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
protein 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 L.-O. 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 [D. Eaton et al., Biochemistry, 25, pp. 505-12 (1986)].
Haemophilia 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
et 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
causative 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 Antihae- mophilic 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
our preferred embodiment have a deletion 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:C- like polypeptides produced by the DNA sequences of this invention, and various methods of using those compositions in haemophilia treatment- therapy 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 adeno- virus 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 S1 analysis of Factor VIII:C mRNA isolated from transfected BMT10 cells.
Figure 6 depicts the results of a Southern analysis of plasmid DNA isolated from transfected BMT10 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 (1' 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 ("A"), guanine ("G"), cytosine ("C"), and thymine ("T"). The four RNA bases are A, G, C, and uracil ("U").
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 ("Leu"), 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 "F"), leucine ("Leu", "L"),
isoleucine ("He", "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 ("Gin", "Q"), asparagine ("Asn:N"), lysine ("Lys:K"), aspartic acid ("Asp", "D"), glutaiαic acid ("Glu", "E"), cysteine ("Cys", "C"), tryptophane ("Trp", "W"), arginine ("Arg", "R") and glycine ("Gly", "G"). 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 α-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.
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 plasmid 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 repli- cate 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, e.g., 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, e.g., 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.
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 β-lactamase system, the trp system, the tac and trc systems, the major operator and promoter regions of phage λ, 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, e.g., Pho5, the promoters of the yeast α-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 VHI-clotting activity, antihemophilic globulin (AHG), antihemophilic factor (AHF), and antihemophilic factor A [see W. J. Williams et al., Hematoloqy, 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 7). 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.
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:C- like polypeptide" includes proteins having the biological activity of modified factor VIII:C. It also includes proteins having an amino terminal methionine, e.g., 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-Iike 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.
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 whicϊt.permit the production of modified factor VIIl:C and modified factor 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 Vlll: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-liked glycosylation
sites 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 SV40, known bacterial plasmids, e.g., plasmids from E.coli including col E1, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., KP4, phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM 989, and other DNA phages, e.g., M13 and Filamenteous single stranded DNA phages, yeast plasmids such as the 2μ 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 [R. Cate et al., Cell, 45, pp. 685-98 (1986)].
In addition, any of a wide variety of expression control sequences — sequences that con
trol 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 λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-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, BSC40, 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
example, 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 Vlll: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, L.-O. Andersson et al., PNAS (USA), 83, pp. 2979-83 (1986)].
After purification the modified factor VIII:C and modified factor VIII:C-like polypeptides
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 form 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 calcuim 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., e.g., human serum albumin or plasma preparations. See, e.g., Remington's Pharmaceutical Sciences (E. 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 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.
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 discussion in Williajns, 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 [W. I. Wood et al., Nature, 312, pp. 330-37 (1984); (Figure 7)]. The bar represents the coding sequence. Below the restriction 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.
These libraries were made using oligo (dT) as first- strand primer and λgt10 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 single- stranded cDNA sequences double-stranded by the technique of Gubler and Hoffman [U. 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 genomic 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.
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 Pstl site at 5163 to the Pstl site at 5755. We inserted this fragment into clone 4.7573 at the Pstl site at 5755 thereby extending clone 4.7573. This Pstl 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 Ndel 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 EcoRV 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 Sall site in tetracycline resistance and at the Sacl site in the sequence encoding the signal peptide in the insert of clone 2.82 and inserted the synthetic duplex
This ligation resulted in the introduction of a Sall-Nrul-Ncol polylinker immediately 5' to the start
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 1). 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 Sall in the polylinker to Aval at 731. We isolated Fragment 2 from the insert in the λgt10 recombinant 1.7977. Fragment 2 extended from Aval 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 Ndel site at 5522. We isolated fragment 5 from the above-described derivative of clone 4.7573. Fragment 5 extended from Ndel at 5522 to Ncol 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 Ncol 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
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 Ncol. We then treated the resultant Ncol restriction fragment with nuclease S1 to create a blunt end. We ligated this fragment to Smal-digested pBG312. pBG312 is an animal cell expresion vector whose construction has been described elsewhere [R. Cate et al., Cell, 45, 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 [S. 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 [R. J. Kaufman, and P. A. Sharp, J. Mol. Biol., 159, pp. 601-21 (1982)]; a polylinker containing sites. for HindlII, Xhol, EcoRI, Smal, Ndel, Sstl, and Bglll; the SV40 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 Ncol 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
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 acides 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 one aliquot of the expression plasmid for the full-length factor VIII:C gene with EcoRI. This endonuclease cleaves between the codons for Gin 744 and Asn 745. We removed the 5'AATT overhang with nuclease S1, and then subjected the plasmid to complete digestion with Pvul within the ampicillin resistance gene. We partially digested another aliquot with BamHI, which cleaves between the codons for Leu 1562 and Asp 1563 (see Figure 7). We filled out the 5'GATC overhang with the Klenow fragment, and again digested the plasmid with Pvul within amp. We then combined the two mixtures of fragments and ligated them with T4 DNA ligase. A BamHI site between the codons for Gin 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
terminus of the light chain, Glu 1649 (Figure 7). The 908 amino-acid maturation polypeptide is thus, replaced by a 90 amino-acid maturation polypeptide, with the protease substates 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:
(1) 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 S1, and subsequently digesting with Kpnl which cleaves uniquely between the codons for Tyr 586 and Leu 587.
