0 0 0 0 0* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SOC 00 *0 0 0 00 0 00 00 0 0 .00 0 0 0 0 0 0 0 ~r -I 42 GAG AAA GAA GAT GAT AAA GTC TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG AAT GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT CAT GTG GAC CTG GTA AAA GAC TTG AAT TCA GGC CTC ATT GGA GCC CTA CTA OTA TOT AGA GAA GGG AGT CTG CCC AAG GAA AAG ACA CAC ACC TTG CAC AAA TTT ATA CTA CTT TTT GOT GTA TTT GAT GAA GGG AAA AGT TGG CAC TCA GAA ACA AAG AAC TCC TTG ATG CAG GAT AGG GAT GCT GCA TCT GCT CGG GCC TGG CCT AAA ATG CAC ACA GTC AAT GGT TAT GTA AAC AGG TCT CTO(CTA) CCA GGT CTG ATT OGA TGC CAC ACG AAA TCA GTC TAT TGG CAT GTG ATT GGA ATG GGC ACC ACT CCT OAA GTG CAC TCA ATA TTC CTC GAA GGT CAC ACA TTT CTT GTG AGO AAC CAT CGC CAG GCG TCC TTO GAA ATC TCO CCA ATA ACT TTC CTT ACT GCT CAA ACA CTC TTG ATG GAC CTT OGA CAG TTT CTA CTC TTT TOT CAT ATC TCT TCC CAC CAA CAT OAT GGC ATO GAA OCT TAT GTC AAA OTA GAC AGC TOT CCA GAG GAA CCC CAA CTA CCA ATO AAA AAT AAT OAA GAA GCG GAA GAC TAT OAT CAT CAT CTT ACT OAT TCT OAA ATG GAT GTG GTC AGO TTT OAT OAT CAC AAC TCT CCT TCC TTT ATC CAA ATT CGC TCA CTT CCC AAG AAG CAT CCT AAA ACT TOG OTA CAT TAC ATT OCT OCT CAA GAO GAO GAG TGG GAC TAT OCT CCC TTA GTC CTC 0CC CCC OAT CAC AGA ACT TAT AAA ACT CAA TAT TTO AAC AAT GGC CCT CAG CGG ATT GGT AGG AAC TAO AAA AAA OTC CGA TTT ATG GCA TAC ACA CAT CAA ACC TTT AAC ACT COT GAA GCT ATT CAG CAT CAA TCA GGA ATC TTG GGA COT TTA CTT TAT GGG GAA OTT GGA CAC ACA-CTC TTC ATT ATA TTT AAO AAT CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT CAC GGA ATC ACT CAT OTC CGT OCT TTO TAT TGA AOG AGA TTA CCA AAA GGT OTA AAA CAT TTO AAC OAT TTT CCA ATT CTG CGA GGA OAA ATA TTG AAA TAT AAA TGG ACA GTG ACT OTA OAA OAT GGG CCA ACT AAA TCA OAT CCT CGG TGC CTO ACC CGC TAT TAO TOT ACT TTC OTT AAT ATG GAG AGA CAT GTA OCT TCA OGA CTC ATT GGC COT CTC CTC ATC TGC TAC AAA OAA TOT OTA OAT CAA AGA OGA AAC CAG ATA ATG TCA GAG AAC AGO AAT CTC ATC CTG TTT TOT OTA TTT OAT GAO AAC CGA AGC TGG TAC CTC ACA CAG AAT ATA CAA CGC TTT CTC CCC AAT CCA GCT OGA GTO CAG OTT GAG CAT CCA GAO TTC CAA 0CC 0. 41, 1 1. 0 0.0 0 0* 43 TCC AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT TTT GAT AGT TTG CAG TTG TCA CTT TGT TTG CAT GAG GTG GCA TAC TGG TAC ATT CTA AGC ATT GGA GCA CAG ACT GAC TTC CTT TCT GTC TTC TTC TCT GGA TAT ACC TTC AAA CAC AAA ATC GTC TAT GAA GAC ACA CTC ACC CTA TTC CCA TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA AAC CCA C3T CTA TGG ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC ATG ACC GCC TTA CTG AAG GTT TCT ACT TGT GAC AAG AAC ACT GGT GAT TAT TAC GAG GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA AAC AAT GCC ATT CAA CCA AGA AGC TTC TCC CAG GAT CCT CTT GCT TGG GAT AAC CAC TAT GGT ACT CAG ATA CCA AA1A GAA GAG TGG AAA TCC CAA GAG AAG TCA CCA GAA AAA ACA GCT TTT AAG AAA AAG GAT ACC ATT TTG TCC CTG AAC GCT TGT GAA AGC AAT CAT CCA ATA CCA CCA ATA AAT GAG GGA CAA AAT. AAC CCC CAA ATA CAA GTC ACC TCG CCA AAC CAA GGT AGG ACT GAA ACG CTC TCC TCT CAA AAC CCA CCA CTC TTC AAA CGC CAT CAA CGG GAA ATA ACT CGT ACT ACT CTT CAC TCA CAT CAA GAG GAA ATT CAC TAT CAT CAT ACC ATA TCA CTT CAA ATC AAC AAC GAA GAT TTT CAC ATT TAT CAT GAG CAT CAA AAT CAC AGC CCC CCC AGC TTT CAA AAG AAA ACA CCA CAC TAT TTT ATT CCT CCA CTG GAG AGG CTC TGG GAT TAT GGG ATC ACT ACC TCC CCA CAT CTT CTA ACA AAC ACG CCT CAC ACT GGC ACT CTC CCT CAC TTC AAC AAA CTT CTT TTC CAC CAA TTT ACT CAT GGC TCC TTT ACT CAC CCC TTA TAC CGT GGA CAA CTA AAT CAA CAT TTG GGA CTC CTG GGG CCA TAT ATA AGA CCA CAA CTT CAA CAT AAT ATC ATG GTA ACT TTC AGA AAT CAC CCC TCT CCT CCC TAT TCC TTC TAT TCT ACC CTT ATT TCT TAT GAG CAA CAT CAC AGC CAA CGA CCA CAA CCT ACA AAA AAC TTT CTC AAC CCT AAT GAA ACC AAA ACT TAO TTT TGG AAA GTC CAA CSAT CAT ATG GCA CCC ACT AAA CAT GAG TTT CAC TCC AAA GCC TGG CCT TAT TTC TCT CAT CTT GAC CTG GAA AAA CAT GTG CAC TCA GGC CTC ATT GGA CCC CTT CTG GTC TCC CAC ACT AAC ACA CTC AAC CCT CCT CAT GGG AGA CAA GTG ACA CTA CAG GAA TTT GCT CTC TTT TTC(CTC) ACC ATC TTT CAT GAG ACC AAA ACC TGG TAC TTC ACT GAA AAT ATC GAA AGA AAC TGC AGG GCT CCC TGC AAT ATC CAG ATG GAA CAT CCC ACT TTT AAA GAG AAT TAT CGC IAA( 0 Opa 0 0 0 9 S O. 5 SS S S S S I i 44 TTC CAT GCA ATC AAT GGC TTA AGC GGA GCA ATG ATT GTG GGA GGA TCA AAG ACC CAG ACT GGC CCT TAT GAT GCA AAT CTC CCA GTC AGC GGC GTT TCT CCC CTG ATG TGG GCA ACC GAT GTA ATG CAT CTG TTA GGC TAC CAC CAG ATC GTG CAG TTT TAT AAT CCA AGC TTA ATA ATG CAA AAA ACA ATG CAT TTT CTA CAG GGC GCC GAC AGA TCA CAC ATG GGC GTG TAC CCA GAG AGC ATT TGG AAT GAT GGT ATC CGA GTG ATT ATT AAT TCA TTT GGG GAG GGA TAT CAG CAG GAC AGT TGC ACC TAT TTT GTC CTT GCT AGC TTC AAT TCC CAT AAT AGA GCC GCC CTG GCC ATC GGA GAT ATT CGC AGT GAT GCC AGG TGG GTA GTG TGG GGA CCA TGG GAG AGA ATG CCT GTG TTC CAG AAT ACT CTC AAA CTA AAG GAT CCA TGG TTG CGT ATG AAT TCA GCT AGC TGC GCA ACC AGT CTG ACT AAG ACT AAT CCG GTG GCA AGA CAA CCT TAC AAC GAT GAA GTA TAT GCT CAT TGT TTT AAG AGC GCA CAG TAT TCC TCT CGA ACT AGC CAG TGG AAT CAA ACT GAG CTC CAA TTA CAC CAG TAC AGT AGA AAA ATC TAC CAA AAC CGA CCA GGA GCT CAG CAG <',TG ACC CCA AAG AGT ACT GGG TAC CTT ATG ATT TCT GCC GTG CAG TTC TTT GAC CTG CAG GAC TAC GAT GTG AAG GCT ATA AGG ATC AAA GGT ATT GGG ACT ATT GCC AAG ATG TTC CTT GGA ATA ATC CGC CCA ACT CCT TGG GAC GGA CTC TTT TCC ACT ATT CTC CTG CTC CCA ACT AAG ATG GAT ATT CGA CAT TCT AAA GAG GTT TTT TGG CGG ATG AGC CCC CTG ACA GCT AGA CTT GAG CCC ATT ATT TCC AGC GAT GGG ACC TTA AAA CAC CGT TTG ATG GAG TTG GGA GCT TCA TCA AAA AGA CCT TTC CAG GTA AAA ATC TCC CAG AAT TTC ACA CGC TAC GCC CTG TAC; GGT GCA GGT GAG AAA TCT CTG TTT CCA AGG ACA TGG ATT GAG GAG GTG ACA GGA TCA CAT TTT CAC CTC AAG ATG AAT CAC TTG ATG TCC GCT CAG AAG TCT AGC GGC CCT CTT AGG GTG CTG TTT GTA CCA CTA TAT CAT TAT ACA GAA CTT ATG GGA TAT TCT GGC TAC AAG GTC ATT CCA ATG GAG TAC CGA GTG ACA CTG AGT AAA GTG CGA ATG GAA CCT CCA GAA CCC CCT CTG TTC AAA GTG TGC TTT GCT CAA TCC TGG ATC ATC TGG TTC TTT ACT GGC AGT TTT CTT AAT ATG CTT CAA GTA GTG ATT GAG CTG GTG TTC TTC TGG GGC CTC AGT ATG GAA CTT CTG TCT TAT GGA ATC AAG TCT CAG TTT AAC CAT TGT AAA ACC CAC AAT AAA ACC GAT AAG AAC CAC GTT TCA GAC AAC ACG ATG *4 a Va* 4, a. 0,4 a a a a a a a a a.. a. Va. a a 9 a a a a a a a a. 9 P a.. a a a.. a.. P a l~n~ r_ i; GGT GTG OTT GCT GAT GTC OTT TTG GGG ATA TCA TCT GTA AAA GAA AGG TTO CTA GAA CTA GAT GAC AAG GAG AGA ATT GAT OGA CTO ATO AGO ATT GTA CGC TCA CTG GTO CAT GAA AAA CTG ACC AAT AGT OTA GAA OCT AAC TCA GTG AAO OTT OTG GCT OGA OTT AAO OAT GAO AGT GGT GAA ATO TTG TAO AGA OTG OAA TAT GGA OTA ATT GCT TAT GTC AAA TAO TCA OTG OTT ACA CGG AGG GTO CAC OAT ACT TTT TAT ATG ACT TCT CCT TGG TAT AGG ACC TTG ATT OCT TTA OCA GAT TAO CTC GGT ACA OTT GAT TTC GAG TCA GGC GCC TTT AAG GCC TCT TAT TCA CGC OCT TGT GTC AAA OAT CCT AAA GAO AAA AAG TTT GGA ATA CAC OCA OGA GGG TCT ATT OCT ACC OTT AAO GGT OTA OAT CAG CCT GGT AAT GGT TAT OTT CTC ATT AAG OAA GCT GTA AAO TCC TGG CCT CTG(CTA) TGG OAT ATA TTO CAG GCG CAA ACA OAT ATO AAA GTA AAT AAT TCT GAA TCC TTT ACT TGG TAT GCT AGT CAA TAO AAA AAO ACT CCT TTA TTT AAG GGA ATO AAA GGT GAA ATA OCA ACT AGT TTO GGC CCT 45 ATC CAG OCT GAG GTT TAT C AAO ATO GOT TCC CAT OCT C TCC TAO TGG AAA GOT TOT ACC AGT CAA AGO GAG AAA OGA AGC OAT ACA TAT GTO OCA ATG GCC TOT GAC CCA TCT OAT GTG GAC CTG GTA OGA GCC OTA OTA OTA TGT AAO ACA CAC ACC TTG CAC TTT OAT GAA GGG AAA AOT TTG ATO CAG OAT AOG OAT AAA ATO CAC ACA GTC AAT OCA GOT CTG ATT OGA TGC GTG ATT OGA ATG GGC ACC CTC GAA OGT CAC ACA TTT TCC TTG OAA ATO TCG CCA CTC TTG ATO GAC CTT OGA TCT TCC CAC CAA OAT OAT GAO AGO TGT OCA GAO GAA GAA OAA GCG GAA GAO TAT ATO GAT GTG GTO AGO TTT ATO CAA ATT CGC TOA OTT OTA CAT TAO ATT OCT GCT CCC TTA GTC CTC GCC CCC TAT TTO AAO AAT GGC CCT AAA GTC COA TTT ATG GCA CGT OAA OCT ATT CAG OAT OTT TAT GGG GAA OTT OGA AAT CAA GCA AGO AGA OCA ACT OAT GTC COT OCT TTO GTA AAA OAT TTG AAG OAT TTC AAA TAT AAA TGG ACA AAA TOA GAT OCT CGG TGC OTT AAT ATO GAG AGA OAT CTC CTC ATO TGC TAO AAA ;AT ACA 'TC AGT 3AG GGA 'AA OAT PrOG CAG 2TG TGC %AA GAC kGA GAA kAA TTT rGG CAC GCT GCA GOT TAT CAC AGO ACT OCT CTT GTG ATA ACT CAG TTT GGC ATO CCC CAA OAT OAT OAT OAT 0CC AAO GAA GAO OAT GAO CAG CGG TAO ACA GAA TCA OAC ACA TAT AAO TAT TCA TTT OCA GTG ACT CTG ACC OTA OCT OAA TCT S S.. S C S S S S S 5 SS 0 6 5 S@ O C S S C 6 S C. 5 5 *0O S I h- -L %-JJ-J f L WlUl~l. LMLL'dV= L4JM LWAZ% I 46 GTA GTC CTC GTG CAC GTT GGA ACC TTC CCA AAC AAG TCA GAA GAC TTT TTT AGG AGA GTT TTA CCA TTC CTT AGA TGG GAC AAA CAC GTA ACC AGG GAG ACA TGG GAT ATC ACA CAG AGC TGT GC7A TTC CCA GGT AGA AAC GCA ATA TAT GAC CAA CTC AAC GTT TAC TAT AGA ATT A-kL AAA TGC GAT ACT CAG AAA GCT AAT CTA TAT CAA CTG GAG CTT ATC TTG CAG AAA TTC CTA GGC ACT TAC ACT GAT ATT AAG TGG AGG TTC CGT ATA AAT TCT AAC GTG AAA GTG AAC GAA AGC Ccc TAT CCT CTG AGA TTT AAT GAG AAT CAT ACT CAC TCA TGG ATG GGT TTG CGT GAT TAT AAA GAT GCT CAG GGA AGA CAG TAT TTT CAA GCC CAC ACA TTT TGG