Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
  • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Factor VIIIR Factor VIIIR
• its recombinant DNA-directed synthesis and its use in the treatment of coagulation disorders, such as von illebrands's disease.
• This host is typically an-Escherichia coli (,E. coli) cell line; however, depending upon the desired product, eukaryotic hosts may be utilized. Clones incorporating the recombinant vectors are isolated and may be grown and used to
• RNA-directed DNA polymerase also called reverse transcriptase.
• Reverse transcriptase synthesizes DNA in the 5'-3' direction, utilizes deoxyribonucleoside 5'-triphosphates as precursors, and requires both a template and a primer strand, the latter of which must have a free 3'-hydroxyl terminus.
• Reverse transcriptase products whether partial or complete copies of the mRNA template, often possess short, partially double-stranded hairpins ("loops") at their 3' termini. In tne second reaction, these "hairpin loops" can be exploited as primers for DNA polymerase.
• Preformed DNA is required both as a template and as a primer in the action of DNA polymerase.
• the DNA polymerase requires the presence of a DNA strand having a free 3'-hydroxyl group, to which new nucleotides are added to extend the chain in the 5'-3' direction.
• the products of such sequential reverse transcriptase and DNA polymerase reactions still possess a loop at one end.
• the apex of the loop or "fold-point" of the double-stranded DNA, which has thus been created, is substantially a single-strand segment.
• this single-strand segment is cleaved with the single-strand specific nuclease Sl to generate a "blunt end" duplex DNA segment.
• This general method is applicable to any mRNA mixture, and is described by Buell, et al. , J. Biol. Chem. , 253; 2483 (1978).
• the resulting double-stranded cDNA mixture (ds-cDNA) is inserted into cloning vehicles by any one of many known techniques, depending at least in part on the particular vehicle being used. Various insertion methods are discussed in considerable detail in Methods In Enzymology, 68:16-18, and the references cited therein. 1
• the cloning vehicle is used to transform a suitable host. In a common form of this technique, these cloning vehicles can impart an antibiotic resistance trait to the host.
• Such hosts are generally prokaryotic or eukaryotic cells. Generally at this point, only a few of the transformed or transfected hosts will contain the desired cDNA, and the sum of all transformed or transfected hosts constitutes a gene "library". In the principle, the overall ds-cDNA library created by this method
• oligonucleotide sequence it can be used to identify clones of interest as follows. Individual transformed or transfected cells are i ⁇ grown as colonies on nitrocellulose filter paper. These colonies are lysed; the DNA released is rendered single strands and then strongly bound to the filter paper after heating. Such a sheet is then incubated ' with a labeled oligonucleotide probe which is complementary to the
• the probe hybridizes with the cDNA for which it is complementary, and hybrids are identified by autoradiography.
• the corresponding clones are characterized in order to identify one, or a combination of clones which contain all of the structural information for
• the nucleic acid sequence coding for the protein of interest is isolated and reinserted into an expression vector.
• the expression vector brings the cloned • gene under the regulatory control of a specific prokaryotic or eukaryotic control element which allows the efficient
• vWF or Factor VIIIR The von illebrand Factor
• ⁇ j c a large, adhesive plasma glycoprotein which is instrumental in mediating the attachment of platelets to damaged areas of • the circulatory system.
• v f function there . is a defect in formation of the "platelet plug" of primary hemostasis, and a bleeding disorder, von
• vWf circulates as a series of self-aggregated structures ranging from dimers of a 225 kd 5 subunit to polymers containing more than 50 such subunits [Legaz, et a _, J. Biol. Che . , 248: 3946-3955 (1983); Hoyer and Shainoff, Blood, 55: 1056-1059 (1980); Ruggeri and Zimmerman, J. Clin. Invest., 65: 1318-1325 (1980)].
• the results of various studies have established that the largest 0 structures are most important in the intrinsic hemostatic role of vWf (Zimmerman, e_t al.
• Factor VIIIR biosynthesis in endothelial cells is the major source of plasma vWf.
