JP2525438B2 - Factor-8: C manufacturing method - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a DNA sequence encoding an activation factor VIII: C-like polypeptide and a method for producing the polypeptide using the DNA sequence. More particularly, the present invention relates to the production of activated Factor VIII: C, and the production of activated Factor VIII: C-like polypeptides exhibiting the biological activity of Factor VIII: C. Furthermore, the polypeptides of the invention are produced in higher yields than previously produced Factor VIII: C-like polypeptides and are more easily purified to biochemically pure mature Factor VIII: C.

BACKGROUND OF THE INVENTION Factor VIII: C, a type of high molecular weight plasma glycoprotein, functions as a procoagulant component of Factor VIII, which plays a central role in multistage blood coagulation reactions (generally WJ Williams et al. ., Hematology, pp. 1085
-90, McGraw-Hill, New York (1972)). Factor VIII: C circulates in the blood by forming a complex with Factor VIII R: Ag (also known as von Willebrand factor protein), which is a high molecular weight protein involved in platelet aggregation and adhesion.

Factor VIII: C is synthesized as a single-chain, high molecular weight precursor that is then cleaved to produce the “mature” factor.
This produces the fragments that make up VIII: C. Maturation factor VIII: C is a double strand crosslinked by calcium ions,
That is, the amino terminal H chain consisting of 740 amino acids and 684
It consists of a carboxyl-terminal L chain consisting of 4 amino acids. The primary translation product of Factor VIII: C is single chain, in which the H chain of mature Factor VIII: C is
“Matured polypeptide” consisting of 908 amino acids (mat
uration polypeptide).
Excision of this matured polypeptide results in Arg 1648-Glu 1649
The primary translation product is proteolytically cleaved and initiated by an unknown or as yet unidentified protease that acts on binding. The H chain produced by the first cleavage becomes shorter than its C-terminus due to subsequent proteolytic cleavage.
The result is a mature H chain of 740 amino acids,
It joins the 684 amino acid L chains together and matures (m
ature) Factor VIII: C [L.-O. Anderson et
al. “Isolation and Characterization of Human Fact
or VIII: Molecular Forms In Commercial Factor VIII
Concentrate, Cryoprecipitate, and Plama, "PNAS (US
A), 83, pp. 2979-83 (1986)]. The resulting complex is then activated by cleavage of the Arg 1689-Ser 1690 bond with thrombin. [D. Eaton et al., Biochemistry, 25, pp. 505-1
2 (1986)].

Hemophilia A is a concomitant hemorrhagic disease that develops when either the amount of Factor VIII: C or the biological activity is defective.
Acute hemorrhagic symptoms in hemophiliacs are treated with conventionally purified Factor VIII obtained from normal serum. Various methods have been described in the literature for purification [Zimmerma
net al., USP 4,361,509; Saundrey et al.USP 4,578,21
8; EGDTuddenhem et al., “The Properties of Facto
r VIII Coagulant Activity Prepared By Immunoadsorb
ent Chromatography, Journal of Laboratory Clinical M
edicine, 93, pp.40-53 (1979); DEG Usten, "The Ch
romatographic Separation of Factor VIII on Aminohe
xyl Sepharose, "British Journal of Hematology, 43, p
p.669-74 (1979); M. Weinstein et al., “Analysis of
Factor VIIII Coagulant Antigen In Normal, Thrombin
−treated, and Hemophilic Plasma, “PNAS (USA), 78, p
p.5137-41 (1981); PJFay et al., “Purification A
nd Characterization of Highly Purified Human Facto
r VIII Consisting of Single Type of Polypeptide Ch
ain, "PNAS (USA), 79, pp.7200-04 (1982); CAFulche
r and TSZimmerman, “Characterization of The Huma
n Factor VIII Procoagulant Protein with A Heterolo
gous Precipitating Antibody, “PNAS (USA), 79, pp.16
48-52 (1982); F. Rotblat et al., Thromb. Haemostasi.
s, 50, p.108 (1983); CAFulcher et al., Blood, 61, pp.
807-11 (1983)].

However, the purification was carried out using a relatively low serum concentration of Factor VIII, and a larger Factor VIII R: A
It has proved difficult due to its tight association with g and its sensitivity to serum proteases. Therefore, when Factor VIII: C is purified from plasma, various proteolytic reactions occur, and a uniform mixture of H chains having a chain length of 1648 amino acids to 740 amino acids is mixed in. Anderson et al., Supra, p.298
3). Due to the heterogeneous chain length mixture of peptides found in Factor VIII: C purified from plasma, it was almost impossible to recover substantially pure mature Factor VIII: C. Moreover, the conventional method of treating hemophilia using Factor VIII purified from plasma has fatal drawbacks. Among other things, the treatment includes hepatitis pathogens or AIDS (Acqu
ired Immune Deficiency Syndrome) The annoying situation of virus transfer can occur.

Given the importance of treating hemophilia, many attempts have been made to produce large amounts of Factor VIII: C by genetic engineering [eg Genetics Institute, PCT Appli.
cation W085 / 01961; Genentech European Patent Applic
ation 160,457; Chiron European Patent Application 1
50,735; JJToole et al., “Molecular Cloning of ac
DNA Encoding Human Antihaemophilic Factor "Nature, 3
12, pp.342-47 (1984) and WIWood et al., Nature, 31
2, pp.330-37 (1984)].

However, it turned out that these attempts were not as successful as desired. One of the causes is that the recombinantly obtained 2332 amino acid Factor VIII: C is susceptible to proteolysis at many positions on the chain. Another difficulty is that it is difficult to produce Factor VIII: C in a sufficiently high yield by a recombinant method.

SUMMARY OF THE INVENTION The present invention seeks to solve the problems set forth above by providing a DNA sequence encoding an activated Factor VIII: C and an activated Factor VIII: C-like polypeptide. Factor VI, which has been recombinantly produced up until now, has been constructed with DNA sequences encoding these polypeptides.
II: C, and 20 times the amount of Factor VIII: C are produced,
More easily purified to biologically pure maturation Factor VIII: C.

Encoding activation factor VIII: C according to the present invention
DNA sequences are produced and expressed in high yield. As is apparent from the specification and the examples that follow, the activating factor VIII: C and activating factor VIII: C-like polypeptides of the present invention lack the major portion of the matured polypeptide of factor VIII: C. The characteristic is that they are lost.
In our preferred embodiment, the DNA sequence lacks substantially all of the nucleotides encoding the matured polypeptide. In our most preferred embodiment, the DNA sequence encoding the matured polypeptide is completely deleted. When our DNA sequence is expressed, the maturation factor V
The III: C H chain is directly linked to the L chain. When one proteolytic cleavage occurs, mature Factor VIII: C is formed.

Finally, according to the present invention, the activation factor VIII: C and the activation factor VIII: C produced by the DNA sequence of the present invention.
Various anti-hemophilia compositions containing such polypeptides are provided. It also provides various uses of these compositions for the treatment of bloody disease A, which causes acute or chronic bleeding.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing of a restriction enzyme digestion map of Factor VIII: C cDNA.