TGC CC GGC CTC GGA TCT ATA GAT GGC GAG GAC AAA GGA ATT ACC GAT CTG ACT ACC GAT ACA TAT CAG GAA GAA GCA GCC GAG GTC CAT TGG TCA CTG GCT TAC AAT TTC TTA AGC AAC GTA CAA CCA TAT GTG TTC ATG GAA CTG GC TAT AGT ACT ATA GAG CGA GGG AGT TTT CTA GAA TCT GAA AAG CAT GCT GGC AAC CTG TTC ATC CAT GTA ATG CAG TTT CGC GAG GTT GCA CTT GTC ACT GGG TTA TAC AAA CTT TCA GAT CAC ATG GGC ACT AAT GTT CGT GAT CCT ATG TAT CTG CCT TTT ACT CAG GCA ATG GGC ATA GAT TTT TTC TTT TAC TCT TAT GTC TGC CTG GAG AAC CAG GTT GAA TAT AGT AGT GAT GAA GAA CCC CAG AAT GCA TTC ATT GCT TTC GAA ATG ATC GCT AGC ATG GAG CTC CAA GAT TGG GTC GAA TTC CAC AAG GAC AAT TCA GAA AAT TTT AGC GTC GGC CAT GAT TAT AGG GAA CCC TCT GGA CAT (CTC AAT GAA AAT CAG AAT TCA GAC AAC CGA I CCC AAT GCC TCC AGT TTG( TAC ATT TTC TTC 9J GAC ACA ATG TCG1 AAC TCAC GTT TCT2 AGT TATC GCC ATT GAT CAA ATG AAG2 CAG AGC ATT GCT TCC CCA CCT CAG TCC TTT2 TTG GGA AAT ATC TCC TTC CAA GGA ACC AAA ACT AAA GAT GTT CCC CTT GGG AGA ACC ATC ATG GAA GAT CCC GGC TAC GAT CAA GAA AAC AG .GC ~CA AC !AG [CT ;AC k.GT ;AA ;,A ;AG kAG ;CA AT PTC k.CT ::TC k.TG rAT 3CA kCT GAT GAC CTG CAA TT A.GA A.CT ATA AiGG ATC AGG AAT TGG TAC GCT GGA ATC ATG TTG TCA Z GC ATT GGA TAT ACC CTA GAA AAC TTT CGG TGT GAC GAT ATT CCA AGA GAA ATT GAA GAT CGC AGC GTG GAG GTT CTA AAG AAA CAG CCC CTG GGG GTA ACT TCT AGC GAA CCT TAC TT7' GAG TT'P CTG GAA GTC TGC GTG ACA P GAT GAG AAC TGC TTT AAA ATG GAT ATT CGA CAT TCT 0 0 0 S OS. 09S9 S S *Sq 5 5 5 S S

5055. 5 5 S 5 5 5 5 5 5 5 5 0 005 55 5.* 55 0 5 6 00 0 4* S S S 4, 5 S 55 S S S 000 500 5 S. S S S S 5 -t 47 ATT GAG GAG GTG ACA GGA TCA CAT TTT CAC CTC AAG ATG AAT CAC TTG ATG TCC GCT CAG AAG TCT AGC GGC CCT CTT AGG GCC GAC AGA TCA CAC CTG GTC CAT CAT TAT ACA GAA CTT ATG GGA TAT TCT GGC TAC AAG GTC ATT CCA ATG GAG TAC CGA GTG ACA CTG AGT AAA GTG CGA ATG ACC TAT TTT GTC CTT CTA ATT GCT TTC AAA GTG TGC TTT GCT CAA TCC TGG ATC ATC TGG TTC TTT ACT GGC AGT TTT CTT AAT ATG CTT CAA GTA GTG ATT GAG AGA ATG CCT GTG TTC GGT ACA GTT AGT ATG GAA CTT CTG TCT TAT GGA ATC AAG TCT CAG TTT AAC CAT TGT AAA ACC CAC AAT AAA ACC GAT AAG AAC CAC GTT AGA CAA CCT TAC AAC CCT CTT GGT GGA GCA ATG ATT GTG GGA GGA TCA AAG ACC CAG ACT GGC CCT TAT GAT GCA AAT CTC CCA GTC AGC GGC GTT TCT CCC CTG TAC AGT AGA AAA ATC ACC AAG GTA CAT CTG TTA GGC TAC CAC CAG ATC GTG CAG TTT TAT AAT CCA AGC TTA ATA ATG CAA AAA ACA ATG CAT TTT CTA CAG GGC TAC GAT GTG AAG GCT ATO AAC TCC GTG TAC CCA GAG AGC ATT TGG AAT GAT GGT ATC CGA GTG ATT ATT AAT TCA TTT GGG GAG GGA TAT CAG CAG GAC AGT TGC CTG CTC CCA ACT AAG CAG ATG TAC TTC AAT TCC CAT AAT AGA GCC GCC CTG GCC ATC GGA GAT ATT CGC AGT GAT GCC AGG TGG GTA GTG TGG GGA CCA TGG GAG GGT GGT AAA CTG CCA GCT GCT TGG ACT CTC AAA CTA AAG GAT CCA TGG TTG CGT ATG AAT TCA GCT AGC TGC GCA ACC AGT CTG ACT AAG ACT AAT CCG GTG GCA GCA GAG TCT TTT AGG GAG TCC AAA GTA TAT GCT CAT TGT TTT AAG AGC GCA CAG TAT TCC TCT CGA ACT AGC CAG TGG AAT CAA ACT GAG CTC CAA TTA CAC CAG GTG CTG TTT GTA CCA GTT CAT GCT CGA CCA GGA GCT CAG CAG CTG ACC CCA AAG AGT ACT CG TAC CTT ATG ATT TCT CC GTG CAG TTC TTT GAC CTG CAG GAC GAA CCT CCA GAA CCC TAT CCT TCT AAA CGT ATT GGG ACT ATT GCC AAG ATC TTC CTT GGA ATA ATC CC CCA ACT CCT TGG GAC GGA CTC TTT TCC ACT ATT CTC CTG GTG TTC TTC TGG GAT GTC GAG AAA GAG GTT TTT TGG CGG ATG AGC CCC CTG ACA GCT AGA CTT GAG CCC ATT ATT TCC AGC GAT GGG ACC TTA AAA CAC CGT TTG ATG GAG TTG GGA GCT TCA TCA AAA AGA CCT TTC CAG GTA AAA ATC TCC CAG AAT TTC ACA CGC TAC GCC CTG TAC; arnd TCA TGG GAC GCA AAC ACC ACG OAT ATG GGT ACA GTG AGT CTT GGA GCT 0 0 0 00. *ge~ 0 see 0 0 0 0 0* *00 0 0 *@00* S S S 0B Be 9 @0 000 0000 S 000 0 000 9 SO 5* 0 0 48 GAA AAA CTG ACC AAT AGT OTA GAA GCT AAC TCA GTG AAC CTT CTG GCT CGA CTT AAC CAT GAC AGT GGT GAA ATC TTG TAC AGA CTG GAA TAT GGA GAT ATC ACA TAT GTC AAA TAC TCA CTG CTT ACA CGG AGG GTC CAC CAT ACT TTT TAT ATG ACT TCT CCT TGG TAT AGG ACC TTG ATT CCT TTA CCA GAT TAC CTC CAA CTG GAG GAT TTC GAG TCA GGC GCC TTT AAG GCC TCT TAT TCA CGC GCT TGT GTC AAA GAT CCT AAA GAC AAA AAG TTT GGA ATA CAC CCA GGA GGG TCT ATT AGA TTT AAT GAT CCT AAT TAT CTC AAG GCT AAC TGG CTG TGG ATA CAG CAA CAT AAA AAT TOT TCC ACT TAT AGT TAC AAG CT TTT GGA AAA GAA CCA AGT GGC G6A TOT ATA CAG GGT GGT CTT ATT GAA GTA TCC CCT CTA) CAT TTC GCG ACA ATC GTA AAT GAA TTT TGG GCT CAA AAA ACT TTA AAG ATC GGT ATA ACT TTC CCT AAC GTA CA.A ACC AGT CAA AGG GAG AAA GAA GGA AGC CAT ACA TAT GTC TGG CCA ATG GCC TCT GAC CCA CTG TCT CAT GTG GAC CTG GTA AAA GGA GCC CTA CTA GTA TGT AGA AAG ACA CAC ACC TTG CAC AAA TTT GAT GAA GGG AAA AGT TGG TTG ATG CAG GAT AGG GAT GCT AAA ATG CAC ACA GTC AAT GGT CCA GGT CTG ATT GGA TGC CAC GTG ATT GGA ATG GGC ACC ACT CTC GAA GGT CAC ACA TTT CTT TCC TTG GAA ATC TCG CCA ATA CTC TTG ATG, GAC CTT GGA CAG TCT TCC CAC CAA CAT GAT GGC GAC AGC TGT CCA GAG GAA CCC GAA GAA GCG GAA GAC TAT GAT ATG GAT GTG GTC AGG TTT GAT ATC CAA ATT CGC TCA GTT GCC GTA CAT TAC ATT GCT GCT GAA CCC TTA GTC CTC GCC CCC GAT TAT TTG AAC AAT GGC CCT CAG AAA GTC CGA TTT ATG GCA TAC CGT GAA GCT ATT CAG CAT GAA CTT TAT GGG GAA GTT GGA GAC AAT CAA GCA AGC AGA CCA TAT ACT GAT GTC CGT CCT TTG TAT GTA AAA CAT TTG AAG GAT TTT TTC AAA TAT AAA TGG ACA GTG AAA TCA GAT CCT CGG TGC CTG GTT AAT ATG GAG AGA GAT CTA CTC CTC ATC TGC TAC AAA GAA CAG ATA ATG TCA GAC AAG AGG TTT GAT GAG AAC CGA AGC TGG CGC TTT CTC CCC AAT CCA GCT ;AT GAT AG GTC E'GC CTT 3AC TTG 3AA CCC TTT ATA CAC TCA GCA TCT TAT GTA AGG AAA CCT GAA GTG AGG ACT TTC TTT CTA ATG GAA CAA CTA GAT GAT GAT CAC AAG AAC GAG GAG GAC AGA CCC ATT ACA GAT TCA GGA ACA CTG AAC ATC TCA AGG CCA ATT ACT GTA ACC CGC GCT TCA TCT GTA AAT GTC TAC CTC GGA GTG 0 0*0 V:ee 0. .0 0. See 0 50 e a S Ce see e eec e S e e eec e e e.g 0 See See S S 49 CAG CTT ACC ATC TGT TTG CCA CAG TTC AAA CCA TTC GGT CTA AGA GGC AAC ACT GCA TAC ATA ACT TAT GAT CAC ATT CAA AAG CTC TG AAC AGG GTT TTC TAG GGT TAT ATA AGA AAT ATT TCT AAA AAG AAA GTG TGC AAA GAT GTG ACT AAG GAG GAA AAA AGG GCT GGG AAT TAT CTA GGT TAT GTG GAT TTC TAT AAA AGA GTG GAG AAT CAT ACT GAG TCA TGG ATG GGT TTG CGT GAT TAT AAA CAT GGT GAG GGA AGA GAG TAT TTT CAA GCC GAG ACA TTT TGG TGG CGC GGC CTC ACT ATG GAA GAT GGG GAG GAG AAA GGA ATT ACC GAT CTG ACT ACC GAT ACA TAT CGAG GAA CAA GCA GGG GAG GTC CAT TGG TGA GTG GGT TAG AAT TTG TTA AGC GGA GCA ATG GGA TAT GTG TTC ATG GAA GTG GGG TAT AGT AGT ATA GAG GGA GGG AGT TTT CTA GAA TGT GAA AAG CAT GGT GC AAC CTG TTG ATG CAT GTA ATG CAT CTG TTA GAG GTT GGA CTT GTC ACT GGG TTA TAG AAA GTT TGA GAT GAG ATG CGC ACT AAT GTT CGT GAT GCT ATG TAT CTG GGT TTT ACT GAG GGA ATG GCC GTG TAG CCA TTG TTT TAG TGT TAT GTG TGG GTG GAG AAG GAG GTT GAA TAT AGT AGT GAT GAA GAA CCC GAG AAT GCA TTG ATT GGT TTG GAA ATG ATC GCT AGC TTC AAT TC CAA GAT TGG GTG GAA TTG GAG AAG GAG AAT TGA GAA AAT TTT AGC GTC GGG CAT GAT TAT AGG GAA CCC TGT GGA CAT (CTC AAT AAT GAG AAT ACT GTC AAA GCC TGG ACT TTG TAG ATT TTG TTG GAG ACA ATC TG AAC TGA CTT TGT AGT TAT GGC ATT GAT CAA ATG AAG GAG AGC ATT GCT TCC CCA CCT GAG TGC TTT TTG GGA AAT ATC TGC TTC CAA GGA ACC AAA ACT AAA CAT GTT CCC GTT GC AGA )ACC AT ATG GAA GAT CCC GGC TAG GAT CAA GAA AAC CTA CCA TAT GGA GCT GGA AAG ATC ATG GAG GAG TTG TCA CTT GTA AGC ATT GGA TGT GCA TAT ACC GTC ACC CTA TTC ATG GAA AAC CCA GAG TTT CGG A.AG AGT TGT GAG AAC GAA GAT ATT TCA GAA CCA AGA CAA GAG GAA ATT GAG AAC GAA CAT TTT CCC CCC ACC TTT GGA GTG GAG ACC CAT GTT CTA ACA TTC AAC AAA CTT ACT GAG CCC TTA GTC CTG CCC CCA ATC CTA ACT TTC TAT TCT ACC CTT CCA CAA CCT AGA ACT TAG TTT TG CAT GAG TTT GAG GAG CTC CAA AAA CG GC TGC GAG CAA GTC ACA GTA C TTT CAT GAG ACC AGA AAC TGG AGC ACT TTT AAA GAG ATA ATG CAT ACA ACC ATT CCA TG ATG CAT TCT ATT AAA AAA GAG GAG GCT GTT TTT GAG ATT TGG CCC CTC 0 a 00 9 S S 0 0 0 ee S 0 55 0 SS 5@ S S S *50 S S 555 5 Se S S e S 50 GAA TGC CTT ATT GGC GAG CAT CTA CAT GCT GGG ATG AGC ACA CTT TTT CTG GTG TAC AGC AAT AAG TGT CAG ACT CCC CTG GGA ATG GCT TCT GGA CAC ATT AGA GAT TTT CAG ATT ACA GCT TCA GGA CAA TAT GGA CAG TGG GCC CCA AAG CTG GCC AGA CTT CAT TAT TCC GGA TCA ATC AAT GCC TGG AGC ACC AAG GAG CCC TTT TCT TGG ATC AAG GTG GAT CTG TTG GCA CCA ATG ATT ATT CAC GGC ATC AAG ACC CAG GGT GCC CGT CAG AAG TTC TCC AGC CTC TAC ATC TCT CAG TTT ATC ATC ATG TAT AGT CTT GAT GGG AAG AAG TGG CAG ACT TAT CGA GGA AAT TCC ACT GGA ACC TTA ATG GTC TTC TTT GGC AAT GTG GAT TCA TCT GGG ATA AAA CAC AAT ATT TTT AAC CCT CCA ATT ATT GGT CGA TAC ATC CGT TTG CAC CCA ACT CAT TAT AGC ATT CGC AGC ACT CTT CGC ATG GAG TTG ATG GGC TGT GAT TTA AAT AGT TGC AGC ATG CCA TTG GGA ATG GAG AGT AAA GCA ATA TCA GAT GCA CAG ATT ACT GCT TCA TCC T TTT ACC AAT ATG TTT GCC ACC TGG TCT CCT TCA AAA GCT CGA CTT CAC CTC CAA GGG AGG AGT AAT GCC TGG AGA CCT CAG GTG AAT AAT CCA AAA GAG TGG CTG CAA GTG GAC TTC CAG AAG ACA ATG AAA GTC ACA GGA GTA ACT ACT CAG GGA GTA AAA TCT CTG CTT ACC AGC ATG TAT GTG AAG GAG TTC CTC ATC TCC AGC AGT CAA GAT GGC CAT CAG TGG ACT CTC TTT TTT CAG AAT GGC AAA GTA AAG GTT TTT CAG GGA AAT CAA GAC TCC TTC ACA CCT GTG GTG AAC TCT CTA GAG CCA CCG TTA CTG ACT CGC TAC CTT CGA ATT CAC CCC CAG AGT TGG GTG CAC CAG ATT GCC CTG AGG ATG GAG GTT CTG GGC TGC GAG GCA CAG GAC CTC TAG.