• the 225 kd vWf subunit found Q in plasma and in endothelial cell culture medium is first synthesized in endothelial cell cultures as a 240 kd intracellular proprotein precursor (provWf) [Wagner and Marder, J. Biol, Chem., 258:.2065-2067 (1983); Lynch, et al. , Proc. Natl. Acad. Sci., 80: 2738-2742, (1983)].
• provWF 5 may be in-itially detected as a monomer, it rapidly dimerizes, apparently by disulfide bond formation.
• ProvWF dimers are the predominant intracellular form of the vWf gene product in endothelial cells. They do not self-associate further and are not secreted [Lynch, t ⁇ al. , J. Biol. Chem. 258: Q 12757-12760 (1983)]. However, provWF dimers do undergo a series of post-translational modifications involving addition of a sulfated moiety and cleavage of sequence(s) specific to
• the invention provides replicable expression vectors incorporating a DNA sequence encoding human Factor VIIIR or fragment thereof and a self-replicating host cell system transformed or transfected thereby.
• the host system 0 is usually of prokaryotic, e.g., E ⁇ coli, B. subtilis, or eukaryotic cells.
• the human Factor VIIIR or fragment thereof is produced by a process which comprises (a) preparing a replicable expression vector capable of expressing the DNA 5 sequence encoding human Factor VIIIR in a suitable host cell system; (b) transforming said host system to obtain a recombinant host system; (c) maintaining said recombinant host system.under conditions permitting expression of said Factor .VIIIR-encoding DNA sequence to produce human Factor Q VIIIR protein; (d) recovering said human Factor VIIIR protein or fragment thereof.
• the Factor VIIIR-encoding replicable expression vector is made by preparing a double-stranded complementary DNA (ds-cDNA) preparation representative of 5 Factor VIIIR mRNA and incorporating the ds-cDNA into replicable expression vectors.
• the preferred mode of recovering the human Factor VIIIR comprises reacting the proteins expressed by the recombinant host system with a reagent composition comprising at least one binding protein Q specific for Factor VIIIR.
• the invention provides a process for the cloning of human factor VIIIR in a self-replication recombinant host system which comprises: a) providing a heterogeneous mixture of mRNA enriched in mRNA encoding for human VIIIR; b) preparing ds-cDNA complementary to said enriched mixture; c) incorporating said ds-cDNA into a vector; and e) identifying those vectors in which said ds-cDNA has been incorporated.
• the invention provides a process wherein ds-cDNA encoding, factor VIIIR is put into a replicable expression vector capable of expressing the DNA sequence encoding human Factor VIII-R in a suitable self-replicating recombinant host system; b. transforming said host system to obtain a recombinant and host system; c. maintaining said recombinant host system under conditions permitting expression of said Factor VIII-R encoding DNA sequence to produce human
• the invention provides a cDNA comprising a region whose polynucleotide is substantially CTG CAG TAT GTC AAG GTG GGA AGC TGT AAG TCT GAA GTA GAG GTG GAT ATC CAC TAC TGC CAG GGC AAA TGT GCC AGC AAA GCC ATG TAC TCC ATT GAC ATC AAC GAT GTG CAG GAC CAG TGC TCC TGC TGC TCT CCG ACA CGG ACG GAG CCC ATG CAG GTG GCC CTG CAC TGC ACC AAT.
• the invention provides vectors containing cDNAs encoding VIII-R and host cells transformed thereby.
• the invention provides probes 5 useful for identification and isolation of DNA fragments containing the encoding region of human Factor VIII-R comprising the vector of Claim 31.
• probes 5 useful for identification and isolation of DNA fragments containing the encoding region of human Factor VIII-R comprising the vector of Claim 31.
• Such probes have utility as a means for recovering from a mixture of cDNAs by hybridization those cDNAs containing full length copies of 0 the VIIIR gene, which then may be cloned and expressed in a suitable vector system.