FIG. 2 is a schematic representation of the construction of a recombinant DNA molecule with a QD deletion.

3A and 3B are schematic representations of the construction of recombinant DNA molecules with RE deletions.

Figure 4 SV40 replication / enhancer origin, adenovirus major late promoter, RE deletion factor VIII: C c
Mammalian cell expression vector showing the positions of DNA, 3'untranslated region of factor VIII: C mRNA and polyadenylation site
It is a drawing of a restriction enzyme cleavage map of the RE deletion inserted in pBG312.

FIG. 5 depicts the S1 analysis of Factor VIII: C mRNA isolated from transfected BMT10 cells.

FIG. 6 shows the results of Southern analysis of plasmid DNA isolated from transfected BMT10 cells.

Figure 7 shows the published sequences of Factor VIII: C DNA and amino acids (EPO 160,457).

DETAILED DESCRIPTION OF THE INVENTION In order that the present invention may be more fully understood, it is described in detail below.

 The following terms will be used in the description.

Nucleotide: A monomer unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate ester and a nitrogen-containing heterocyclic base.
The base is attached to the sugar moiety via the glycosidic carbon (the 1'carbon of the pentose) and this combination of base and sugar is called a nucleoside. Bases characterize nucleotides. There are four DNA bases, adenine (A), guanine (G),
Cytosine (C) and thymine (T). The four RNA bases are A, G, C and uracil (U).

DNA sequence: A single chain of nucleotides linked by a phosphodiester bond between the 3'and 5'carbons of phosphopentose.

Codon: A DNA sequence consisting of three nucleotides encoding an amino acid, a translation start signal or a translation stop signal through mRNA. For example, the nucleotide triplets of TTA, TTG, CTT, CTC, CTA and CTG encode leucine (Leu), TAG, TAA and TGA are translation stop signals, and ATG is a translation initiation signal.

Amino acid: A monomeric unit of peptide, polypeptide or protein. 20 amino acids, namely phenylalanine (Phe or F), leucine (Lue or L), isoleucine (Ile,
I), methionine (Met, M), valine (Val, V), serine (Ser, S), proline (Pro, P), threonine (Thr,
T), alanine (Ala, A), tyrosine (Tyr, Y), histidine (His, H), glutamine (Gln, Q), asparagine (Asn, N), lysine (Lys, K), aspartic acid (Asp,
D), glutamic acid (Glu, E), cystine (Cys, C),
There are tryptophan (Trp, W), arginine (Arg, R) and glycine (Gly, G).

Reading Frame: The grouping of mRNAs for translation into amino acid sequences.

An accurate reading frame must be maintained during translation. For example, GCTGGTTGTAAG can be expressed in three open reading frames or phases, each giving a different amino acid sequence.

GCT CCT TGT AAG-Ala-Gly-Cys-Lys G CTG GTT GTA AG-Leu-Val-Ual-GC TGG TTG TAA G-Trp-Leu- (STOP) Polypeptide: α-amino acid of adjacent amino acids A single chain of amino acids linked by a peptide bond between a group and a carboxyl group.

Genome: The total DNA of a cell or virus. Among them,
Included are structural genes that encode the polypeptide of the substance, as well as operator genes, promoter genes, ribosome binding and interaction sequences, including various sequences such as the Shine-Dalgarno sequence.

Gene: DN that encodes an amino acid sequence unique to a specific polypeptide through a template or messenger RNA (mRNA)
A array.

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

Translation: The process of producing a polypeptide from mRNA.

Expression: The process of producing a polypeptide from a gene or DNA sequence. Subleasing and translation are paired.

Plasmid: A non-chromosomal double-stranded DNA sequence consisting of complete replicon (plural units), which is replicated in host cells. Placing a plasmid in a unicellular organism can alter or transform the properties of the organism due to the DNA of the plasmid. For example, when a cell previously carrying tetracycline sensitivity is transformed with a plasmid carrying a gene responsible for tetracycline resistance (TET ^R ), the cell becomes resistant. Cells transformed with the plasmid are called transformants.

Phage or bacteriophage: Many bacterial viruses consist of a DNA sequence contained within a protein bag or shell (CAPSID).

Cloning medium: a plasmid capable of replicating in a host cell,
Phage DNA, cosmid or other DNA is a method in which the DNA sequence can be confirmed in a certain manner, and
A label suitable for use in identifying transformed cells which can be cleaved without loss of essential biological functions of, for example, duplication, production of coat proteins, or loss of promoter genes or binding sites, and the like, For example, it is characterized by having one to several endonuclease recognition sites including resistance to tetracycline or ampicillin. Cloning vehicles are often also referred to as vectors.

Cloning: by asexual reproduction, one organism or DNA
The process of obtaining multiple identical organisms or DNA sequences from a sequence.

Recombinant DN molecule or Hybrid DNA: A molecule consisting of a DNA segment obtained by ligating different genomes outside a living cell, with the ends linked together, and capable of maintaining them in an adult cell.

Expression control sequence: When the expression control sequence binds to the above gene,
Nucleotide sequences that control and regulate gene expression. These include lac system, β-lactamase system, trp system, tac system, trc system, main operator gene and promoter gene region of phage λ, fd coat protein regulatory region, SV40 early and late promoter genes, polyoma virus. And adenovirus-derived promoter gene, metallothionein promoter gene, 3-phosphoglycerin ester kinase or other glycolytic enzyme promoter gene, acid phosphatase such as Pho5 promoter gene, yeast α-hyperfactor promoter gene and other prokaryotes Alternatively, there are known sequences that control the expression of eukaryotic cells and their viruses or their combined genes.

Factor VIII: C: A polypeptide having the amino acid sequence of FIG. 7, which, when matured and activated, can function as a cofactor for Factor lXa-dependent Factor X maturation in a hemagglutination reaction by a multi-step mechanism. The factor VIII: C used in the present invention is a factor VIII aggregation precursor activating protein, factor VIII coagulation active protein, anti-hemopathic globulin (AHG), anti-hemophilia factor (AHF) and anti-hemophilia. It contains a glycoprotein, also known as factor A [W.
J. Williams et al., Hematology, pp. 1056, 1074 and 108.
1].

Matured polypeptide: The matured polypeptide of Factor VIII: C consists of 908 amino acids from the serine at position 741 to the arginine at position 1648 (see FIG. 7).

The maturation of Factor VIII: C begins with a cleavage between amino acids 1648 and 1649, which results in a C-less L chain. A series of cleavages then occur to produce the mature N-terminal heavy chain.

Maturation Factor VIII: C: The maturation Factor VIII: C used in this application is a C-terminal L chain (Glu 1649-Tyr 2332) and an N-terminal H chain linked via an alkali metal, eg calcium bridge ( Ala 1-Arg 740).

Activating Factor VIII: C: As used in this application, “Activating Factor VIII: C” is a deletion of a major portion of the mature polypeptide of Factor VIII: C. For example, activation factor VIII: C lacking the entire mature polypeptide
Contains a protein in which the N-terminal mature H chain and the C-terminal mature L chain of Factor VIII: C are linked together in a single chain.