4. The recombinant DNA molecule according to any one of claims 1-3, wherein the DNA sequence coding on expression for the modified factor VIII:C-like polypeptide is operatively linked to an expression control sequence. The recombinant DNA molecule according to claim 4 wherein the expression control sequence is selected from the group consisting of the lac system, the trn system, the tac system, the trc system, major a, S *g Cc OS 55o S: g e* g* S S S S S S S A yp' S S Se S :0 5 0 0 6i:- I I 51 operator and promoter regions of phage the control region of fd coat protein, the early and late promoters of SV4O, promoters derived from polyoma, adenovirus and simian virus, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of yeast acid phosphatase, the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses. 6. A modified factor VIII:C-like polypep- tide, having a formula selected from the group consist- ing of: met ala trp asp ala arg thr ser asp his gly leu val val leu his ala glu asp lys val leu leu thr leu asn gly ser ile leu ser glu ser ala val asn ser val val his asn his leu thr thr tyr phe val ieu leu ile ala tyr val lys tyr ser leu leu abr arg tyr ser arg ala arg met pro va 1 phe gly thr val asp phe glu s er gly ala phe lys ala s er trp ile gln gin arg gin pro tyr asn pro ieu gly asp pro asn tyr leu lys ala asn trp leu his phe ala thr tyr ser arg lys ile thr lys va 1 gin gly gly leu ile giu va 1 ser pro pro val ieu ser leu tyr leu gly asp leu gly val pro lys lys thr leu ala lys pro ile gin ala asn met ala ser tyr trp thr ser gin gly ser his pro met ala ser his val gly ala leu lys thr gin phe asp glu leu 'met gin lys met his gly leu ile ile gly met al gly his leu glu ile leu met asp ala giu ser phe arg glu s er lys arg thr ser asp leu thr gly asp thr gly gly thr ser leu val leu phe va 1 pro va 1 his ala glu tyr asp leu va 1 leu lys arg va 1 cys thr phe pro gly glu pro pro glu pro tyr pro ser lys va 1 pro va 1 cy s his ser asp asn his thr leu lie gin leu va 1 phe phe trp asp va 1 g lu glu trp leu lys arg lys trp ala gly arg pro va 1 thr phe 5 er asp asn thr met thr ser gly asp gin cys asp glu phe his ala try lys glu arg phe leu a 6 *6 4@ *O 6 6 *606.. 9 6 @6 6 6 SO *OS B 66 @6 6* B B S 0*O a .me B B* 66 6 52 leu phe cys ala tyr val arg met lys ieu thr asp asn ser pro his pro lys asp trp asp ser tyr lys gly arg lys giu thr phe ile ieu gly leu ile ile tyr pro his arg ieu pro leu pro gly glu asp gly tyr tyr ser gly ieu ile asn gin arg ile leu phe thr glu asn gin leu glij ser ile asn cys leu his ala gin thr phe lys his pro phe ser gly leu trp arg gly met asn thr gly ala tyr leu phe ser gin gin ile pro giu lys thr asn ala cys his ile lvs val asn asn ser glu ser phe thr trp tyr ala ser gin tyr lys lys thr pro leu phe lys gly ile lys gly giu iie pro thr ser phe gly pro gly asn ser val ile gin asp pro gly tyr giu val asp phe lys met gly glu ile ieu thr ala asp tyr leu ser asp pro lys giu ala phe glu ser 5 er asp glu met i le va 1 pro tyr lys arg leu asn thr va 1 phe lys val 1 eu gin phe arg glu va 1 ala leu va 1 thr gly leu tyr lys leu glu lys asn ser his ser cys glu ala asp val gin ile his tyr leu val leu asn val arg glu ala tyr gly gin ala asp val lys his lys tyr ser asp asn met leu ile ile met asp glu phe leu phe qin phe asp tyr trp ser val tyr glu val phe cys his leu lys glu asp asn asn ala trp trp lys lys lys gin his asp pro glu glu glu asp tyr val arg phe arg ser val ile ala ala leu ala pro asn gly pro phe met ala ile gin his glu val giy ser arg pro arg pro leu leu lys asp lys trp thr pro arg cys glu arg asp cys tyr lys ser asp lys asn arg ser pro asn pro ala ser asn ser leu gin tyr ile leu phe phe ser asp thr leu met ser met asn ser asp val ser ser ser tyr glu ala ile glu asp asn his ser gin glu asp thr ile gly pro asp asp ala glu asp gin tyr glu asp tyr tyr phe va 1 1 eu leu glu arg trp ala ile leu ser gly thr giu phe cys asp pro tyr lys leu met gin asp asp lys glu asp arg thr ser thr asn ser pro thr thr ala ser asn tyr gly met ser ile tyr 1 eu asn arg asp ile arg g ly ser ser glu leu asp asp lys glu arg ile asp gly leu i le arg ile val arg ser va 1 va 1 leu val his val gly thr phe pro asn lys s er ser thr pro leu his ala ile ala ala ile asn giu L 0 go 4 0 v :e h 00 0 06 0006 0 C 0 Ga. 0 0 e.g 0 9g 000 6 0 K 7 53 gly gin asn lys pro giy arg thr giu arg lys arg his gin arg asp gin glu giu ile met iys iys giu asp gin ser pro arg ser iie ala ala val glu ser pro his val ieu pro gin phe iys lys ser phe thr gin pro leu giy leu ieu gly asn ile met val thr ser phe tyr ser ser gin gly ala giu pro thr lys thr tyr phe thr iys asp giu phe asp val asp ieu glu pro leu ieu val cys gly arg gin val thr thr ile phe asp giu met giu arg asn cys asp pro thr phe lys gly tyr ile met asp asp gin arg iie arg giu asn ile his ser val arg lys lys giu tyr pro gly val phe ala gly iie trp arg his ala gly met ser cys gin thr pro ieu phe gin iie thr ala lys leu ala arg ieu ser thr lys giu pro ala pro met iie ile giu ieu giu asp phe phe arg arg va 1 leu pro phe leu arg trp asp lys his val thr arg giu thr trp ile giu giu va 1 thr gly ser his phe his ile cys ile tyr asp gin ieu asn va 1 tyr tyr arg ile lys lys cys asp thr gin lys asn ieu tyr his tyr thr glu ieu met gly tyr ser gly giu ser thr asp i le lys trp arg phe arg ile asn ser asn va 1 lys va 1 asn glu s er pro tyr pro leu phe lys va 1 cys phe ala gin s er trp ile ile va 1 gin arg asp tyr lys asp ala gin gly arg gin tyr phe gin ala his thr phe trp cys arg gly ieu ser met giu ieu leu ser tyr gly ile lys ser thr trp asn pro thr thr thr ile asp glu thr arg tyr gly gin ser glu phe giu ieu ala giu ala ser glu giu val lys his his trp ala ser gly leu asn ala leu tyr phe asn ile phe his ieu val ser met giy his ala leu met ieu ile gly val tyr gly his gly gin ser ile lys val thr gin gin phe ala pro ieu ser asp his met gly thr asn va 1 arg asp pro met tyr leu pro phe thr gin ala met gly va 1 tyr pro giu ser ile trp asn asp gly ile lys gin val ieu gin ser val giu giu asn tyr phe ser ser ser val asp gly glu his glu asp Vco tyr gin arg asn gl" ala pro phe ser ile giy 4la his phe (ieu) glu asn met giu ile asn ala gin ser asn phe thr asn ieu ser lys his leu asn lys arg asp ala pro ala trp leu leu ala arg ile met gin lys phe ser ser leu tyr a a o 4, a a a a a a A a a a a a a a a a a a ,h a I a a a a *c. a. a a a a. a a a a a a a 0t* a a a a a a a a 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 0 **9 9 @9 9*e 9 9 9 9 9.. 9* 54 tyr ser ser arg thr ser gin trp asn gin thr giu ieu gin ieu his gin ala asp arg ser his -1eu va 1 his giu lys leu thr asn ser 1 eu giu ala asn ser thr giy tyr ieu met ile ser ala va 1 gin phe phe asp 1 eu gin asp thr tyr phe va 1 leu leu ile ala tyr va 1 lys tyr ser leu ieu thr arg arg ieu giy le ile arg pro thr pro trp asp gly ieu phe ser thr iie ieu arg met pro va 1 phe gly thr va 1 asp phe g iu ser gly ala phe iys ala ser asp gly thr ieu lys his arg ieu met giu ieu giy ala ser ser iys arg pro phe gin val lys le ser gin asn phe thr arg tyr aia leu tyr; arg tyr gin ser pro arg tyr iys asn iie pro thr ieu :iys gJly vai asp gin pro gly asn gly tyr ieu leu iie lys giu ala val asn ser trp pro leu pro lys met asn his ieu met ser ala gin lys ser ser giy pro ieu arg tyr asp val lys aia le asn ser thr gly pro ser gly iys phe ieu lys gly lys va 1 le pro met g lu tyr arg vai thr 1 eu ser iys va i arg met ieu ieu pro thr lys gin met tyr s er ser met his ala thr asp met met leu trp phe phe thr giy ser phe ieu asn met leu gin va 1 va i iJ.