(2) 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 5' Glu lle Thr Arg Thr Thr Leu Gin Ser Asp 1649 1657 (3) 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 Pstl which cleaves between the codons
for Ala 1702 and Val 1703, to obtain the 135 bp 5' fragment.
(4) pUC18 digested with Kpnl and Pstl.
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 Pstl. 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 dephos- phorylating the 5' GTAC overhang with calf intestinal phosphatase, and then partially digesting the plasmid with Pstl.
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 by this recombinant molecule cell is secreted, and may be purified as a single chain, i.e., 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
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 (i.e., 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:
(1) the 462 bp fragment, obtained by digesting the expression plasmid for the full-length gene with HindlII between the codons for Arg 740 and Ser 741, removing the 5' AGCT with nuclease SI, and subsequently digesting with Kpnl which cleaves uniquely between the codons for Tyr 586 and Leu 587.
(2) the synthetic oligonucleotide duplex fragment:
5' pAGC TTT CAA AAG AAA ACA CGA CAC TAT TTT ATT GCT GCA TCG AAA GTT TTC TTT TGT GCT GTG ATA AAA TAA CGp 5' Ser Phe Gin Lys Lys Thr Arg His Tyr Phe He Ala Ala 1690 1702 (3) pUC18 digested with Kpnl and Pstl.
In this fusion, we recreated the HindiII site between the codons for Arg 740 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 Pstl 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 [R. D. Gerard and Y. Gluzman, Mol. Cell. Biol., 5, pp. 3231-40
(1985)] with the supercoiled expression plasmid- We used the DEAE-dextran technique [L. M. Sompayvac and K. J. Danna, PNAS, 78, pp. 7575-78 (1981)] and chloroquine [H. Luthman and G. Magnusson, Nucleic Acids Research, 11, pp. 1295-1308 (1983)] to trans
fect 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 into 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 S1 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
with the QD deletion was 1.46% plasma level and that for the RE deletion was 1.30% plasma level.
Thus, we observed that BMT10 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 S1 ANALYSIS OF FACTOR VIII.C mRNA
In order to determine the levels of mRNA in each construction, we conducted a nuclease S1 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 7.5) - 5 mM EDTA - 1% SDS containing 100 μg/ml proteinase K for 20 minutes at 37°C. We transferred the lysate to a 50 ml conical tube containing 3 ml of phenol and then mechanically sheared the DNA for 15 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°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°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 SI analysis by digesting the QD expression plasmid with Espl. We labelled the 5' ends of the Espl fragments with [γ- 32P]ATP and T4 polynucleotide kinase, and annealed 10 μg RNA to 5000 cpm of the
32 P-antisense strand of the 477 nucleotide Espl frag
ment isolated on a 5% strand separation gel [A. M. Maxam and W. Gilbert, Methods In Enzymology, 65, pp. 499-560 (1980)]. We incubated the RNA overnight at 48°C in 10 μl 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 37°C by adding 190 μl nuclease S1 at a concentration of 100 units/ml in 0.28 M NaCl - 50 mM NaOAc (pH 4.6) - 4.5 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 gel to Kodak XAR-5 X-ray film backed by a Lightning-Plus intensifying screen (Dupont) overnight at -70°C. The 477 nucleotide Espl fragment has one end within the hybrid intron spliced out from the 5' untranslated region of the factor VIII:C mRNA [R. 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 μg RNA in order to verify that the single- stranded 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 μg of input RNA, and Figure 5B is the analysis for 1 μg 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 S1 digestion; that is, 10% of the input to each hybridization reaction. Lane 2 contained RNA
isolated from BMT10 cells transfected with the signal- minus construct; lane 3, BMT10 cells transfected with the full-length factor VIII:C cDNA (construct 8.1); 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 Hinfl and labeling their 3' ends with [α- 32P]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 BMT10 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.