Activating Factor VIII: C-Like Polypeptide: As used in this application, "activating Factor VIII: C-like polypeptide" comprises a protein having the biological activity of Activating Factor VIII: C. The polypeptide is characterized in that it retains substantially the biological activity of activating factor VIII: C even if a protein containing amino-terminal methionine, such as f-Met factor VIII: C and other amino acids is deleted, added or substituted. Containing a protein having

“Activation factor VIII: C-like polypeptides” within the scope of the above definition also include naturally occurring allelic variants that may be present or occur in each polypeptide. Furthermore, it is possible to include an activation factor VIII: C-like polypeptide in which the degree of glycosylation and the position of the sugar chain or other post-translational modification differs depending on the production host or the cellular environment of the tissue.

The present invention relates to a method for producing activating factor VIII: C and activating factor VIII-like polypeptide. More particularly, the invention provides DNA sequences capable of producing activation factor VIII: C and activation factor VIII: C-like polypeptides in high yield in a suitable host. The polypeptides produced by the DNA sequences of this invention are useful in treating hemophilia A.

Compared to Factor VIII: C, activated Factor VIII: C produced by the DNA sequence of the present invention is Factor VIII: C.
The major part of the matured polypeptide is deleted. The DNA sequence of the present invention surprisingly exhibits activation factor VIII: C.
Is expressed in much higher yield than the DNA sequence encoding Factor VIII: C.

While not wishing to be bound by theory, we find that the DNA sequences of the present invention are high in yield of activation Factor VIII due to the absence of most or all of the mature polypeptide. I believe in producing: C. For example, the mRNA of the activating gene is translated more efficiently because the long RNA that encodes the matured polypeptide need not be translated. Furthermore, when Factor VIII: C is in the cell,
Although it undergoes protein cleavage attack at many sites, activation factor VIII: C is less susceptible to such attack because there is no protein cleavage site in the matured polypeptide. Furthermore, in the absence of the matured polypeptide, 19 of 25 N-linked glycosylation sites of native Factor VIII: C were missing, leaving only 6 N-glycans on the activated polypeptide.
Only the glycosylation site remains (3 on the H chain and L
(3 places on the chain). Apparently there are few glycosylation sites, which simplifies the production and purification of activation factor VIII: C.

In the method of the present invention, the generator modified the DNA sequence encoding Factor VIII: C to remove a major portion of the DNA sequence encoding the matured polypeptide. Having prepared a DNA sequence with the desired deletion site, we used it in various expression vectors and hosts to produce the activation factor VIII: C encoded by the sequence. For example, a great variety of expression vectors are used to express the activation factor VIII: C coding sequence of the present invention. It will also be appreciated that DN sequences encoding activation factor VIII: C-like polypeptides are similarly produced by the present 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 SV40, known bacterial plasmids such as colE1,
Plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, a broader range of host or plasmids such as RP4, phage DNA such as phage λ, numerous derivatives of NM989 and other DNA phages such as M13. ,
And filamentous single-stranded DNA phage, yeast plasmids such as the 2μ plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNA, such as plasmids modified using phage DNA or other expression control sequences. In a preferred embodiment of the invention, the pBR327-related vector pBG312 is used [R. Cate et al., Cell, 45, pp, 685-98 (1986)].

A wider range of expression control sequences can be used in these vectors to express the DNA sequences of the present invention, ie, sequences that control the expression of engineered linked DNA sequences. Examples of useful expression control sequences include SV40 early and late promoter genes, lac system, trp.
System, TAC or TRC system, main operator and promoter gene of phage λ, control region of fd coat protein, promoter gene of 3-phosphoglycerin ester kinase, or other glycolytic enzyme, acid phosphatase, for example, promoter gene of Pho 5 , The yeast α-mating factor promoter gene and other prokaryotic or eukaryotic cells, and their known sequences controlling the expression of viruses or their combined genes. In a preferred embodiment of the invention, adenovirus-2 major late promoter gene expression control sequences are used.

A wide variety of host cells may also be activated factor VII of the present invention.
Useful for I: C production. These host cells include well-known nucleated and prokaryotic cell hosts such as E. coli., Pseudo.
Included are monas, Bacillus, Streptomyces bacteria, fungi such as yeast, and animal cells such as CHO cells, African green monkey cells such as COS1, COS7, BSC1, BSC40 and BMT-10, and human and plant cells in tissue culture. In a preferred embodiment of the invention, BMT10 African Green Monkey cells are used.

Of course, it is understood that not all vectors and expression control sequences function equally well to express the activating DNA sequence of the invention and produce the activating factor VIII: C of the invention. Should be. Also, not all hosts function equally well in the same expression system. However, a person skilled in the art can obtain unreasonable experimental results or depart from the scope of the present invention by appropriately selecting the above-mentioned vector, expression control sequence and host. For example, in choosing a vector, the host must be considered because the vector is replicated in the host. The vector's copy number, copy number control ability, and expression of other proteins encoded by the vector, such as antibiotic resistance markers, should also be considered.

In selecting an expression control sequence, various factors must be considered. Among these are, for example, the relative strength of the system, its controllability and its compatibility with the DNA sequence encoding the activation factor VIII: C of the invention, especially possible secondary structure. In selecting a host, compatibility with the selected vector, toxicity of our activating factor VIII: C to the host, secretory properties of the host, ability to fold proteins correctly, fermentation conditions, activating factor VIII: C of the present invention Consideration must be given to the easiness and safety of purifying the bacterium from the host.

Within these parameters, those skilled in the art have to choose various vector / expression control system / host combinations which will produce useful amounts of the activation factor VIII: C of the invention in fermentation. I won't. For example, in a preferred embodiment of the present invention, the adenovirus 2 major late promoter expression system pBG312 vector is used in BMT10 African Green Monkey cells.

Activating Factor VIII: C and Activating Factor VIII: C-like polypeptides produced by the present invention can be purified by a variety of conventional methods and procedures. Useful purification methods include those used to purify native and recombinant Factor VIII: C (eg L.-O. Andersso
n et al., PNAS (USA), 83, pp. 2979-83 (1986)).

After purification, the activating factor VIII: C and activating factor VIII: C-like polypeptides of the present invention are useful as compositions and methods for the treatment of hemophilia A, and for uncontrolled bleeding. On the other hand, it is useful as various therapeutic agents.

The activation factor VIII: C and the activation factor VIII: C-like polypeptide of the present invention can be a composition in the form of a produced single chain polypeptide, or can be administered by various methods. Within the range of activation factors
It will be understood that it is possible to administer VIII: C after proteolysis. Activation factor
VIII: C is cleaved in vitro to mature H of maturation factor VIII: C.
It can be broken into chains and L chains and linked with calcium or other alkali metal crosslinks before, during or after purification.