-a giu giy iys ieu pro ala ala trp gin his ala va 1 ieu gin glu gin his le gin phe asn his cys iys thr his asn lys thr asp lys asn his va 1 thr gly pro tyr asp ala asn leu pro va i ser giy va 1 ser pro leu aia val giu ieu ser phe phe val arg pro giu val ser his iys ala arg giu thr tyr ser asp asp ieu ieu val thr ieu giy iys asp arg thr val gly (Zy5 .49 tyr asn pro ser leu le met gin lys thr met his phe leu gin giy giu pro pro giu pro tyr pro ser iys va 1 pro va 1 cys his ser asp asn his arg va 1 le le asn ser phe giy giu g ly tyr gin gin asp ser cys ieu va 1 phe phe trp asp val giu giu trp ieu lys arg lys trp ala gly arg gly asp le arg ser asp ala arg trp va 1 vai trp gly pro trp glu ser asp asn thr met thr e r gly asp gin cys asp glu phe his ala try lys asn ser ala ser cys ala thr ser ieu thr lys thr asn pro va 1 aia trp ala thr asp gly va 1 ieu ala asp va 1 leu ieu giy ile ser ser val s er 9 9 9 9 9@9 9 9 9 9 0 9 9 9 9 C 9 9 9 9 9 9 9 9 9 9 9 9 9 9 *9* *99 9 999 9 99 9 9 9 9 9 55 va 1 his his thr phe tyr met thr s er pro trp tyr arg thr ieu ile pro leu pro asp tyr ieu gin leu glu ieu ile leu gin lys phe 1 eu gly thr tyr tyr s er arg ala cys val lys asp pro lys asp lys lys phe g ly i le his pro g ly g ly s er ile arg phe asn glu asn his thr his ser trp met g ly leu trp his ile phe gin ala gin thr his le lys val asn asn ser glu ser phe thr trp tyr ala ser gin tyr lys lys thr pro ieu phe lys giy iie lys gly giu iie pro thr ser phe gly pro gly asn ser val iie gin asp pro gly tyr glu val asp phe lys met gly giu le leu thr ala asp tyr leu ser va 1 leu ser leu s er asp glu met i le val pro tyr lys arg ieu asn thr val phe lys va 1 leu gin phe arg glu val ala ieu va 1 thr g ly leu tyr lys le gly glu gly leu giu leu met ser his ser cys giu ala asp val gin ile his tyr ieu vai leu asn val arg glu ala tyr gly gin ala asp vai lys his lys tyr ser asp asn met ieu le le met asp glu phe ieu phe gin phe asp tyr trp ser val tyr glu val phe cys his ieu lys glu asp asn asn met gly thr thr his thr phe ieu le ser pro le asp leu gly gin gin his asp gly pro glu glu pro glu asp tyr asp val arg phe asp arg ser val ala le ala ala glu leu ala pro asp asn gly pro gin phe met ala tyr le gin his glu giu val gly asp ser arg pro tyr arg pro leu tyr ieu lys asp phe lys trp thr val pro arg cys leu giu arg asp ieu cys tyr lys glu ser asp lys arg asn arg ser trp pro asn pro ala ala ser asn ile ser leu gin leu tyr ile ieu ser phe phe ser gly asp thr leu thr met ser met giu asn ser asp phe val ser ser cys ser tyr giu asp ala le giu pro pro glu val val arg asn thr phe ieu phe leu ieu met giu ala gin ieu arg asp asp ieu asp asp asn lys lys his glu glu asp asp arg ser arg iie gly thr asp glu ser giy iie thr ieu leu asn ile tyr ser arg arg pro ile leu thr val gin thr arg tyr ala ser gly ser val asp asn val le tyr leu thr gly val gin met his ser ser val cys ile gly ala tyr thr phe len phe pro asn pro gly arg asn arg asp iys asn ile ser ala arg ser phe S S S S S S S S S S S S S S S S S S S 5 555 SS S S 5 55 S S S S S S S.. S 555

5 S S S S S S 56 ser gin asp pro ieu ala trp asp asn his tyr gly thr gin ile pro lys giu giu trp lys ser gin giu lys ser pro giu lys thr ala phe lys lys lys asp thr ile ieu ser leu asn ala cys glu ser asn his ala ile ala ala ile asn giu gly gin asn lys pro glu ile glu val thr trp ala iys gin gly arg thr glu arg ieu cys ser gin asn pro pro val ieu lys arg his gin arg glu ile thr arg thr thr ieu gin ser asp gin glu giu iie asp tyr asp asp thr ile ser val glu met iys lys glu asp phe asp ile tyr asp glu asp giu asn gin ser pro arg ser phe gin lys iys thr arg his tyr phe ile ala ala val glu arg ieu trp asp tyr gly met ser ser ser pro his val ieu arg asn arg ala gin ser gly ser val pro gin phe lys lys val val phe gin giu phe thr asp giy ser phe thr gin pro ieu tyr arg gly glu leu aar' giu his ieu gly leu ieu gly pro tyr ile arg ala glu val giu asp asn ile met val thr phe arg asn gin ala ser arg pro tyr ser phe tyr ser ser ieu ile ser tyr giu giu asp gin arg gin gly ala glu pro arg lys asn phe val lys pro asn glu thr lys thr tyr phe trp lys val gin his his met ala pro thr lys asp giu phe asp cys lys ala trp ala tyr phe ser asp val asp leu glu lys asp val his ser gly leu ile gly pro ieu ieu val cys his thr asn thr ieu asn pro ala his gly arg gin val thr val gin giu phe ala leu phe phe(leu) thr ile phe asp glu thr lys ser trp tyr phe thr glu asn met glu arg asn cys arg ala pro cys asn ile gin met giu asp pro thr phe lys giu asn tyr arg phe his ala ile asn gly tyr ile met asp thr leu pro gly ieu val met ala gin asp gin arg ile arg trp tyr leu leu ser met gly ser asn glu asn ile his ser ile his phe ser giy his val phe thr val arg lys lys glu glu tyr iys met ala ieu tyr asn leu tyr pro gly val phe giu thr val glu met ieu pro ser lys ala gly ile trp arg val glu cys leu ile gly giu his ieu his ala gly met ser thr ieu phe ieu val tyr ser asn lys cys gin thr pro ieu gly met ala ser gly his ile arg asp phe gin ile thr ala ser gly gin tyr gly gin trp ala pro iys V* 57 ieu thr pro lys ser thr gly tyr leu met ile er ala val1 gin phe phe asp leu gin asp met trp ala. thr asp gly Val1 leu ala asp val1 leu 1 eu gly ala arg ieu lys giu pro met ile ile phe ser ser ieu asp gly gly thr leu ile lys his ile arg ieu arg met glu pro ieu gly thr ala ser pro 5er lys trp arg pro asp phe gin gly val lys leu ile ser phe gin asn ser phe thr thr arg tyr ile ala ieu leu tyr; ala thr arg asp tyr met arg phe pro ser val val his leu phe leu ieu gly Val iie thr his ala val giu tyr asp lys val phe leu iys glu thr tyr ser asn ser gly ser leu ala his tyr phe ser his gly ieu tyr lys lys met Val asn ile his pro ieu met met giu ser tyr ala arg gin val lys thr ser ieu ser ser gly lys pro val ieu arg arg met ser gly trp ile ile lys ile ser trp gin phe phe phe asn thr his gly cys er lys phe thr leu his asn asn met iys leu thr gin asp val lys val asn ile his glu val ser lys thr gin thr g ly pro ty r asp ala asn leu pro val1 ser gly val ser pro leu gly gly lys ieu pro al1a al1a trp gin his ala val1 leu gln ile val1 gin phe tyr asn pro ser ieu ile met gin lys thr met his phe ieu gin gly ala giu ser phe arg giu c ar lys arg thr ser asp 1 eu thr asn ala asp ieu gly ala ile iie arg gly val asp ile ile ile arg asn ser ser asp phe ala giy arg glu trp gly val tyr val gin trp gin gly asp pro ser trp cys glu val giu ieu pro phe pro Val giu pro pro val tyr his pro ala ser glu lys tyr val asp pro ieu Val val cys ieu his trp ieu arg met asn s er ala ser cys ala thr ser leu thr lys thr asn pro Val1 ala leu val phe phe trp asp Val1 glu glu trp leu lys arg lys ser ala gin tyr ser ser arg thr ser gin trp asn gin thr giu 1 eu gin 1 eu his gin ser asp asn thr met thr ser gly asp gin cys asp glu phe arg gin pro tyr asn pro 1 eu gly asp pro asn tyr ieu lys tyr tyr leu ser asp ieu arg Val pro lys lys thr ile ala lys thr iie gin lys asn met val ser tyr gin thr ser gly gly ser gly pro met leu ser his ile gly ala glu lys thr 0 0 0 0 0 9 000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 000 *0 000 0* 0 0 00 0 0 0. 0 0 0 0 00 0 0 0 000 000 0 0 00 0 0 0 58 ile ser ser va 1 s er va 1 asn ieu leti ala arg leti asn his asp ser gly giti ile ieu tyr arg ieu giti tyr gly asp i le thr gin ser cys ala phe pro leu giu ala asn va 1 his his thr phe tyr met thr ser pro trp tyr arg thr leti ile pro 1 eu pro asp tyr leti gin l~eu giu leu ile ieu gin lys phe ieu thr arg arg tyr ser arg ala cys va 1 lys asp pro lys asp lys lys phe gly ile his pro gly gly s er ile arg phe asn giti asn his thr his ser phe lys ala ser trp le gin gin his lys asn ser s er thr tyr s er try lys pro phe gly lys giu pro ser gly gly ser ile asp gly glu asp lys gly ala asn trp leti his phe ala thr ile va 1 asn giti phe trp ala gin lys thr ieu lys ile gly ile thr phe pro asn va 1 gin pro tyr Val1 phe met glu va 1 ser pro pro va 1 leti ser ieu ser asp giu met ile val pro tyr iys arg 1 eu asn thr val phe lys va 1 leu gin phe arg giu va 1 ala ieu va 1 thr phe ieu lys gly ile giu leti ieu ser ser glu asp gin his ieu ieu va 1 giu tyr gin asp lys lys ser asn leti i le -asr phe phe phe tyr ser tyr va 1 asp met met leti gly gly giu met his cys ala va 1 ile tyr va 1 asn arg ala gly ala va 1 his tyr asp met ile met g it 1 eu gin asp trp va 1 glu phe glu gin his le met his ile asp gin pro giu va 1 arg le 1 eu asn phe i le giti ser arg ieu lys pro glu cys ser asn pro ala ser t-yr phe asp met gly asp thr gly gly thr ser leti his giu asp arg ser ala ala gly met gin val arg pro lys trp arg arg tyr asp arg asn ser ieu ile phe thr ser lys ser arg asp val asn cys his thr thr phe ieu pro ile gly gin asp gly giti pro tyr asp phe asp val ala ala giu pro asp pro gin ala tyr his giu gly asp pro tyr leu tyr asp phe thr val cys ieu asp ieu lys giti lys arg ser trp pro ala asn le gin ieu leti ser ser gly leu thr met glu trp his ala ala giy tyr arg lys pro giu val arg thr phe phe ieu met glu gin ieu asp asp asp asp lys lys glu giu asp arg arg le thr asp ser gly thr leu asn ile ser arg pro le thr val thr arg ala ser ser val asn val tyr ieu gly val met his ser val le gly tyr thr leu phe asn pro A> S.0 00' 04 S S S. S S 5 9 5 S *SS S S S S S 9 S I 59 gly arg asn ala ile tyr asp gin 1 eu asn va 1 tyr tyr arg ile lys lys cys asp thr gin lys ala asn lea tyr his tyr thr glu lea met gly tyr s er lea gly thr tyr thr asp ile lys trp arg phe arg ile asn s er asn va 1 lys va 1 asn glu ser pro tyr pro lea phe lys va 1 cys phe ala gin ser trp trp ile leu gly met thr ala lea gly asp tyr tyr lea lea ser lys arg thr thr ieu asp thr ile ser tyr asp glu asp lys thr arg his asp tyr gly met ala gin ser gly gin gla phe thr gly gla leu asn arg ala gla val gin ala ser arg tyr gia gla asp phe Val iys pro gin his his w ala trp ala tyr his ser gly lea thr lea asn pro phe ala leu phe trp tyr phe thr cys asn ile gin arg phe his ala gly lea val met leu ser met gly ser gly his val met ala leu tyr gla met leu pro leu ile gly giu leu val tyr ser ser gly his ile tyr gly gin trp giy ser ile asn ile lys val asp cys his asn ser asp phe arg asn leu lys val gla asp ser asn asn ala gin ser asp val gla met glu asn gin tyr phe ile ser ser ser ser val pro asp gly ser gla his lea glu asp asn pro tyr ser gin arg gin asn gla thr ala pro thr phe ser asp ile gly pro ala his gly ser ser cys asp lys tyr gla asp ile ser ile gia pro arg glu gin glu gla ile asp lys lys glu asp phe ser pro arg ser phe ala ala val gla arg pro his val leu arg gin phe lys lys vai phe thr gin pro lea gly lea lea gly pro ile met val thr phe phe tyr ser ser ieu gly ala gla pro arg lys thr tyr phe trp lys asp glu phe asp val asp lea glu lys ieu leu val cys his arg gin val thr val phe(leu) thr ile phe asp gla thr glu met i le ala ser phe asn ser his asn arg ala ala leu asn met glu glu asp pro asn gly tyr gin asp gin asn gla asn thr val arg lea tyr pro lys ala gly lea his ala lys cys gin asp phe gin pro lys lea trp ser thr lea ala pro arg thr ile arg ile lys gly ile gly thr ile ala lys met asn cys arg phe lys gla met asp thr ile arg trp his ser ile lys gia glu val phe glu trp arg val met ser thr pro lea gly thr ala ser arg lea his gla pro phe ile ile his V: :5 9. 