• Figure 1 provides a diagramatic illustration of a restriction map of pDL34. For simplicity, only relevant sites in the insert cDNA are shown. "Eco” and “Ori” refer to the direction of the unique EcoRI site and origin or replication, respectively . of the pBR322 vector.. The insert is flanked by PstI sites resultant from the GC tailing method of cloning. Of the o original nine colonies identified with the polysome-generated probe, all had sequences which hybridized to the 250 bp fragment flanked by internal PstI sites and to the 160 bp fragment to the right of the PstI sites.
• FIG. 5 was extracted from a low melting point agarose gel, and ligated to Pstl/SacI digested pSP64.
• the resultant plasmid is designated pSP64-34.
• Figure 2 illustrates the hybridization of 32P-oligo dT to PDL34
• a southern blot was prepared from various restriction digests of pDL34 which had been electrophoresed through a 2% agarose gel. Lane 1, SphI (There is one recognition site in both the vector and the insert.); Lane 2, SacI (there is a recognition site in the insert only.), and EcoRI (there is a recognition site in vector and none in the insert) . Some anomalous cleavage was noted under the conditions of the digestion, leading to the appearance of two additional minor DNA species, one of which hybridized to the probe.); Lane 3, PstI (Recognition sites flank the insert, and there are two sites within the insert); Lane 4, pBR322 DNA digested with Avail and EcoRI (Used as molecular size- markers.) .
• the probe used here was-prepared by labeling oligo (dT) , g with gamma- 32P-ATP and T. polynucleotide kinase. Hybridization was at 33°C in 4XSSC and 0.1% SDS. The probe specifically hybridized to the portion of pDL34 to the right of the SacI site, as displayed in Figure 1.
• Figure 3 illustrates the hybridization of a fragment of pDL34 to Endothelial Cell RNA 10 ug of endothelial cell (Lane 1 and 2) or SV80 cell (Lane 3) total RNA were electrophoresed through a 0.8% agarose gel and transferred to nitrocellulose. Hybridization was with the nick translated, gel purified approximately 800 bp PvuII fragment of pDL34 (see Figure 1) . The markers at the left denote the migration positions of the 28S and 18S rRNAs. The size of the band in the endothelial cell RNA lanes was estimated to be 9.5 kb, based on the migration of the rRNA specj.es.
• Fiqure 4 illustrates the translation of synthetic mRNA derived from pSP64-34 in a rabbit reticulocyte lysate
• Lane 2 a reaction mixture containing approximately 50 ug/ml synthetic pSP64-34 RNA.
• the Figure depicts a fluorogram of
• Figure 5 illustrates the monoclonal anti-vWf Immunoprecipitation of pSP64-34 derived translation products
• Lane 1 Lane 1, rabbit anti-vWf; Lanes 2-4, monoclonal anti-vWf 0 antibodies AVW-3, AVW-2, and AVW-1, respectively, AVW-1 reacts preferentially with the two major species recognized by the polyclonal anti-vWf.
• Factor VIIIR von Willebrand's Factor
• Factor VIIIR produced by cell or cell-free culture systems, in bioactive forms having the capacity to initiate normal blood coagulation as does Factor VIIIR native to the human plasma.
• Different alleles of Factor VIIIR may exist in nature. These variations may be characterized by difference (s) in the nucleotide sequence of the structural gene coding for proteins of identical biological function.
• the location and degree of glycosylation as well as other post-translationa-1 modifications may vary and will depend to a degree upon the nature of the host and environment in which the protein is produced. It is possible to produce analogs having single or multiple amino acid substitutions, deletions, additions, or replacements.
• E ⁇ coli K12 strain HB101 (ATCC No. 33694) is particularly useful. Of course, other microbial strains may be used.
• Vectors containing replication and control sequences which are derived from species compatible with the host cell or system are used in connection with these hosts.
• the vector ordinarily carries an origin of replication, as well as characteristics capable of providing phenotypic selection in transformed cells.
• E ⁇ coli can be transformed by the vector pBR322, which contains genes for ampicillin and tetracycline resistance [Bolivar, et al. , Gene, 2: 95 (1977)].
• the expression vector- may also contain control elements which can be used by the vector f° r expression of its own proteins.