The activation factor VIII: C and the activation factor VIII: C-like polypeptide of the present invention can be formulated by a known method to produce a pharmaceutically useful composition. The composition may also preferably include known pharmaceutically acceptable carriers, or may also include other agents, carriers, adjuvants, excipients and other such human serum albumin or plasma formulations. For example
See Remington's Pharmaceutical Sciences (EWMartin). The resulting formulation has a sufficient amount of activation factor VII to exert sufficient effect to treat uncontrolled bleeding in the recipient.
Contains I: C. Administration of these polypeptides or pharmaceutically acceptable derivatives thereof may be carried out by any of the methods conventionally accepted as Factor VIII administration methods. They include parenteral, subcutaneous, and intravascular administration.

The composition of the present invention used in the treatment of hemophilia can have various dosage forms. The preferred form depends on the intended mode of administration and treatment. The dosage regimen and dosage will vary depending on various factors, such as whether to treat acute bleeding patients or to use them prophylactically. However, Factor VIII: C levels must be high enough to prevent bleeding and promote epithelialization (Williams, Hematolog
y, pp. 1335-43).

In order to better understand the present invention, the present invention will be described below with reference to Examples. This example is for illustrative purposes only, and the scope of the present invention is not limited thereby.

EXAMPLES The present inventors have found that a cD encoding an activation factor VIII: C molecule lacking major or all of the mature polypeptide.
Created an NA array. To test the limitations of the present invention, the present invention also generated a cDNA sequence encoding a polypeptide that is more absent than the mature Factor VIII: C polypeptide.

A. Construction of complete Factor VIII: C cDNA In Figure 1, Factor VIII: C based on published sequence.
A restriction enzyme map of cDNA is shown [WIWood et al., Nature,
312, pp.330-37 (1984); Fig. 7].

Double lines represent coding sequences. Below the restriction map, the maturation factor by calcium cross-linking
VIII: C depicts the maturation factor VIII: C amino terminal heavy chain attached to the carboxyl terminal light chain. The oligonucleotide probe (indicated by *) used to screen the human placenta, liver and kidney cDNA libraries is shown below a concatenated protein model that matches the restriction map. These libraries have ol as the first chain primer
was made using igo (dT) and λgt10 as the vector.

On this secondary linkage is the oligonucleotide primer (left arrow) that the inventor used to initiate first-strand cDNA synthesis using human kidney mRNA as a template. The present inventors have described these single-stranded cDNA linkages as Gubler and Hoff.
Double-stranded by man's technology [U.Gubler, and BJHo
ffman, Gene, 25, pp. 263-69 (1983)]. PBR these DNA
In 322, it was cloned at the EcoRV site with dC at the end. The kidney cDNA plasmid library thus obtained was screened with an oligonucleotide probe located 5'of the linked oligonucleotide primer.

Under the primer / probe linkage in Figure 1, the partial factor VIII: C isolated by the inventors from these libraries is shown.
The cDNA and genomic subclones are shown. Together, they encode a full-length cDNA gene.

Further details of these clones are shown in Table 1.

Table 1 Summary of Factor VIII: C Genomic and cDNA clones Isolated from genomic library composed of cosmid pTCF [Grosveld, FGet al., Nucleic Acids Research, 10, p.
p.6715-6732 (1982)] Subclone Length Tissue pUC 19.2874 2874 bp 48, XXXX Human lymphoblasts Before assembling the complete cDNA gene, the inventor constructed two intermediate plasmids. This is Factor VII
This is necessary because the I: C cDNA is too long. In the first pre-assembly, the inventor found that from the 5163th PstI site,
A fragment from clone 7.7475 that spans the 5755th PstI site was isolated. This fragment was inserted into clone 4.7573 at the 5755 PstI site, thereby extending clone 4.7573. This PstI site is a clone
7.7475 and clone 4.7573 are shared by both inserts. By extending clone 4.7573 by this method, the inventor provided a unique NdeI site in the insert of this clone 4.7573 derivative. The inventor had to create this Nde site because it required a unique site to extend the linkage of this insert below 5 '.

As a second pre-assembly, the inventors have introduced a polynucleotide linker into clone 2.82 at the 5'position immediately adjacent to the translation initiation codon of the Factor VIII: C signal sequence. Insertion of clone 2.82 occurs at the EcoRV site of pBR322, which is in the opposite direction of tetracycline resistance. The insert 5'end in clone 2.82 is at position -133 in the 5'untranslated leader sequence. The inventor cleaved clone 2.82 at the SalI site in the tetracycline resistance gene, SacI in the sequence encoding the signal peptide in the insert of clone 2.82 and introduced the synthetic duplex.

As a result of this ligation, the SalI-NruI-NcoI polylinker
The Factor VIII: C signal sequence is introduced immediately 5'to the translation initiation codon. These three restriction enzymes do not cleave the full length Factor VIII: C cDNA gene.

A complete Factor VIII: C was assembled in a 6-fragment ligation reaction using these two intermediate constructs (Fig. 1, minimum bottom). The present inventors have confirmed that complete DNA in a single-strand clone state
Since it has never been isolated, this method is used to
Had to build. The inventor has isolated fragment 1 from the above-described derivative of clone 2.82. Fragment 1 runs from SalI in the polylinker to AvaI at position 731. Fragment 2 is a λgt10 recombinant gene
Isolated from insert in 1.7977. Fragment 2 is 731
It runs from AvaI in 2nd place to EcoRI in 2289th place. Fragment 3 was isolated from the genomic cosmid recombinant subclone pUC 19.2874. The fragment runs from EcoRI at position 2289 to BamHI at position 4743. Fragment 4 was isolated from clone 7.74575, starting from the BamHI site at position 4743 and continuing to NdeI at position 5522. Fragment 5 was isolated from clone 4,7573 described above.

Fragment 5 extends from NdeI at 5522 to NcoI at 7991. Fragment 6 is an assembly vector containing the E. small li. Origin of replication and the ampicillin resistance gene as a selectable marker. Fragment 6 was isolated from pAT.SV2.tPA. pAT.SV2.tPA was 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. The inventor is pAT.SV2.tP
S that cleaves A within the tetracycline resistance marker gene
Enzymatic digestion was performed using alI and NcoI, which cleaves within the SV40 early region.

Analysis of 96 recombinants revealed that 32 contained all 5 restriction fragments of Factor VIII: C. The present inventor has determined the DNA sequence of one of these clones and confirmed that there are two changes from the published sequence.
One is Leu242, where CTG is CTA, and the other is at amino acid residue 1880, where TTC is CTC (Phe is Leu) (see Figure 7).

B. Insertion of full-length cDNA into mammalian cell expression vector The present inventor excised the complete Factor VIII: C cDNA gene from the recombinant vector using NcoI. The obtained NcoI
The restriction fragment was treated with nuclease S1 to form blunt ends. This fragment was SmaI-digested pBG312
Combined with. pBG312 is an animal cell expression vector whose structure has been described by other researchers [R. Cate e.
al., Cell, 45, pp.685-98 (1986)]. EcoRI to BamHI
Up to pBG312 sequence (clockwise) is SV40 origin of replication, adenovirus-2 major late promoter and complete triparticle leader sequence [S.Zain et al., Cell, 16, pp.851.
-61 (1979)]; hybrid splice signal [RJKa consisting of adenovirus-5 splicing donor and immunoglobulin variable region gene splicing acceptor.
ufman, and PASharp, J. Mol. Biol., 159, pp.601-21 (19
82)]; HindIII, XhoI EcoRI, SmaI, NdeI, SstI and BglII
A polylinker containing a site for use; an SV40 t antigen intron flanked by splicing donors and acceptors,
And SV40 having the polyadenylation site.