5S :0 *5 S S S SO S S S S S S S.. S SSO S OS SS@ S gly le iys thr gin giy ala tyr lys val le pro met giu tyr arg va 1 thr ieu ser iys val arg met aia asp arg ser his leu va 1 his giu lys ieu thr asn ser ieu giu aia le ser gin trp gin thr phe phe giy phe asn pro thr his tyr giy cys asp ser lys ala phe thr asn ieu his ieu asn asn pro met lys vai ieu thr ser gin asp gly vai lys val val asn ser ile his pro giu val ieu thr arg arg tyr met gin phe pro pro val val tyr ieu phe asn leu gly pro iie thr ieu ala val giy tyr asp asp val phe pro lys giu asn tyr ser tyr ser gly ieu ieu ala lys leu phe ala thr lys asn arg ala trp phe tyr asn pro ser leu ile met gin iys thr met his phe leu gin gly tyr 5 er arg lys le thr lys val gin giy gly leu le glu va 1 ser pro ile ile arg giy val asp le le le arg asn ser ser asp phe ala gly arg giu trp gly val tyr vai gin trp gin gly asp pro ser trp cys giu tyr leu asp ieu val pro lys thr ala lys le gin asn met ser tyr thr ser gly ser pro met ser his gly ala lys thr phe asp ieu met iys met arg met asn ser ala ser cys ala thr 5 er leu thr lys thr asn pro vai ala gly gly lys ieu pro ala ala trp gin his ala val ieu gin glu gin his gin iys tyr ser ser thr ser gly arg tyr thr ieu ser met gin le trp ser asn ala gin val thr gin giu phe ieu phe gin asp ieu leu his gin gin asp ala val giu ieu ser phe phe val arg pro giu val ser his lys ala arg giu thr tyr ser asp asp leu ieu val thr ieu gly lys asp arg thr val phe ieu gly ile le arg pro thr pro trp asp gly ieu phe ser thr le ieu giu pro pro giu pro tyr pro ser lys va 1 pro va 1 cys his ser asp asn ser ser ieu asp gly lys thr ieu met lys his asn arg ieu his met giu ieu ieu gly met ala ser ser ser iys ala arg pro gin phe gin lys vai lys ser le ser ser gin asn gly phe thr pro arg tyr leu ala ieu arg tyr; and ieu ser trp val asp ala phe asn thr phe thr asp trp met gly asp thr val val ser ieu giu gly ala giu asp asp trp gin val leu cys ieu lys asp ieu arg giu giy iys phe le trp his ser ala ala ser gly tyr vai 0

6 SO S SOS 0 0 050 0 0 0 5 S 00 SO S 0 0 S 0*e*S 0 0 5 5 500 S. 00 S 90 50 ~3 0* S 5 O 000 S SOS 0 050 0# S p 61 asn arg Val tyr his ser his arg thr ala phe cys tyr val met lys thr asp ser pro pro lys trp asp tyr lys arg lys thr phe ieu gly ile ile pro his ieu pro pro gly asp gly tyr ser ieu ile gin arg ieu phe giu asn leu glu ile asn leu his gin thr lys his phe ser leu trp gly met thr gly ser trp i le gin gin his lys asn ser ser thr tyr s er try lys pro phe gly ].ys giu prc 5 er gly gly ser ile asp gly glu asp lys gly i le thr asp ieu his phe ala thr ile val1 asn giu phe trp ala gin lys thr ieu lys le gly le thr phe pro asn Val1 gin pro tyr Val phe met giu leu ala tyr pro gly leu ile val le gly met ieu glu gly his ser leu giu ile ieu leu mnet asp ser ser his gin asp ser cys pro giu giu ala glu met asp val val le gin ile arg Val his tyr ile pro leu val leu tyr ieu asn asn lys val arg phe arg giu ala ile leu tyr gly glu asn gin ala ser thr asp val arg val iys his ieu phe lys tyr lys iys ser asp pro Val asn met giu leu leu ile cys gin ile met ser phe asp giu asn arg phe ieu pro giu phe gin ala val phe asp ser ala tyr trp tyr leu ser val phe Val tyr giu asp thr val phe met gly cys his asn ieu ieu lys Val tyr glu asp ser gly gly thr ser ieu his glu asp arg ser ala ala gly met gin Val1 arg pro lys trp arg arg tyr asp arg asn 5 er ieu ile phe thr ser s er ser tyr cys his thr thr phe leu pro ile gly gin asp gly glu pro tyr asp phe asp val ala ala giu pro asp pro gin ala tyr his glu giy asp pro tyr ieu tyr asp phe thr val cys ieu asp ieu lys giu iys arg ser trp pro ala asn ile gin ieu leu ser ser gly leu thr met glu asp phe ser cys glu asp arg pro val thr phe met gin asp asp lys glu asp arg thr ser thr asn ser pro thr thr ala ser asn tyr gly met ser ile tyr ieu asn arg asp ile lys giu arg phe ieu giu leu asp asp lys giu arg le asp gly ieu iie arg iie val1 arg ser Val1 val1 1 eu val1 his val1 gly thr phe pro asn lys s er ser val1 asn ieu ieu ala arg ieu asn his asp ser giy g iu le ieu tyr arg ieu giu tyr giy asp ile thr gin ser cys ala phe pro gly arg asn ala 0 5 0 05 00 SC S 55 C CC e.g C 5 *00 6 0 6 S Cw*~ C C S 0 C 9 C C C S *.@CCC S C S SOC 00 seeC 0 CO C CCC C e.g C CCC S C S 62 tyr thr asp ile lys trp arg phe arg ile asn ser asn va 1 lys va 1 asn glu ser pro tyr pro ieu phe lys va 1 cys phe ala gin ser trp ile ile trp ieu leu ser arg thr thr asp thr ile tyr asp gLlu lys thr arg asp tyr gly ala gin ser gin glu phe gly glu leu arg ala giu gin ala ser tyr glu glu phe val lys gin his his ala trp ala his ser g-iy thr leu asn phe ala leu trp tyr phe cys asn ile arg phe his gly leu val leu ser met ser gly his met ala leu glu met leu ieu ile gly ieu val tyr ser gly his tyr gly gin gly ser ile ile lys val lys thr gin ser gin phe gin thr tyr lys leu ser asp his met gly thr asn val arg asp pro met tyr leu pro phe thr gin ala met gly val tyr pro giu ser ile trp asn asp gly i le arg asn asn ala ile giu pro arg giu ile gin ser asp gin glu glu ile asp tyr val glu met lys lys giu asp phe asp giu asn gin ser pro arg ser phe gin tyr phe ile ala ala val giu arg leu ser ser ser pro his val leu arg asn ser val pro gin phe lys lys val val asp gly ser phe thr gin pro leu tyr giu his leu gly ieu ieu gly pro tyr giu asp asn ile met val thr phe arg pro tyr ser phe tyr ser ser leu ile gin arg gin gly ala giu pro arg lys asn giu thr lys thr tyr phe trp lys ala pro thr iys asp giu phe asp cys phe ser asp val asp ieu glu lys asp ile gly pro ieu leu val cys his thr ala his gly arg gin val thr vai gin phe(leu) thr ile phe asp giu thr lys giu asn met glu arg asn cys arg ala met giu asp pro thr phe lys giu asn ile asn gly tyr ile met asp thr leu ala gin asp gin arg ile arg trp tyr ser asn giu asn ile his ser ile his phe thr val arg lys lys glu glu tyr asn ieu tyr pro gly val phe giu thr ser lys ala gly ile trp arg val glu his leu his ala gly met ser thr leu asn lys cys gin thr pro ieu gly met arg asp phe gin ile thr ala ser gly ala pro iys leu ala arg leu his tyr ala trp ser thr lys giu pro phe ser ieu ieu ala pro met ile ile his gly ala arg gin lys phe ser ser leu tyr ile. met tyr ser ieu asp gly lys lys gly asn ser thr gly thr leu met val C C S C CS CCCC S C CCC C C S C C 603665 C C C CCCSC C C C C OCS CC CCC C CC C C S CCC C C C CCC C S SC. S CCC s.c C A 63 phe phe thr gly ser phe leu asn met leu gin val val ile glu phe asn his cys lys thr his asn lys thr asp lys asn his val gly asn val asp ser ser gly pro pro ile ile ala arg tyr tyr ser ile arg ser thr leu asp leu asn ser cys ser met ala ile ser asp ala gin ile asn met phe ala thr trp ser leu gin gly arg ser asn ala pro lys glu trp leu gin val val thr gly val thr thr gin ser met tyr val lys glu phe gly his gin trp thr leu phe val phe gin gly asn gin asp ser leu asp pro pro leu leu pro gin ser trp val his gln leu gly cys glu ala gin asp ile lys ile arg arg met pro leu thr ala pro ser trp arg asp phe gly val leu ile phe gin ser phe thr arg ile ala his leu glu gly ser lys pro gin lys ser asn thr tyr leu asn ile his pro leu met met glu ser tyr ala arg gin val lys thr ser leu ser ser gly lys pro val leu arg arg met leu tyr.

7. A modified factor VIII:C-like polypep- tide, comprising the N-terminal heavy chain of mature factor VIII:C linked directly to the C-terminal light chain of mature factor VIII:C, said polypeptide being essentially free of other serum proteins.

8. A process for producing a polypeptide, comprising the step of proteolytically cleaving the modified factor VIII:C-like polypeptide of claim 7 into the N-terminal heavy chain of mature factor VIII:C and the C-terminal light chain of mature factor VIII:C.