• Common prokaryotic control elements used for expression of foreign DNA sequences in E_ ⁇ coli include the promoters and regulatory sequences derived from the B-galactosidase and tryptophan (trp) operons of E_ ⁇ coli, as well as the pR and pL promoters of bacteriophage lambda. Combinations of these elements have also have been used (e.g., TAC, which is a fusion of the trp promoter with the lactose operator) .
• Other promoters have also been discovered and utilized, and details concerning their nucleotide sequences have been published enabling the skilled worker to combine and exploit them functionally.
• Host cells can prepare human Factor VIIIR proteins which can be of a variety of chemical compositions.
• the protein is produced having methionine as its first amino acid (present by virtue of the ATG start signal codon naturally existing at the origin of the structural gene or inserted before a segment of the structural gene) .
• the protein may also be intra- or extracellularly cleaved, giving rise to the amino acid which is found naturally at the amino terminus of the protein.
• the protein may be produced together with either its signal polypeptide or a conjugated protein other than the conventional signal polypeptide, the signal polypeptide of the conjugate being specifically cleavable in an intra- or extracellular environment.
• Factor VIIIR may be produced by direct expression in mature form without the necessity of cleaving away any extraneous polypeptide. This.would include any post-translational modifications required for biologic activity.
• Recombinant host cells refer to cells which have been transformed with vectors constructed using recombinant DNA techniques. As defined herein. Factor VIIIR is produced as a consequence of this transformation. Factor VII ⁇ R is produced by such cells are referred to as "recombinant Factor VIIIR". Recombinant and Screening Methodology
• the procedures below are but some of a wide variety of well established procedures to produce specific reagents useful in the process of this invention.
• the general procedure for obtaining a messenger RNA (mRNA) mixture is to prepare an extract from a tissue sample or to culture cells producing the desired protein, and to extract from a tissue sample or to culture cells producing " the desired protein, and to extract the mRNA by a process such as that disclosed by Chirgwin, et al. , Biochemistry, 18: 5294 (1979).
• the mRNA is enriched for poly(A) mRNA-containing material by chromatography on oligo (dT) cellulose or poly(U) Sepharose, followed by elution of the poly(A) containing mRNA-enriched fraction.
• the above pol (A) containing mRNA-enriched fraction is used to synthesize a single-strand complementary cDNA (ss-cDNA) using reverse transcriptase.
• ss-cDNA single-strand complementary cDNA
• reverse transcriptase reverse transcriptase
• the resultant double-strand cDNA (ds-cDNA) is inserted into the expression vector by any one of many known techniques. In general, methods, etc., can be found in Maniatis, supra, and Methods in Enzymology, Vol. 65 and 68 (1980); and Vol. 100 and 101 (1983). In general, the vector is linearized by at least one restriction endonuclease, which will produce at least two blunt or cohesive ends.
• the ds-cDNA is ligated with or joined to the vector insertion site.
• prokaryotic cells or other cells which contain substantial cell wall material are employed, the most common method of transformation with the expression vector is calcium chloride pretreatment as described by Cohen, R.N., et al. , Proc. Nat'l. Acad. Sci. USA, 69: 2110 (1972) . If cells without cell wall barriers are used as host cells, transfection is carried out by the calcium phosphate precipitation method described by Graham and Van der Eb, Virology, 62: 456 (1973). Other methods for introducing DNA into cells such as nuclear injection or protoplast fusion, have also been successfully used. The organisms are then cultured on selective media and proteins for which the expression vector encodes are produced. 1 Clones containing part or all of the gene for
• oligonucleotide probes are prepared by oligo (dT) lg primed reverse transcription of polysome-isolated poly A RNA in the presence of
• Such clones are detected using in situ colony hybridization. Northern blotting, and Southern blotting. These procedures 0 are described in Maniatis, supra.
• Clones containing the entire sequence of Factor VIIIR may be identified using specific antibodies directed against part or all of the Factor VIIIR protein. This - requires that the ds-cDNA be inserted into a vector 5 containing appropriate regulatory nucleic acid sequences adjacent to the insertion site. These regulatory sequences initiate transcription and translation of those ds-cDNA molecules inserted in the vector. Those clones containing - Factor VIIIR cDNA sequences correctly positioned relative to o tne regulatory sequences synthesize part or all of the Factor VIIIR protein.