The present inventor has demonstrated two independent clones encoding factor VIII: C containing the signal sequence in the polylinker part of pBG312: 8.1 and 8.2 cDNA. Clone 8.1 differs from clone 8.2 in the 3'untranslated region,
6 is fused to the SmaI site of pBG312, whereas in clone 8.2 the C of the NcoI site at position 7990 is fused. Furthermore, the present inventors have found that within the sequence encoding the signal peptide of Factor VIII: C, the cD encoding Factor VIII: C is
Another clone in which the NA gene was fused to pBG312 was also isolated.
This clone, which the present inventor calls Signal Minus, is a transient expression test (transient expression test) described below.
assay) was used as a negative control.

C. GLN744-ASP1563 (abbreviated as QD) Construction of Absence
741-1648) QD excluding the portion of the DNA sequence encoding expression
Here's how to make the lack. QD deficiency leaves approximately 90 amino acids of the mature polypeptide (4 amino acids at the N-terminus and 84 amino acids at the carboxyl terminus of the mature polypeptide).

Figure 2 shows the configuration without QD. The inventor has used a certain amount of an expression plasmid of the complete Factor VIII: C gene for EcoRI.
It was partially disassembled. This endonuclease is Gln744
Cut between the codon and the Asn745 codon. The inventor is 5'AA
The TT overhang was removed with nuclease S1, and the resulting plasmid was completely digested with PvuI within the ampicillin resistance gene. The inventor also took a fixed amount and partially decomposed it with BamHI which cuts between the Leu1562 codon and the Asp1563 codon (see FIG. 7). The 5'GATC overhang was filled in with the Klenow fragment and the resulting plasmid was re-filled with PvuI.
disassembled in amp. Two mixtures of fragments were combined and ligated with T4 DNA ligase. Gln744 codon A
The BamHI site between the sp1563 codons was created in this fusion.

The activating polypeptide produced upon expression as a result of QD deficiency is the four C-terminal amino acids attached to the carboxy-terminal Arg740 of the mature H chain and the amino terminal Glu1 of the L chain.
908, leaving the 86 N-terminal amino acids attached to 649
Of the mature polypeptide of 1 amino acid, 818 amino acids are missing (FIG. 7). Thus, a matured polypeptide of 908 amino acids is produced by 90% of both protease substrates used in the initial maturation of the primary translated polypeptide and subsequent maturation of the unaltered heavy chain. Is replaced by a matured polypeptide consisting of

D. Construction of a lack of ARG740-GLU1649 (abbreviated as RE) This chapter describes how to create a RE deficiency except for the entire DNA sequence encoding the mature polypeptide.

Figures 3A and B show how this RE-deficient fusion was obtained in two steps. In the first step, the four fragments are ligated to obtain one intermediate plasmid. The four fragments are (1) 462 bp fragment: The full-length gene expression plasmid was digested with HindIII between Arg740 and Ser741 codons, 5'AGCT was removed with nuclease S1, and specifically Tyr586 and Kp that cuts between the Leu587 codons
(2) Synthetic oligonucleotide duplex fragment obtained by digestion with nI (3) First, the expression plasmid for the full-length gene is Sau3A.
Digestion with to give a 135 bp fragment. The inventor is S
between er1657 and Asp1658 codons and Glu1794 and Asp1795
A 411 bp fragment was isolated by digesting with Sau3A between the codons. The 411 bp fragment was then digested with Ala1702 and Vall70.
Digestion with PstI, which cuts between 3 codons, yielded a 135 bp 5'fragment.

(4) pUC18 digested with KpnI and PstI.

The inventor obtained a fragment encoding the RE fusion from this intermediate plasmid. For this purpose, an intermediate plasmid generated by ligating four fragments was used as Asp718 and PstI.
I cut it. With the fragment encoding the RE fusion
The corresponding fragment in the expression plasmid for QD fusion was replaced. The resulting 624 bp fragment encoding the RE fusion was first completely cleaved from the QD internal deletion expression plasmid at the unique Asp718 site, followed by phosphorylation of the 5'GTAC overhang with calf intestinal phosphatase, and The resulting plasmid was ligated with a mixture partially digested with PstI.

A map of the RE-less insert introduced into pBG312 is shown in Figure 4.
90 for activated polypeptides produced by gene expression
The mature polypeptide of 8 amino acids has been completely removed. Good polypeptides produced by cells containing such recombinant species are secreted and
It may be purified as a single chain, such that the chain is directly attached to the L chain.

In this absence, the peptide bond between Arg at position 1648 and Glu at position 1649, which is normally cleaved during the first cleavage of the full-length primary translation product, is conserved, so that the primary translation product of this internal deletion is It is cleaved by the same proteolytic enzyme that initiates cleavage of the full-length primary translation product. Thus, the mature molecule of the Factor VIII: C H chain is directly produced.

We confirmed by Western analysis (data not shown) that RE activation factor VIII: C encodes a single-chain molecule that is processed into 90K H and 80K L chains in culture.

The resulting L chain contains a peptide consisting of Glu at position 1649 to Arg at position 1689, which allows the double-stranded complex to engage the von Willebrand protein.

As such, this recombinant product will bind to von Willebrand proteins present in cell culture when secreted from animal cells.

Similarly, when injected, it will complex with von Willebrand protein in serum and circulate in the body. 1689
Cleavage between Arg at position 1 and Ser at position 1690 by thrombin activates double-chain maturation factor VIII: C, dissociates it from von Willebrand protein, and causes factor IX on the platelet surface.
It will form a ternary complex with a and Factor Xa.

Construction of E.ARG740-SER1690 (abbreviated as RS) Lack To test the external limits of these deficiencies, the inventor
Encodes the matured polypeptide and other missing polypeptides (ie, the inventor has identified L of maturation factor VIII: C).
Encodes the expression of 41 amino acids at the N-terminus of the chain
A plasmid was constructed (lacking the DNA sequence).

The present inventor created this RS fusion by the two-step method described in RE fusion. In the first step, three fragments were ligated to construct an intermediate plasmid. The three fragments used were: (1) 462 bp fragment: a full-length gene expression plasmid was cleaved with HindIII between Arg 740 and Ser 741;
5'AGCT was removed with nuclease S1, followed by specific Tyr
It is obtained by cutting with KpnI which cuts between the codons of 586 and Leu 587.

(2) Synthetic double-stranded oligonucleotide fragment: (3) pUC18 cut with KpnI and PstI. This fusion recreates the HindIII site between the Arg 740 and Ser 741 (now Ser 1690) codons.