9. The process according to claim 8, further comprising the step of linking together by an alkaline metal bridge, the N-terminal heavy chain of mature factor VIII:C and the C-terminal heavy chain of mature factor VIII:C. I /7 ~~JT 6 9 m o o S i a S #o S 50 b S S 0@ @05 5 S S@ S a 0 1 5 04 a a.. 0 S i, O 4 *5 A 0 a a S L~L~1~L 64 A process for producing a modified factor VIII:C-like polypeptide comprising the step of culturing a host transformed with a recombinant DNA molecule as defined in claims 1 through 11. The process according to any of claims 8, 9 or 10, wherein the host is selected from BMT10, BSC1, COS1, COS7, CHO cells, as herein defined, and other animal and human cells in culture. 12. A pharmaceutical composition comprising a polypeptide, produced according to the process of any of claims 8, 9, 10, or 11, in an amount effective as a coagulant and a pharmaceutically acceptable carrier. 13. A pharmaceutical composition comprising a modified factor VIII:C-like polypeptide as defined in claim 6 or 7 in an amount effective as a coagulant and a pharmaceutically acceptable carrier. 14. A method for treating haemophilia comprising the step of treating a human with the pharmaceutical composition as defined in claims 12 and 13. A recombinant DNA molecule according to claim 4, selected from the group consisting of the recombinant DNA molecules contained in transformed host E.coli HBO01(RE), E.coli HBO01(RD) and E.coli HBO01(RSD), as herein defined. 16. A modified factor VIII:C-like polypeptide produced by a host transformed with a recorbinant DNA molecule selected from a group consisting of recombinant DNA molecules contained in transformed host E.coli 3 3 0 06 0 B# Lee ste 6 0ie 00960 0 c e SO 0 s *e 0 5 9 t I S e 4 5 0 0 06 6 .ii- i -ui: iirs -x~i*rr- I *CI' 65 HBO01(RE), E.coli HBO01(RD) and E.coli HB101(RSD), as herein defined. 17. A process for producing a modified factor VIII:C-like polypeptide comprising the step of culturing a host transformed with a recombinant DNA molecule selected from a group consisting of recombinant DNA molecules contained in transformed host E.coli HBO01(RE), E.coli HB101(RD) and E.coli HBIC1(RSD), as herein defined. 18. A pharmaceutical composition comprising a polypeptide produced according to the process of claim 17 in an amount effective as a coagulant and a pharmaceutically acceptable carrier. 19. A method for treating haemophilia comprising the step of treating a human with a pharmaceutical composition according to claim 18. DATED this 24th day of January 1990 BIOGEN, INC. AND MARK P. PASEK By their Patent Attorneys G.R. CULLEN CO. 4 S 9@ S r S 5 S 5*5 5 5 S S S 5 5 5 5 55 5 55 S .5.5 SS S S S A Uo RESTlJCr1OA' NOOAYL/CLASE A-AP w OF T1l FACT0A3:C cOA ECORI PSTI SPJII BALI A1A3 BCL 1 STU NSII ECOR/ /PAI 14IND3 0AMI T7Ty ND~f SrAI MMB/AI A/A3 BALI PY12 SAMI AFL2 P1102 ECOB 411A3 Nl I kMV f STU BAL I XUvI PSTI NCO f CORI PVU2 XAI KPNI ECOR'; SACN NDEI BCL i SnII PST APA I SPY AS02 5711 8ALI M911 SCI iii' UIN~oj h~/A~ly 1 A3 PA1 XI WI BG6) 1 2ALI PST I NDVE XMl/1 AIA3X A) XIAf ND3 9.0/I1 SACI A1I (D Iff 14103 96i 2 111N ,03 Y111 11 14 319 561 09 1379 1V5 2227 2658 3174 3736 4210 464 J16.3 5522 6/38 6569 6945 7339 7868 8230 8683 214 567 I40 16A9 2289 29763307 3774 44/9 5390 57S5 6/98 6585 6975 7359 7912 8441 C377 1852 2427 2991 3786 4538 5604 6208 6532 7268 7991 w 402 /869 2444 3245 3797 4743 5793 6860 7269 U) 462 2616 3933 6977 7435- 6/0 3935 7062 7463 -4 l=s--:1I= C ci) -I N 82 7-9 77 8386 74 75 73 I----------pUC192874 /.7977 10,797783 774-- 475 COglR AL41 NDE! NIDEf BAMI SAC NOE! B4MI1l ECORI SAC/ IVRD6 AA! SM/I AOEI lCORl A/C! 84mAM1 lI I I I I I I I I I II III7 I 14 4a 71 1869 2289 299/ 3774 410/ 4743 S522 6927 799/ 8441 214 561 3245 3786 8230 Vn I- i- I- 00 FIG. 00 WO 88/00831 WO 8800831PCT/US87/0 1814 z/z$ FIG2 SAC .7 yind X B 4 ,m W'( fPt- 'T 6Laffq F(9Z F Sa he -6 /3) -SUBSTITU -E SH T WO 88/00831PC/S7081 PCT/US87/01814 462 1frag, e,71 115 ,149 27$hp /657 Gle, frag.~n~SerAsi FIG 3A SUBSTITUTE SHEET WO 88/00831 WO 8800831PCT/US87/0 1814 '1/gs 151f40 FIG 38 746 pzy iol 7 p_ 71ev 44/4 1702 PS I SUBSTITUTE SHEET WO 88/00831 WO 8800831PCT/US87/0 1814 I 5/28 FIG 4 /VCO f245 4E.:2/ 1 f266

7249-521t 6 843 p A-Rn 2e,5.9 6/25 54C 5245 Xba 'SUBSTITUTE SWEET i I WO 88/00831 PCT/US87/0 1814 0b 4 SUBSTITUTE SHEET r-- WO 88/00831 WO 8806831PCT/US87/0 1814 7/28 4.w..fp. FIG. 5 B SUBSTITUTE SHEET I WO 88/00831 PCT/L'S87/0 1814 WO 88/00831 WO 8800831PCTr/US87/0 1814 ~P SUBSTITUTE SHEET WO 88/00831 PTLS7011 PCT/US87/01814 Cl/Z8 1 ala thr arg arg tyr tyr leu gly ala val glu leu ser GCC ACC AGA AGA TAC TAC CTG GGT OCA GTG GAA CTG TCA trp, asp tyr met gin ser asp TGG GAC TAT ATG CAA AGT GAT asp ala arg phe pro pro arg GAC GCA AGA TTT CCT CCT AGA phe asn thr ser val val tyr TTC AAC ACC TCA GTC GTG TAC ecoRI glu phe thr asp his leu phe GAA TTC ACG GAT CAC CTT TTC pro pro trp met gly leu leu CCA CCC TGG ATG GOT CTG CTA glu val tyr asp thr val val GAG GTT TAT GAT ACA GTG GTC ala ser his pro val ser leu OCT TCC CAT CCT GTC AGT CTT leu gly CTC GOT val pro GTG CCA lys lys AAA AAG asn ile AAC ATC gly pro GOT CCT ile thr ATT ACA 100 his ala CAT OCT glu leu GAG CTG pro val CCT GTG lys ser phe pro AAA TCT TTT CCA s0 thr leu phe val ACT CTG TTT GTA ala lys pro arg GCT AAG CCA AGO thr ile gin ala ACC ATC CAG GCT leu lys asn met CTT AAG AAC ATG val gly val ser GTT GOT OTA TCC FIG7 N WO 88/00831 WO 8800831PCT/US87/0 1814 hindlI I tyr trp lys ala ser TAC TGG AAA .GCT TCT 120 thr ser gin arg glu ACC AGT CAA AGO GAG 110 glu gly ala glu tyr asp asp gin GAG GGA GCT GAA TAT GAT GAT CAG lys glu AAA GAA 130 asp asp, lys val- p-he-pro CAT GAZ AAA GTC. CCT trp, gin. val ieu iys- gui TGO CAG. G7C. T' AAA* .GAG gly gly ser his thr COT GGA AGC CAT ACA tyr val TAT GTC 150 asn gly pro met ala AAT GGT CCA ATG GCC 160 ser tyr leu ser his TCA TAT CTT TCT CAT 170 ser gly leu ile gly TCA GGC CTC ATT GGA ser ieu ala lys glu AGT CTG GCC AAG GAA 200 ile ieu ieu phe ala ATA CTA CTT TTT GCT 210 his ser glu thr lys CAC TCA GAA ACA AAG ser asp TCT GAC val asp GTG GAC ala ieu GCC CTA lys thr AAG ACA pro leu cys ieu thr tyr CCA CTG TOC CTT ACC TAC ecoRl leu val lys asp leu asn CTG GTA AAA GAC TTG AAT 180 leu val cys arg glu gly ("TA GTA TGT AGA GAA CG 190 gin thr leili his lys phe CAC ACC TTG CAC AAA TTT val phe GTA TTT asp giu gly lys ser trp GAT GAA COG AAA AGT TGG 220 leu met gin asp arg asp TTG ATG CAG CAT AGO CAT asn ser AAC TCC FIG7(c on t'd) WO 88/00831 WO 8800831PCT/US87/0 1814 /1#/8 ala ala GCT OCA asn gly AAT GOT ser ala TCT GCT try val TAT GTA 2.50 arg lys AGO AAA thr pro ACT CCT arg ala CGG GCC 240 asn arg AAC AGO trp pro TGG CCT ser leu TCT CTG 230 lys met AA.A ATG pro gly CCA GGT cys his TGC CAC gly thr GOC ACC ser val TCA GTC glu val GAA GTG tyr trp TAT TGG his ser CAC TCA 280 asn his AAC CAT his val CAT GTG 270 ile phe ATA TTC hi s thr CAC ACA phe leu TTT CTT val arg GTG AGO arg gln CGC CAG his thr val CAC ACA GTC leu ile gly CTG ATT GOA 260 ile gly met ATT GOA ATG leu glu gly CTC GAA GOT ala ser leu GCG TCC TTG gln thr leu CAA ACA CTC 310 cys his ile TGT CAT ATC tyr val lys TAT GTC AAA arg met lys CGA ATG AAA 290 glu ile GAA ATC 300 leu met TTG ATG ser ser TCT TCC ser pro TCG' CCA ile thr ATA ACT phe leu TTC CTT thr ala ACT GCT asp ieu GAC CTT his gin CAC CAA gly gin GGA CAG his asp CAT GAT 330 pro glu CCA GAG phe leu TTT CTA 320 gly met GGC ATG leu phe CTG TTT~ hindlI I glu ala GAA GCT val asp GTA GAC ser cys AGC TGT giu pro GAA CCC gin. leu CAA CTA F IG 7(cont'd) WO 88/00831 WO 8800831PCT/US87/O 1814 340 asn asri AAT AAT asp ser GAT TCT ser pro TCT CCT glu glu ala glu asp tyr GAA CAA GCG GAA GAC TAT glu met asp va. val arg CAA ATG GAT GTG GTC AGO 370 ser phe ile gin ile arg TCC TTT ATC CAA ATT CC 380 lys thr trp va]. his tyr AAA ACT TGG GTA CAT TAC asp asp ;AT GAT 360 phe asp TTT GAT ser val TCA GTT asp GAT 350 leu thr CTT ACT asp CAT ala CC asp asn CAC AAC lys AAG lys AAC 390 glu GAG his pro CAT CCT ile ala ATT CCT 400 val leu CTC CT-_ ala CCT glu GAA glu asp GAG GAC trp asp tyr ala pro leu TCG GAC TAT CCT CCC TTA ala CC pro CCC asp -GAT 410 asp arg ser tyr lys ser gin tyr leu asn asn gly pro CAC ACA AGT TAT AAA ACT CAA TAT TTG AAC AAT CCC CCT 420 gin arg CAG CGG 430 ala tyr GCA TAC ile gly arg lys tyr iys ATT GOT AGG AAG TAC AAA lys val AAA GTC arg CGA 440 glu GAA phe TTT met ATG thr asp glu thr phe lys ACA CAT GAA ACC TTT AAC 450 glu ser gly ile leu gly CAA TCA GGA ATC TTG GGA thr arg ACT CGT pro leu CCT TTA ala GCT ile ATT gly GGG gin his CAG CAT ieu tyr CTT TAT FI/G 7(cont'1 S M "Z A J :Z rJr z :T WO 88/00831 PCT/US87/O 1814 I 5/28 glu val GAA GTT 470 ala ser GCA AGC asp val GAT GTC val lys GTA AAA ile phe ATA TTC pro thr CCA ACT ser ser TCT AGT leu ile CTC ATT 560 asp gin GAT CAA 460 gly asp thr leu leu ile GGA GAG ACA CTG TTG ATT arg pro tyr asn ile tyr AGA CCA TAT AAC ATG TAG arg pro leu tyr ser arg CGT GCT TTG TAT TGA AGG 500 his leu lys asp phe pro CAT TTG AAG GAT TTT CCA 510 lys tyr lys trp, thr vai AAA TAT AAA TGG, ACA GTG lys ser asp pro arg cys AAA TCA GAT GCT CGG TGC 540 phe val asn met glu arg TTC GTT AAT ATG GAG AGA 550 gly pro leu ieu ile cys GGC GCT GTC GTC ATC TGC arg gly asn gin ile met AGA GGA AAC GAG ATA ATG ile phe lys asn gin ATA TTT AAG AAT CAA 480 pro his gly ile thr CGT CAC OGA ATC ACT 490 arg leu pro lys gly AGA TTA CCA AAA GGT ile leu pro gly giu ATT GTG CCA GGA GAA 520 thr val giu-asp gly ACT GTA GAA GAT GGG 530 leu thr arg tyr tyr CTG ACC CGC TAT TAG asp ieu ala ser gly GAT GTA GCT TCA GG3A tyr lys giu ser val TAG AAA GAA TCT GTA 570 ser asp lys arg asn TCA GAG AAG AGO AAT F G 7(cont *di,) SUBSTITUTE SH1EET WO 88/00831PC/S7084 PCT/US87/01814 va2. ile GTC ATC pnI tyr leu TAG CTC 600 ala gly GCT GGA leu CTG thr ACA val GTG 580 phe ser val phe asp glu TTT TCT GTA TTT GAT GAG 590 glu asri ile gin arg phe GAG AAT ATA CAA CGC TTT b amH I gin leu glu asp pro glu GAG GTT GAG GAT GGA GAG 620 his ser ile asn gly tyr GAG AGG ATC AAT GGG TAT 630 ser val cys leu his'glu TGA GTT TGT TTG GAT GAG ser ile gly ala gin thr AGG ATT GGA GGA GAG ACT asn AAG leu CTG phe TTG arg CGA pro GGG gin CAA ser AGG as-n AAT 610 ala GGG trp TGG pro GGA ser TGG asn ile AAG ATG leu gin TTG GAG met ATG 1 eu TTG 640 1 eu GTA val GTT val GTG phe TTT ala GGA asp ser GAT AGT tyr trp TAG TGG 650 leu ser GTT TGT tyr ile TAG ATT asp GAG 660 his GAC phe TTG val phe GTC TTG phe TTG ser gly tyr thr phe lys TGT GQA TAT AGG TTC