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 [B. Hirt, J. Mol. Biol., 26, pp. 365-69 (1967)]. For each construction, we digested 0.5 A260 units with Dpnl to distinguish newly-replicated (Dpnl- resistant) 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°C in 1 M NaCl - 50 mM Tris-HCl (pH 7.5) - 0.1% sodium pyrophosphate - 0.2% polyvinyl- pyrrolidone - 0.2% Ficoll - 0.2% BSA - 1% SDS using 10 cpm/ml denatured probe. We then washed the filter at 65°C with the same buffer and exposed it overnight at -70°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 Espl fragment isolated from the RE expression plasmid (see Figure 4) and 32P-labeled to a specific activity of 109 cpm/μg by the random hexadeoxynucleotide primer method of Feinberg and Vogelstein [A. 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 Dpnl; lane 5: Dpnl digest of 0.5 A260 units
Hirt fraction obtained from BMT10 cells transfected with the signal-minus construct; lane 6: transfected
with the full-length factor VIII:C cDNA (construct 8.1); 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 Dpnl (lanes 5-9), 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 10 3 in the approximately 10 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 [L. Dailey et al., J. Virol. 54, pp. 739-49 (1985)] at the unique Bcll 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 Bcll site to generate plasmid 411.Bcll, which contains the Bcll site on the Asp 1658 side of the 411 bp insert (i.e., 5' to the sequence encoding Asp 1658). Plasmid 411.BclI may be linearized uniquely with Bcll, resulting in a 5' GATC overhang which consists of the GAT codon for Asp 1658 and the first base of the CAA codon for Gin 1659.
We also digested plasmid QD with HindlII 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 Bcll- linearized 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 HindlII fragment. RD.411 contains a Pstl 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 Pstl. 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 Pstl to generate a 601 bp fragment spanning the RD fusion. We then ligated this fragment to the Asp718-cleaved, Pstl-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 59 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
sequence coding on expression for Ser 741 to Gin 1656 of the mature polypeptide. Initially, we constructed plasmid 411.Bcll and linearized it with Bcll as described in example "J". Subsequently, we digested plasmid QD with Hindlll, 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 HindlII fragment to Bcll-linearized 411.Bcll, 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 HindlII fragment and a Pstl site 3' to the fusion site within the 411 bp Sau3A fragment.
We then prepared Asp718-cleaved, Pstl- partially cleaved RE plasmid DNA as described in example "D". Subsequently, we cleaved plasmid RSD.411 with Asp718 and Pstl and ligated the resulting 604 bp fragment spanning the RSD fusion to the Asp718-cleaved, Pstl-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 59 light chain.
L. TRANSFECTION OF AFRICAN
GREEN MONKEY KIDNEY CELLS
We first produced African green monkey kidney cell line 6L by cotransfecting cell line BSC40 (BSC1 African green monkey kidney cells which have been adapted to grow at 40°C), [W. Brackman and D. Nathan, Proc. Natl. Aead. Sci. USA, 71, pp. 942-46 (1974)] with pLTRtsA58 and with pY3, which has a transcription unit for hygromycin B phosphotranferase [K. Blochlinger, and A. Diggelmann, Mol. Cell Biol. 4, p. 2929-31 (1984)]. Plasmid LTRtsA58 contains a transcription unit for a temperature sensitive SV40 T-antigen allele. A mutant tsA58 virus is a temperature-sensitive mutant of SV40 which does not produce progeny at 39°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 [H. Tegtmeyer et al., J. Virol 16, pp. 168-78 (1975). The resulting cell line 6L inducibly expresses SV40 T-antigen at 33°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 "F". We then incubated the transfected cells at 33°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 D), RD and RSD expression constructs for factor VIII:C production after transfection and incubation at 33°C for three
days 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°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
Type Culture Collection, in Rockville, Maryland on July 27, 1987, and identified there as:
Ad.RD.2 [E.coli HB101 (RD)], having ATCC accession number 67475; and Ad.RSD.1.2 [E.coli HB101 (RSD)], 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.