A fragment encoding the RS fusion was isolated from this intermediate plasmid and used in the second step to generate QD
The corresponding fragment in the fusion expression plasmid was replaced. In this second step, we code for an RS fusion 50
A 1 bp fragment was isolated. Intermediate plasmid Asp718
Cleavage with PstI and the fragment encoding the RS fusion was isolated. The related fragment in the expression plasmid for QD fusion was then 501b using the method used for RE fusion.
It was replaced with p fragment.

Glu 1649-Ar was further removed by removing all matured polypeptides.
DNA encoding the g 1689 peptide, von Willebrand
The binding putative domain deletion was performed without RS. Therefore, this recombinant molecule circulates when it is secreted from animal cells into the culture medium or injected into the receptor.
Does not bind to llebrand protein.

F. Transfection of African Green Monkey Kidney Cells The present inventor has developed BMT10 cells [RD Gerard and Y. Gluzman, Mo.
l.Cell.Biol., 5, pp.3231-40 (1985)] was transfected with the supercoiled expression plasmid. The DEAE-dextran method [LMsompayvac and KJDanna, PNAS, 78, pp.7
575-78 (1971)] and chloroquine [H. Luthman and GM
anusson, Nuleic Acids Research, 11, pp.1295-1308 (19
83)] was used to transfect the cells. Transfectants
Since the SV40 T antigen is inducibly supplied in trans by BMT10 cells and binds to the replication origin of SV40 linked to the activation factor VIII: C gene in the expression plasmid, the introduced expression plasmid can be collected in high replication. It is known to replicate at a rate. However, this method is inefficient, as only a few% of the transfected cells actually associate with the DNA.

The resulting transfectant contained 120 activation factors VIII: C.
Secreted within the time. In most experiments, cm ² / ml ratio of about
5.5, ie a fusion monolayer of BMT10 transfectants in a Petri dish (55 cm ² ) with a diameter of 100 mm is covered with 10 ml of culture medium.

G. Factor VIII: C activity measurement The present inventors double-transfected into BMT10 cells and then
Luminus, 8.1, QD, RE and RS expression construct factor VII
I: C production activity was analyzed. Coates made by Kabi Vitrum
t , Factor VIII: C kit with 96 wells
Used with For S1 analysis using one Petri dish
H RNA to be used for Southern analysis from the other one
irtDNA was prepared. After culturing for 120 hours,
Actor VIII: C activity was analyzed. The result is the factor
-VIII: C plasma concentration, based on about 200ng / ml
Expressed as a percentage of.

Upon repeated transfections, Factor VIII: C activity of both the signal-minus construct (negative control) and RS-deficient was undetectable.

This may be explained by the lack of the von Willebrand protein binding domain in RS-deficient bodies.

As shown below in a 120-hour experiment, cells transfected with the full chain length gene showed about 5% activity lacking QD and lacking RE. The activity observed in the absence of QD was 1.46% plasma level and in the absence of RE 1.30% plasma level.

BMT10 thus transfected in the absence of QD and RE
The cells produce 20-fold more Factor VIII: C than cells transfected with the full chain gene.

H. Factor VIII: C mRNA Nuclease S1 Analysis Nuclease S1 analysis was performed to analyze mRNA levels in each construct. This analysis is based on our QD and RE
Helps determine why expression levels were improved in the absence.

After transfection of BMT10 cells for 120 hours in 100 mm diameter Petri dishes, RNA was then published from W. Schleun, which has not yet been published.
It was isolated using the method of ing and J. Bertonis. This method is briefly described. 500 mM Tris-HCl (pH 7.5) -5 mM EDTA-1% SD containing 100 μg / ml proteinase K.
BMT10 cells were lysed for 20 minutes at 37 ° C. with 3 ml of S solution. The lysate was transferred into a 50 ml conical tube containing 3 ml phenol and centrifuged at high speed for 15 seconds in a Polytron (Brinkmann). Extract the aqueous phase with ether, and
Adjusted to 0.25 NaCl. The same volume of isopropanol was added to precipitate the nucleic acid portion at 4 ° C., the precipitate was recovered by centrifugation, and the precipitate was redissolved in 3 ml of water. The solution was adjusted to 2.8 M LiCl and kept at 4 ° C. overnight to selectively precipitate RNA.

The amount of activation factor VIII: C mRNA for each construct was quantified. The QD expression plasmid was cut with EspI and the probe for S1 analysis was isolated. The 5'end of the EspI fragment was labeled with [γ- ^32p ] ATP and T4 polynucleotide kinase. 10 μg of RNA was isolated on a 5% strand separation gel of 477 nucleosides EspI fragment 5000 ^{cpm 32P-}
Annealed to antisense strand [AMMaxam an
d W. Gilbert, Methods In Enzymology, 65, pp. 499-560
(1980)]. The resulting RNA was treated at 48 ° C. with 10 μl of 80% deionized formamide-400 mM NaCl-40 mM PIPES.
(PH6.4) -Incubate in 1 mM EDTA overnight. The resulting hybrid molecule contained 0.2 nuclease S1 at a concentration of 100 nuits / ml.
8M NaCl-50mMNaOAc (pH4.6) -4.5mM ZnSO 4 solution 170μ
Add l and cut. Stop the digestion by adding EDTA to 10 mM and extract with phenol. The protected fragment is denatured and it is electrophoresed on a 5% strand separation gel. The dried gel was exposed to Kodak XAR-5 X-ray film backed with a Lighting Plus brightness enhancement screen (Du Pont) at -70 ° C overnight. 477 nucleotides Esp
The I fragment has one end in a hybrid intron excised from the 5'untranslated region of Factor VIII: C mRNA [RJ Kaufman and PASharp, J. Mol. Biol., 159, p.
p.601-21 (1981)], and the other end is Ala 62
It is within the codon (Fig. 4).

Activation factor VIII: C mRNA is a single-stranded 300 nucleotides
Detected by protecting the DNA fragment from cleavage. Experiments were performed several times using 1 μg of RNA to demonstrate excess single-stranded probe.

The results of the nucleotide S1 analysis of activation factor VIII: C mRNA for each construct are shown in FIG. Our results show that the activation Factor VIII: C mRNA levels are the same for the three absent and complete Factor VIII: C genes. FIG. 5A shows the analysis results using 10 μg of RNA,
And FIG. 5B shows the analysis results using 1 μg of RNA. Lane 1 in the figure contains 500 cpm as a marker of the labeled 477 nucleotide single-stranded DNA fragment used to protect the activation Factor VIII: C mRNA from S1 cleavage. The amount used for each hybridization
Equivalent to 10%. Lane 2 contains RNA isolated from BMT10 cells transfected with the signal minus construct.
Lane 3 is full length Factor VIII: C cDNA (construct 8.
BMT10 cells transfected in 1); Lane 4 is BMT10 cells transfected with activation factor VIII: C cDNA (lacking QD);
Lane 5 is BMT10 cells transfected with activation factor VIII: C cDNA (RE deficient); lane 6 is cDN from RS deficient.
BMT10 cells transfected with A; Lane 7 contains pinf322 with HinfI
Cleavage with 3'end and [α-32P] dATP and Klenow
Labeled with enzyme (gift from Richard Tizard)
The labeled fragment obtained by. A protective fragment of the expected base length with an equal length of 300 is clearly seen in both figures showing the 8.1, QD, RE, RS constructs. A protected fragment about 220 bases long is clearly visible in both figures in the signal minus construct, lacking the portion of the DNA sequence encoding the signal peptide.