AAA lys AAA met ATG val GTG tyr giu TAT GAA 670 asp thr leu thr leu phe pro phe ser gly glu GAG ACA GTC ACC GTA TTC GGA TTC TGA GGA GAA thr val ACT GTC phe TTC 680 met ser met giu asn pro ATG TGG ATG GAA AAC GGA gly, leu trp GGT GTA TGG 1e ATT F IG 7(cont'd) 0 U ES T iT UT I S ll WO 88/00831PC/S7081 PCTIUS87/01814 690 ieu CTG gly CG 700 cys his asn ser asp phe arg asn arg gly met TGC CAC AAC TCA GAC TTT .CGG AAC AGA GCC ATG 710 thr ACC ala CC ieu TTA ieu CTG lys val ser AAC GTT TCT ser cys asp lys asn thr ACT TCT CAC AAC AAC ACT gly COT 720 asp tyr tyr giu asp ser tyr giu asp ile ser ala CAT TAT TACGCAC CAC ACT TAT CAA CAT ATT TCA CCA 730 hindI tyr TAC ieu TTC ieu CTC ser ACT lys asn asn AAA AAG AAT ala ile giu pro arg ser CCC ATT CAA GGA ACA AC 750 pro ser thr arg gin lys GGT ACC ACT ACO CAA AAG phe TTC ser TCC ecoRI gin asn GAG AAT gin CAA phe TTT asn ala AAT CC 770 asp pro CAC CCT ser arg his TCA ACA CAC 760 thr thr ile ACC ACA ATT trp phe ala TCG =TI CCA lys AAG thr ACT pro giu asn asp ile giti GCA CAA AAT CAC ATA CAC 780 his arg thr pro met pro CAC ACA ACA CCT ATC CCT 790 ser asp ieu leu met leu AGT CAT TTG TTG ATG CTC lys AAA ile ATA gin asn CAA AAT val ser ser CTC TCC TCT 800 pro thr pro CCT ACT CCA leu TTG arg CGA gin ser GAG AGT his gly ieu ser leu ser CAT COG CTA TCC TTA TCT F 7(cont 2dW SUBSTITUTE SHEET WO 880081PCT/US87/O 1814 WO 88/00831 If*2 asp Leu GAT CTC 820 pro ser CCA TCA 810 gin glu ala CAA GAA GCC lys tyr AAA TAT glu GAG thr phe ACT TTT ser TCT 830 ser AGC asp GAT asp GAT pro CCT gly GGA ala GCA ile asp ATA GAC ser AGT asn AAT a sr AAC leu CTG ser TCT glu met GAA ATG asp met GAC ATG 860 leu asn TTA AAT thr ACA val GTA his CAC phe TTT phe TTC 850 thr ACC 840 arg pro gin ieu his his ser giy AGG CCA CAG CTC CAT CAC AGT GGG pro giu CCT GAG ser gly ieu gin ieu arg TCA GGC CTC CAA TTA AGA glu GAG lys AAA leu CTG giy thr GGG ACA thr ACT ala GCA 880 ser AGT ala GCA thr ACA 870 glu GAG ieu TTG lys lys AAG AAA ieu CTT asp GAT phe TTC lys val AAA GTT ser TCT thr ACA ser TCA asn asn AAT AAT ieu ile CTG ATT 890 ser thr ie pro ser asp asn leu ala ala gly TCA ACA ATT CCA TCA GAC AAT TTG GCA GCA GOT thr asp ACT GAT 900 asn AAT thr ACA ser AGT ser leu TCC TTA gly GA 910 pro pro ser met pro CCC CCA AGT ATG CCA val his GTT CAT tyr TAT asp ser gin ieu GAT AGT CAA TTA asp GAT thr ACC 920 thr ieu phe ACT CTA TTT gly GC 'I G. 7(con/'le) SUBSTITUTE SHEET WO 88/00831 WO 8800831PCT/US87/O 1814 17/29 930 ser pro leu thr glu ser gJly gly pro leu TCT CCC CTT ACT GAG GOT GGA CCT CTG lys AAA Jlys ser AAG TCA 940 ser leu ser glu glu asn asn asp ser lys leu..Ieu glu AGC TTG AGT GAA GAA AAT AAT GAT TCA AAG, ,TTA GAA 950 960. ser TCA gly leu GOT TTA met ATG asn ser AAT AGC gin glu CAA GAA 970 ser gly AGT GOT ser. *ser-- trp ,g-Ly Lys-, AGT TCX: .TGG. GGA AAA asn val ser ser thr glu AAT GTA TCG TCA ACA GAG arg leu phe AGO TTA TTT lys gly AAA GG lys AAA ala GCC lys AAA sacI arg ala AGA GCT 990 leu phe TTA TTC thr ser ACT TCC his CAT 980 gly pro GGA CCT ala leu GCT TTG ieu thr lys TkTCY ACT AAA lys AAA val ser GTT AGC ile ser ATC TCT ieu leu lys TTG TTA AAG 1010 asn arg lys AAT AGA AAG asp asn GAT AAT 1000 thr asn ACA AAC thr his ACT CAC asn asn ser ala thr AAT AAT TCA GCA ACT ile ATT asp gly GAT GOC pro CCA 1020 ser leu leu ile TCA LTA TTA AT': glu asn ser GAG AAT AGT pro ser CCA TCA 1040 phe lys TTT A ral GTC 1030 trp gin asn TGG CAA AAT ile leu glu .4er ATA TTA GAA AGT asp thr glu GAC ACT GAG FI/G 7("con/'dV) SURSTITUTE SHEET WO 88/00831 WO 8800831PCT/US87/0 1814 I 8/-Zg 1050 iys val thr AAA GTG ACA pro ieu ile his asp arg met CCT TTG ATT CAT GAC AG~A ATG ieu met asp CTT ATG GAC 1060 lys asn ala AAA AAT GCT lys thr thr AAA ACT ACT 1080 lys lys giu AAA AAA GAG thr ala leu arg ACA GCT TTG AGO 1070 ser ser lys asn TCA TCA AAA AAC gly pro ile pro GGC CCC ATT CCA leu asn his CTA AAT CAT met giu met ATG GAA ATG pro asp ala CCA GAT GCA 1100 leu phe leu CTA TTC TTG met ser asri ATG TCA AAT val gin gin GTC CAA CAG 1090 gin asn pro CAA AAT CCA asp met ser GAT ATG TCG phe phe lys met TTC TTT AAG ATG 1110 ile gin arg thr ATA CAA AGO ACT pro CCA glu ser GAA TCA ala arg trp GCA AGO, TGG 1120 asn ser gly AAC TCT GGG his gly lys CAT GOA AAG, asn AAC leu gly pro TTA GGA CCA gin gly pro ser CAA GGC CCC AGT glu lys ser val GAA AAA TCT GTG 1150 asn lys val val AAC AAA GTG GTA pro lys gin CCA AAG CAA 1140 glu gly gin GAA GGT CAG leu TTA asn AAT ser leu TCT CTG 1130 val ser GTA TCC phe leu TTC TTG ser glu lys TCT GAG AAA val gly lys GTA GGA AAG gly GOT glu phe GAA TTT FI/G. 7(cont 1d) SLnS -1TUTE SHEET WO 88/00831 PCT/US87/0 1814 I 9/za 1160 thr lys asp ACA AAG GAC ser arg asn AGC AGA AAC val gly leu lys glu met OTA GGA CTC AAA GAG ?rTG ieu phe leu thr asn ieu CTA TTT CTT ACT AAC TTG 1170 val phe pro ser GTT TTT CCA AGC 1180 asp asri ieu his GAT AAT TTA CAT 1190 giu asn asn GAA AAT AAT giu ile glu GAA ATA GAA 1210 val leu pro GTT TTG CCT phe met lys TTC ATG AAG val glu gly GTA GAA GGT 1250 gin asp phe CAA GAT TTT thr his asn ACA CAC AAT 1200 lys lys giu AAG AAG GAA gin ile his CAG ATA CAT asn leu phe AAC CTT TTC 1240 ser tyr asp TCA TAT GAC gin glu lys CAA GAA AAA thr leu ile ACA TTA ATC thr val thr ACA GTG ACT 1230 leu leu ser TTA CTG AGC gly ala tyr GGG GCA TAT lys AAA gin CAA gly GGC thr ACT ala, GCT ile gin glu ATT CAG GAA giu asn val GAG AAT GTA 1220 thr lys asp. ACT AAG AAT arg gin asn AGG CAA AAT scaI pro vaj. leu CCA GTA CTT 1260 asn arg thr AAT AGA ACA arg ser leu AGG TCA TTA asn asp ser thr AAT GAT TCA ACA 1270 phe ser lys lys TTC TCA AAA AAA lys lys his AAG AAA CAC thr ala his ACA GCT CAT gly giu giu GGG GAG GAA FIG. 7(cantbd) SUBSTITUTE SHIZET WO 88/00)831 PCT/US87/0 1814 /z giu asn GAA AAC 1280 ieu giu gly ieu giy asn gin TTG GAA GGC TTG GGA AAT CAA thr lys gin ile ACC AAG CAA ATT 1300 iie ser pro asn ATA TCT CCT AAT val giu GTA GAG 129( lys sphI tyr ala cys thr thr arg TAT GCA TGC ACC ACA AGO 1310 thr ser ACA AGC ala leu GCT TTG gin CAG lys AAA leu giu CTT GAA 1340 trp, ser TGG TCC lys AAA gin asn phe val thr gin CAG AAT TTT GTC ACG CAA 1320 gin phe arg leu pro ieu CAA TTC AGA CTC CCA CTA 1330 arg ile ile val asp asp AGO, ATA ATT GTG GAT GAC asn met lys his leu thr AAC ATG AAA CAT TTG ACC 1360 asp tyr asn giu lys giu GAC TAC AAT GAG AAG GAG 1370 pro ieu ser asp cys lieu CCC TTA TCA GAT TCG CTT arg CGT giu GAA ser lys AGT AAG glu thr GAA ACA arg AGA giu GAA thr ACC pro CCG ser thr TCA ACC 1350 s er thr AGC ACC gin CAG lys AAA leu CTC thr gin ACA CAG ile ATA lys AAA g'iy ala GGG CC ile ATT thr gin ACT CAG ser TCT tlir ACG arg ser AGG AGT his CAT 1380 ser ile pro AGC ATC CCT gin ala asn arg ser pro CAA GCA AAT AGA TCT CCA leu TTA 1390 pro ile ala C2CC ATT GCA F/I6G 7(con/Wl) r L -t WO 88/00831 PCT/US87/0 1814 1400 iys val ser ser phe pro ser ile arg pro ile tyr leu AAC GTA TCA TCA TTT CCA TCT ATT AGA CCT ATA TAT CTC 1410 thr arg val leu phe gin ACC ACG GTC CTA TTC CAA 1.420 ala ala ser tyr arg lys GCA GCA TCT TAT AGA AAC asp asn ser GAC AAC TCT ser his TCT CAT ieu pro CTT CCA 1430 gin glu CAA GAA lys asp ser AAA CAT TCT ser ser his phe ieu gin AGC ACT CAT TTC TTA CAA -er ieu ala ile ieu thr TCT TTA CCC ATT CTA ACC 1.460 arg glu val gly ser leu AGA CAG CTT GGC TCC CTC 1470 val thr tyr lys lys val GTC ACA TAC AAG AAA CTT gly ala iys CGA CCC AAA 1450 ieu glu met TTC GAG ATC gly val CCC CTC 1440 lys asn AAA A.AT thr gly ACT GGT asn leu AAC CTT asp gin CAT CAA gly thr ser CCC ACA ACT ala thr CCC ACA asn ser AAT TCA glu asn thr CAG AAC ACT 1480 val leu pro lys GTT CTC CCG AAA pro asp leu pro lys thr CCA CAC TTG CCC AAA ACA 1490 ser gly lys val glu leu leu TCT CCC AAA GTT GAA TTG CTT 1500 pro lys val his ile tyr gin lys asp leu phe pro thr CCA AAA GTT CAC ATT TAT CAG AAG GAC CTA TTC CCT ACG F IG 7t'con/'d2 SUBSTITUTE SAIEET WO 88/00831 WO 8800831PCT/US87/O 1814 1510 glu thr ser asri gly ser pro gly his leu asp GAA ACT AGC AAT GGG TCT CCT GGC CAT CTG GAT 1530 glu gly ser leu leu gin gly thr glu gly ala GAA GGG AGC CTT CTT GAG GGA ACA GAG GGA GCG 1540 trp asn glu ala asn arg pro gJly lys val pro TGG AAT GAA GCA AAC AGA CCT GGA AAA GTT CCC 1550 arg val ala thr glu ser ser ala lys thr pro AGA GTA GCA ACA GAA AGC TCT GCA AAG ACT CCC b amH I 1570 leu leu asp pro leu ala trp, asp asn his tyr CTA TTG GAT CCT CTT GCT TGG GAT AAC CAC TAT 1580 gin ile pro lys glu glu trp lys ser gin glu GAG ATA CCA MAA GAA GAG TcG, AMA TCC CA GAG 1520 leu val CTC GTG ile lys ATT AAG phe leu TTT CTG 1560 ser lys TCC MAG giy thr GGT ACT lys ser AAG TCA 1590 pro glu lys thz ala phe lys lys lys asp thr ile leu CCA GMA AAA ACA GCT TTT MAG AM MAG CAT ACC ATT TTG 1600 1610 ser leu asn ala cys glu ser asn his ala ile ala ala TCC CTG MAC GCT TGT GMA AGC MAT CAT GCA ATA GCA GCA 1620 ile asn glu gly gin asn lys pro glu ile glu val thr ATA MAT GAG GGA CMA MT MAG CCC GMA ATA GMA CTC ACC F 7(cont 'd) WO 88/00831 WO 8800831PCT/US87/0 1814 as/Z8 1630 trp TGG ala lys gin gly GCA AAG CAA GOT arg thr giu arg ieu cys AGO ACT GAA AGO CTG TGC ser gin TCT CAA 1650 ile thr ATA ACT 1640 asn pro pro val ieu AAC CCA CCA GTC TTG lys arg his AAA CGC CAT gin arg giu CAA CGG GAA 1660 arg thr thr leu gin ser asp gin glu giu ile asp tyr CGT ACT ACT CTT CAG TCA GAT CAA GAG GAA ATT GAC TAT 1670 asp asp thr ile ser val giu met lys lys giu asp phe GAT GAT ACC ATA TCA GTT GAA ATG AAG AAG GAA GAT TTT asp GAC phe TTT 1680 ile tyr asp giu ATT TAT GAT GAG gin lys lys thr CAA AAG AAA ACA giu arg ieu trp asp GAG AGO CTC TGO GAT 1720 val ieu arg asn arg GTT CTA AGA AAC AGO 1730 phe lys lys val val TTC AAG AAA GTT GTT asp giu asn GAT GAA AAT. arg his tyr CGA CAC TAT 1710 tyr giy met TAT GGG ATG ala gin ser GCT CAG AGT phe gin giu TTC CAG GAA 1690 gin CAG phe TTT ser pro AGC CCC 1700 ile ala ATT GCT arg ser CGC -AGC ala val GCA GTG ser ser ser pro his AGT AGC TCC CCA CAT gly GC phe TTT ser val AGT GTC pro gin CCT CAG 1740 thr asp gly ser ACT GAT GOC TCC FIG. 7(cont'*d) SUBSTITUTE SHEET FIG. 7(cont'd) WO 88/00831 PCT/US87/018 14 t zqiz& 1750 phe TTT leu TTG asp GAT thr gin pro ACT CAG CCC gly leu leu GGA CTC CTG 1770 asn ile met AAT ATC ATG leu tyr arg TTA TAC CGT 1760 gly pro tyr GG,3 CCA TAT val thr phe GTA ACT TTC giy glu leu GGA GAA CTA ile arg ala ATA AGA GCA arg asn gin AGA AAT CAG 1790 leu iie ser CTT ATT TCT asn AAT glu GAA ala GCC glu his GAA CAT val giu GTT GAA 1780 ser arg TCT CGT pro CCC tyr ser phe TAT TCC TTC tyr ser ser TAT TCT AGC 1800 gly ala glu GGA GCA GAA tyr TAT asp gin arg gin GAT CAG AGG CAA pro