Comparing FIGS. 5A and 5B, it can be seen that the 477 probe used was in excess mole for each construct during hybridization. Although the levels of activation factor VIII: C are at least 20-fold higher in QD and RE deficients compared to RS-deficient and full-constituents, the amount of mRNA in these four constructs is almost identical . Therefore, QD and RE
The reason for the increased expression in the lacking body is due to its post-translational nature.

I. Southern analysis of plasmid DNA from transfected BMT10 cells Quantitative analysis of the DNA levels of newly replicated deletion-engineered activation factor VIII: C plasmids and comparison with that of the full length gene . In addition, this test shows that the activation factor VIII: C
It also helps clarify the reason for the high yield of.

Non-chromosomal DNA isolated from each transfectant in BMT10 cells to control the differences in DNA replication between the respective constructs.
Southern analysis was performed. Hirt method [B.Hirt, J.Mol, B
iol., 26, pp.365-69 (1967)].
DNA was isolated from BMT10 cells cultured in 0 mm diameter Petri dishes for 120 hours. Methylated bacterial DNA input for each construct
Newly replicated (DpnI resistant) DNA from (DpnI sensitive)
0.5 A260 units were cut with DpnI to distinguish DNA fragments were electrophoresed on a 0.7% agarose gel and blocked on Gene Screen Plus to analyze the DNA.
The filter is 1M NaCl-50mM TrsHCl (pH7.5) -0.1% sodium pyrophosphate-0.2% polyvinylpyrrolidone-0.2
% Ficoll-0.2% BSA-1% SDS solution at 65 ° C at 105 cpm / ml
Hybridized with denatured probe. Wash the filters at 65 ° C with the same buffer solution, and
A Kodak XAR-5 X-ray film backed with a ting-Plus brightness enhancement screen (manufactured by Du Pont) was exposed at -70 ° C overnight.

The Factor VIII: C probe was a 2924 bp EspI fragment isolated from the RE expression plasmid (see FIG. 4), Fe
inberg and Vogelstein random hexadeoxynucleotide primer method [AP Feinberg and Vogelstein, An
al. Biochem., 132, pp. 6-13 (1983)] by 109 cpm /
Labeled with 32 _P in μg of non-radioactivity.

As shown in FIG. 6, the newly replicated activation Factor VIII: C plasmid DNA levels are the same for all three deletions and the full length gene. Lane 1 is a chain length size marker having 1 kb as a structural unit labeled with T4 DNA polymerase according to the manufacturer's experimental method obtained from BRL,
Lane 2 is 1 ng of supercoiled RE DNA, lane 3 is 1
0 ng of supercoiled RE DNA, lane 4 is 10 ng RE DNA cut with DpnI, lane 5 is DpnI hydrolyzate of 0.5 A260 unit Hirt portion obtained from BMT10 cells transfected with signal minus construct, lane 6 is full chain Long Factor VIII: C
Transfection with cDNA (construct 8.1), lane 7 with QD deficient, lane 8 with RE deficient, lane 9
Transfected with RS deficient. FIG. 6 shows that after DpnI digestion (lanes 5-9), the supercoiled form of each construct is about the same amount, thus differences in DNA replication may enhance expression of QD and RE deficiencies. Will be eliminated. Lane 2 contains 10 ⁸ molecules of the RE scheme, lane 3 contained 10 ⁹ molecules, the number of replicas, of approximately 10 ⁵ cells successfully transfected, approximately 10 ³ It indicates that there is.

Construction of J.ARG 740-ASP 1658 (hereinafter abbreviated as RD) deficiency In this chapter, a method for producing RD deficiency excluding a DNA sequence encoding the expression between Ser 741 and Ser 1657 is shown. The present invention performed this RD-less fusion in three steps. Sau3, which cuts the plasmid QD (Fig. 2) between the Ser 1657 and Asp 1658 codons and between the Glu 1794 and Asp 1795 codons in the first step
Cut at A. This produces a 411 bp fragment. In addition, the plasmid tsa pML [L. Dailey et al., J. Virol. 5
4, pp.739-49 (1985)] was linearized at a unique BclI site. Then a 411 base pair fragment T4 DNA ligase derived from the plasmid QD (a ligase used for this purpose and in the next example) is linked to the tsa pML linearized at a unique BclI site and the BclI site is Asp 1658 of the 411 bp insert. Plasmid 411 BclI containing the flanking flank (ie, Asp 1658 5'to the coding sequence) was generated. Plasmid 411.Bc
lI can be specifically linearized with BclI and consists of the 5'GAT consisting of the GAT codon of Asp 1658 and the first base of the CAA codon of Gln 1659.
C Providing a protruding part.

In addition, the plasmid QD was cleaved with HindIII, and the same plasmid was
A cleavage between rg 740 and Ser 741 and within the codon of Glu 321, generated a 1258 bp fragment. Then 5'AGCT
The overhang was excised with mung bean nuclease, which was first treated with Klenow enzyme and all four deoxynucleoside triphosphates to create a blunt-ended BclI linearized 411 BclI.
Ligated to the fragment. The thus obtained plasmid RD 4
11 contains Asp 718 at the 5'end of the fusion site within the 1258 bp HindIII fragment. RD.411 is 411 bp Sa
Pst at position 3'to the fusion site in the u3A fragment
Includes I site.

The plasmid PE (Fig. 3B) was subsequently digested with Asp 718 and the Trp
It was cleaved within 585 codons.

The 5'GTAC overhang was then dephosphorylated with calf intestinal phosphatase and further partially digested with PstI. Due to this partial cleavage, the linearized RE plasmid was Ala 1702 and Val 1703.
Cleavage between codons and a 628 bp fragment incorporating the RE fusion is removed.

Further, the plasmid RD.411 was cleaved with Asp 718 and PstI to give R
A 601 bp fragment incorporating the D fusion was generated.
This plasmid fragment was ligated to Asp 718 cleaved, PstI partially cleaved RE plasmid DNA to generate plasmid RD. As shown below, plasmid RD contains Arg 740 and Asp 1
Controls the expression of fusion factor VIII polypeptide between 658. Cleavage of the RD polypeptide after Arg 740 results in a double chain Factor VIII molecule in which the mature H chain is calcium crosslinked to the δ9 L chain, ie, the L chain without the first 9 amino acids at the amino terminus.

Construction of K.ARG 740-SER 1657 (abbreviated as RSD deficiency) In this chapter, the mature polypeptides Ser 741 to Gln 165 are described.
A method for creating an RSD lacking part in which the DNA coding for expression up to 6 is removed is shown.

Initially, plasmid 411.BclI was constructed and linearized with BclI as in Example J. The plasmid QD was subsequently cut with HindIII and cut between the codons of Arg 740 and Ser 741 and within the codons for Glu 321 to create a 1258 bp fragment. AGC codon
The Klenow enzyme and dATP, dGTA and dCTP were used to secure in the 5'AGCT overhang and the remaining 5'T overhang mung bean nuclease was removed. This modified HindIII fragment was BclI linearized 411.Bc previously treated with Klnow enzyme and all four deoxynucleoside triphosphates.
ligated to Il and Asp 718 at position 5'to the fusion site within the 1258 bp HindIII fragment and 411 bp Sau3A
Generates plasmid RSD.411 containing a PstI site 3'to the fusion site within the fragment.

Then Asp 718-cut, as described in Example D,
A PstI partially cleaved RE plasmid DNA was prepared. Subsequently, the plasmid RSD.411 was digested with Asp 718 and PstI, and the obtained 604 bp fragment containing the RSD fusion region was ligated to the Asp 718 digested, PstI partially digested RE plasmid DNA to create plasmid RSD. Upon expression, the RSD plasmid encodes the Factor VIII polypeptide fused between Arg 740 and Ser 1657. Cleavage of the RSD polypeptide after Arg 740 yields a double-stranded Factor VIII molecule with a mature H chain and a Δ8-L chain, ie an L chain lacking the first eight amino acids at the amino terminus. In addition, in the primary translation product, Ser is also located at position 741, so RSD can also see a fusion between Ser 741 and Asp 1658. Cleavage after Ser 741 can give rise to a double-stranded Factor VIII molecule with a Ser 741 terminated H chain and a Δ9-L chain.

L. Transfection of African Green Monkey Kidney Cells The present inventor first described (BSC1 African Green Monkey Kidney cells adapted to grow at 40 ° C.) cell line BS.
C40 [W.Brackman and D.Nathan, Proc.Natl.Acad.Sci.US
A, 71, pp.942-46 (1974)], pLTRtsA58 and hygromycin
B phosphotransferase [K. Blocklinger, and A. Di
ggelmann, Mol. Cell Biol. 4, pp. 2929-31 (1984)], and transfected with both pY3 harboring transcription units to construct an African green monkey kidney cell line 6L. Plasmid
LTRtsA58 contains a temperature sensitive SV40 T-antigen allele. The tsA58 mutant virus is a temperature-sensitive mutant of SV40 that does not produce progeny at 39 ° C. The T-antigen protein identified by the TtsA58 mutant is much more unstable at unacceptable temperatures than wild-type T-antigen protein [H. Tegtmeyer et al., J. Virol. 16, pp. 168- 78 (197
Five)]. The resulting cell line 6L inducibly expresses the SV40 T antigen at 33 ° C.

6L cells were then transfected with the supercoiled expression plasmid RD or RSD. This transfection was performed using the DEAE-dextran method and chloroquine as described in Example F. The transfected cells were cultured at 33 ° C.
During the culture, the transfected cells synthesized the activation factor VIII: C and secreted it into the culture medium. Transfectants secrete activation factor VIII: C for 120 hours. For most of the inspection, cm
^{The 2} / ml ratio was about 5.5. That is, a 100 mm diameter Petri dish (55
A monolayer assembly of 6L-transfected cells in cm ² ) was covered with 10 ml of culture medium.

M. Factor VIII: C activity measurement After transfection and culturing at 33 ° C for 3 days, Kabivitrum
Coates RE with 96 wells using the Factor VIII kit
(Example D), Factor VIII of the RD and RSD expression constructs:
C Production status was inspected. Cells transfected with plasmid RE
Cells produce 0.48% of plasma concentration of Factor VIII in the culture medium.
Produced (normally Factor VIII plasma concentration is about 150 ng / ml).
Cells transfected with plasmid RD had 0.41% plasma concentration
Factor VIII was produced in culture. Plasmid RSD
Cells transfected with γ were treated with Factor VI at 0.71% plasma concentration
Produced II.

In the same test, cells transfected with the plasmid RE or RSD were cultured at 33 ° C. for 3 days and further cultured for 2 days. As a result, the concentration in the cell doubling solution produced Factor VIII as shown below.

Factor VIII: C concentration in culture medium Expressed in% plasma concentration 3 days 5 days RE transfected cells 0.30% 0.77% RSD transfected cells 1.50% 1.16% Microorganisms of the present invention, recombinant DNA molecules, activated Factor VIII: C DNA The coding sequence is E. coli.HB101 at the American Type Culture Collection, American Type Culture Collection, on July 22, 1986 in Rockville, Maryland, USA.
Deposited as (RE). The ATCC accession number of the culture was 53517. The other two cultures were Ad.RD.2 [E.coli.HB101 (RD)] ATCC Accession No .: 67475 and Ad.RD.2 at the American microbial strain preservation organization in Rockville, Maryland, USA on July 27, 1987. RSD.1.2 [E.coli.HB101 (RSD)] ATCC Deposit No: 67476 deposited.

Having described several embodiments of the invention above,
Obviously, the basic constitution of the present invention may be partially modified and applied to other embodiments using the method and composition of the present invention. Therefore, it is to be understood that the scope of the present invention is limited by the following claims, rather than the limited implementations of the examples set forth above.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C12R 1:19) C12R 1:19) (C12P 21/02 A61K 37/54 ACB C12R 1:19) C12R 1:19) (56) Reference JP-A-62-502941 (JP, A) JP-A-62-282594 (JP, A)

Claims (4)

(57) [Claims] 1. Factor VII comprising domains A, B and C
In the I: C polypeptide, the amino acid Ala at the N-terminal of domain A is set as position 1 and the Tyr at the C-terminal of domain C is set at
As an amino acid number is assigned as a position, the H chain consisting of the amino acid sequence from Ala at position 1 to Arg at 740 and the L chain consisting of the amino acid sequence from Glu at position 1649 to Tyr at position 2332 are alkali metal or calcium. In the method for producing maturation factor VIII: C, which is cross-linked with Ala at the 1st position or Met at the -1st position as the N-terminal,
A first polypeptide C-terminal to any amino acid from Arg at position 0 to Gln at position 744, and any amino acid from Asp at position 1563 to Glu at position 1649 to N-terminal; and A second polypeptide having a Tyr at the 2332nd position as the C-terminal is a DNA encoding one polypeptide linked at the C-terminal of the first polypeptide and the N-terminal of the second polypeptide. Culturing animal cells transformed with an expression vector comprising and obtaining said maturation factor VIII: C from this culture. 2. The method according to claim 1, wherein maturation factor VIII: C is directly collected from the culture. 3. The method according to claim 1, wherein maturation factor VIII: C is collected from the culture after treatment with a protein-splitting enzyme and / or crosslinking treatment with an alkali metal or calcium. 4. The polypeptide according to claim 1, wherein the first polypeptide and the second polypeptide are linked to each other, and the polypeptide has any of the following amino acid sequences. The method described.