arg lys CCT AGA AAA asn AAC giu glu GAG GAA phe val TTT GTC 1820 val gin GTG CAA lys AAG 1810 pro asn glu CCT AAT GAA thr lys thr ACC AAA ACT tyr phe trp TAC TTT TGG lys AAA his CAT his met ala CAT ATG GCA pro tlir lys CCC ACT AAA 1830 asp giu phe asp GAT GAG TTT GAC cys lys TGC AAA ala GCC trp ala tyr TGG GCT TAT 1840 phe ser asp val asp lea glu TTC TCT GAT GTT GAC CTG GAA lys asp AAA GAT val GTG 1850 his ser gly leu ile gly pro ieu leu val CAC TCA GGC CTG ATT GGA CCC CTT CTG GTC cys his TGC CAC F I 7(cont Vd) SUBSTITUTE SHEET WO 88/00J831PC/S7081 PCT/US87/01814 1860 1870 thr asn thr leu asn pro ala his gly arg gln va. thr ACT AAC ACA CTG AAC CCT GCT CAT GGG AGA CAA GTG ACA 1880 val gin giu phe ala leu phe phe thr ile phe asp glu GTA CAG GAA TTT GCT CTG TTT TTC ACC ATC TTT GAT GAG 1890 thr lys ser trp tyr phe thr glu asn met gJlu arg asn ACC AAA AGC TGG TAC TTC ACT GAA AAT ATG GAA AGA AAC 1900 1910 cys arg ala pro cys asn ile gin met glu asp pro thr TGC AGO GCT CCC TGC AAT ATC CAG ATG GAA CAT CCC ACT 1920 phe lys glu asn tyr arg phe his ala. ile asn gly tyr TTT AAA GAG AAT TAT CGC TTC CAT GCA ATC AAT GGC TAC 1930 ile met asp thr leu pro gly leu val met ala. gin asp ATA ATO GAT ACA CTA CCT COC TTA GTA ATO GCT CAG CAT 1940 1950 gin arg ile arg trp tyr leu leu ser met gly ser asn CAA £PuW ATT CGA TOG TAT CTG CTC AGC ATG GGC AGC AAT 1960 glu asn ile his ser ile his phe ser gly his val phe GAA AAC ATC CAT TCT ATT CAT TTC AGT OGA CAT GTC TTC 1970 thr val arg lys lys glu glu tyr lys met- ala leu tyr ACT OTA CGA AAA AAA GAG GAG TAT AAA ATG OCA CTG TAG F IG 7(con/ d ISU:STil" 71 WO 88/00831 PCT/US87/o 1814 1. 1980 asn leu tyr pro gly val phe AAT CTC TAT CCA GOT GTT TTT 1990 pro ser lys ala gly ile trp CCA TCC AAA GCT GGA ATT TG glu thr val GAG ACA GTG glu met ieu OAA ATG TTA 2 00 0 cys Ieu ile TGC CTT ATT gly giu his ieu his ala gly OGC GAG CAT CTA CAT OCT GGO 2020 vaJ. tyr ser asn lys cys gin GTG TAC AOC AAT AAG TGT CAG 203 0 ser gly his ile arg asp phe TCT OGA CAC ATT AGA GAT TTT arg val glu COG GTG OAA 2010 met ser thr ATG AOC ACA thr pro leu ACT CCC CTG g',n ile thr CAG ATT ACA 2050 lys leu ala AAO CTO GCC trp, ser thr TG AGC ACC leu leu ala CTG TTG G-CA leu CTT gly OGA ala OCT phe leu TTT CTG met ala ATO OCT 2040 ser gly TCA OGA gin tyr gly gin trp ala pro CAA TAT OGA CG TOG 0CC CCA 2060 tyr ser gly ser ile asn ala TAT TCC OGA TCA ATC AAT 0CC 2070 phe ser trp ile lys val asp TTT TCT TGG ATC AAG GTG OAT arg AGA lys AAG pro CCA leu his CTT CAT glu pro GAG CCC 2080 met ile ATG ATT 2090 ile his gly ile lys thr gin gly ala arg gin ATT CAC GOC ATC AAG ACC CAG OGT 0CC CGT CAG F G. 7(con/ "d) lys phe AAG TTC SUESTITUTE SH1EET WO 88/00831 PCT/US87/0 1814 t4 11 -z7/z7r 2100 ser ser leu tyr ile ser TCC AGC CTC TAC ATC TCT 2110 ieu asp gly lys lys trp CTT GAT GGG AAG AAG TGG 2120 thr gly thr ieu met vaJ. ACT GGA ACC TTA ATG GTC gin CAG gin CAG phe ile ile TTT APTC ATC thr tyr arg ACT TAT CGA met tyr ser ATG TAT AGT gly asn ser GGA AAT TCC 2130 phe TTC phe gly asn TTT GGC AAT 2140 phe asn pro TTT AAC CCT val GTG asp ser GAT TCA ser gly ile lys his asn TCT GGG ATA AAA CAC AAT 2150 ala arg tyr ile arg leu GCT CGA TAC ATC CGT TTG 2160 arg ser thr leu arg met CGC AGC ACT CTT CGC ATC sphI asn ser cys ser met pro AAT AGT TGC ACC ATG CCA i le ATT pro CCA ile ile ATT ATT his CAC pro thr his CCA ACT CAT tyr TAT ser ile ACC ATT glu GAG leu met gly TTG ATG GC 2180 gly met glu CGA ATG GAG 2170 cys asp leu TGT GAT TTA leu TTG ser ACT iys ala AAA GCA 2190 ile ser asp ala gin ile thr ATA TCA CAT CCA CAG ATT ACT ala ser ser GCT TCA TCC tyr TAC phe thr TTT ACC 2200 2210 asn met phe ala thr trp ser pro ser lys ala arg leu AAT ATG TT± CCC ACC TCG TCT CCT TCA AAA CT CGA CTT F I G. 7(conf'd) WO 88jOO83I his ieu gin giy arg ser asn ala trp CAC CTC CAA GGG AGG AGT AAT GCC TGO 2230 asn asn pro lys giu trp, ieu gin vai AAT AAT CCA AAA GAG TGG CTG CAA GTG 2240 thr met lys val thr giy val thr thr ACA ATG AAA GTC ACA GGA GTA ACT ACT PCTIUS87/0 1814 2220 arg pro gin val AGA CCT CAG GTG asp phe gin lys GAC TTC CAG AAG gin gly val lys CAG GGA GTA AAA 2260 giu phe leu ile GAG TTC CTC ATC 2250 ser ieu leu TGT CTG CTT thr ACC ser ser ser TCC AGC AGT gin CAA ser met tyr vai iys AGC ATG TAT GTG AAG 2270 asp gly his gin trp GAT GGC CAT GAG TGO 2280 val lys vai phe gin GTA AAG GTT GAG val val asn ser ieu GTG GTG AAG TGT CTA thr ieu phe phe ACT GTG TTT TTT gin asn gly CAG AAT GOC 2290 ser phe thr TCG TTC ACA lys AAA gly asn gin asp GGA AAT CAA GAG 2300 asp pro pro leu GAC GCA CGG TTA pro GGT ieu thr arg GTG ACT CGC tyr TAG his gin ile GAG GAG ATT ala GCC ecoRl 2310 ieu arg ile his pro gin se~r trp, val GTT GGA ATT CAC CCC GAG AGT TGO GTG 2320 ieu arg met glu vai. ieu gly cys giu CTG AGO ATG GAG GTT GTG GOC TGC GAG 2332 tyr OP TAG TGA F G 7(C0471 d) SUBSTITUTE SHEET 2330 ala gin asp ieu GCA GAG GAG CTG F/ G. 7 cont'd SUBSTITUTE SHEET i INTERNATIONAL SEARCH REPORT International Aoblication N oCr U 07/018 14 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symools apply, indicate all) 3 According to International Patent Classification (IPC) or to both National Classification and IPC IPC(4) A611 35/14,C12P 21/00, C12P 21/02, C12N 15/00 11. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols U.S. 514/2 424/101 530/383 435/68,70,172.3,240,241,253,255,317.1 Documentation Searched other than Minimum Documentation to the Extent that such Documents are included in the Fields Searched 6 Computer 6earcn CAS, BIOSIS, APS, BIONET, under: Sequence, Factor VIII, deletion, mutat!, clon!, variant, mutein, modified DNA, genetic(w) engineering 11l. DOCUMENTS CONSIDERED TO BE RELEVANT 14 Category Citation of Document, id with indication, where appropriate, of the relevant passages i; Relevant t. Claim No. LE Y,P R.L. Burke et al, "The functional domains 1-19 of coagulation factor VII: J. Biological Chemistry, Vol. 261 No. 27, pages 12574-12578, published Sept. 1986 by American Society Biological Chemists Inc. (Washington, USA). See the entire document. Y,P J.J. Toole et al, "A large region 1-19 of human factor VIII is dispen- sable for in vitro procoagulant activity" Proc. Natl. Acad. Sci. Vol. 83, pages 5939-5942 published August 1986 by National Academy of Science, (Washington, D.C. USA). See the entire document. Speclal categories of cited documents: 15 later document published after the international filing date or priority date and not in conflict with the application out document defining the general state of the art which s not cited to understano the principle 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 steo which is cited to establish the publication date of another document of particular relevance: the claimed invention citation or other special reason (as specified) cannot oe considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is comboine with one or more other such docu- other means ments, such comoination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV, CERTIFICATION Daii of the Actual Completion of the International Search I Date of Mailing of this International Search Report 19 OCTOBER 1987 0 3 NOV 1987 international Searching Authority I tur of A horze Officer ISA/US Robin n Form PCT/ISA/210 (second sheet) (October 1981) i ll i--I :i International Aoolication No. PCT/US87/01814 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, i" with indication, wnere aoDroorrate, of the relevant oassaqes Relevant to Claim No i 8 Y,P A WO 86/06101 Published October 23, 1986, Genetics Institute Inc., see the entire document. J. Gitschier et al, "Characterization of the human factor VIII gene", Nature, Vol. 312, pages 326-330, 22 November 1984, MacMillan Journals LTD (London, England) see the entire document. Y W.I. Wood et al, "Expression of active human factor VIII from recombinant DNA clones" Nature Vol. 312, pages 330-336, 22 November 1984, MacMillan Journals LTD (London, England). See the entire docu- ment. Y G.A. Vehar et al, "Structure of human fac- tor VIII", Nature Vol. 312 pages 337-342, 22 November 1984, MacMillan Journals LTD (London, England). See the entire docu- ment Y J.J. Toole et al, "Molecular cloning of a cDNA encoding human anti-haeomophilic factor", Nature, Vol. 312, pages 342-347, 22 November 1984, MacMillan Journals LTD (London, England). See the entire docu- ment. 1-19 1-19 1-19 1-19 1-19 Form PCT ISA 210 (extra sneet) (October 1981) :I- sr--c~ 1 1 ir 'r International Apolication No. PCT/ns R7/ f1 A1 4 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET lnternatlotlal Aoohcatlon NO. PCT/USF R1 4 A D. Eaton et al, "Proteolytic processing of human factor VIII Correlation of specific cleavages with Thrombin, Factor XA, and Activator Protein C With Activation and Inactivation of Factor VIII Coagulant Activity", Biochemicistry, Vol. 25 pages 505-512, published 1986, American Chemical Society (Washington, D.C. USA). See pages 507-512 in particular. 1-19 V.I OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE I This international search report has not been established in resoect of certain claims under Article 17(2) for the following reasons: I. Claim numbers because tney relate to subject matter 1 not required to be searched by this Authority, namely: 2. Claim numoers because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful International search can be carried out i3, specifically: I VIl OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING t1 This international Searching Authority found multiple inventions in this international aoplication as follows: 1.7 As all required additional search fees were timely paid by the applicant, this International search report covers all searchable claims of the international application, 2.1 As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the international application for which fees were paid, soecifically claims: 3.1i No required additional search fees were timely paid by the applicant. Consequently, this international search report Is restricted to the invention first mentioned in the claims: it is covereo by claim numoers: 4. As all searchable claims could be searched without effort justifying an additional fee, the International Searching Authority did not -invite payment of any aoditional lee. Remark on Protest The additional search fees were accompanied by applicant's protest. No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (supplemental sheet (October 1981) r E