• clones containing- the Factor VIIIR cDNA can be confirmed by in vitro transcription of the putative cDNAs.
• the resultant 5 mRNAs are translated _in vitro in rabbit reticulocyte, wheat germ, or Xenopus laevis oocyte translation systems.
• Resultant Factor VIIIR protein production is determined by immunoprecipitation of the translation mixture supernatants using polyclonal or monoclonal antibodies specific for Factor VIIIR. Deposits of a strain useful in practicing the invention
• RNA Endothelial cells were grown in roller bottles for experiments involving production of RNA or polysomes.
• Total cellula-r RNA was isolated by the guanidinium isothiocynate" method of Chirgwin, et al. , [Biochemistry, 18: 5294-5299
• RNA was size fractionated by velocity sedimentation in sucrose gradients as described [Aloni and Attardi, J ⁇ _ Mol. Biol. , 55: 271-276 (1971) ] , and material sedimenting faster than the central C part of the 28S rRNA peak was pooled.
• vWf-specific polysomes were immunoisolated from an endothelial cell lysate prepared with 1% Nonidet P-40 by the general method of Shapiro and Young [J ⁇ _ Biol. Chem. , 256: 1495-1946 (1981) ] . A significant change was that the polysome-antibody mixture was passed over
• vWf-specific poly A-containing RNA was isolated from the eluate of the protein A-Sepharose column by oligl-dT cellulose chromatograph . 5 cDNA Construction and Probe Labeling
• a cDNA library was prepared in pBR322 from poly A-containing RNA isolated from total RNA greater that 28S by the GC tailing method using the detailed procedures described by Maniatis, et_ al. , supra.
• First strand cDNA was 0 synthesized from the RNA which had been poly U-Sephadex selected, using an oligo (dT) lg primer (New England Biolabs) and avian myeloblastosis virus reverse transcriptase (Life Sciences, St. Russia, Fla.). The RNA was then removed by alkaline hydrolysis, and second strand DNA was synthesized by c; hairpin loop priming following addition of E. coli DNA polymerase I large fragment.
• a polysome fraction from endothelial cells was prepared and incubated with monospecific rabbit anti-vWf IgG.
• Antibody-bound polysomes were then purified by immunoaffinity chromotography on protein A-Sepharose.
• Poly A-containing RNA was isolated from the purified polysomes and reverse 0 transcribed in the presence of oligo dT primer and all four alpha- 32-P-deoxynucleotide triphosphates (40 Ci/mmole) at 50 uM.
• the average probe length was approximately 300 bp.
• RNA of approximately 5 x 10 8 cells, 2 x 105 cpm of reverse transcript were synthesized.
• P-oligo (dT) lg was hybridized to pDL34 immobilized on a 5 .Southern blot ( Figure 2) . From these results, it may be seen that a unique hybridization site is detected in pDL34. A single 3.9 kb band was detected in an SphI digest (Lane 1). There is a single SphI recognition site within the pDL34 insert (as shown on the map in Figure 1) as well as one in 0 the pBR322 vector. The probe hybridized to the fragment to the right of the SphI site present in the insert. Two DNA fragments were generated by digestion of pDL34 with the enzymes SacI (which cuts in the insert) and EcoRI (which cuts).
• Lane 2 the probe hybridized to the 3.7 kb fragment, i.e., to the sequence on the right side of the SacI site.
• Lane 3 contains a PstI digest of pDL34, and no hybridization of oligo dT was noted.
• PstI digestion 5 produced three DNA fragments from the insert, as well as an intact pBR322 vector backbone.
• the small ( 160 bp) restriction fragment generated by PstI at the right end of the cloned insert failed to bind to nitrocellulose under the conditions employed for Southern 10 transfer.
• the oligo dT binding region of pDL34 is located in the approximately 50 bp region between the SacI site and the end of the cloned insert (also see Figure 6) . This region was detected in all of the initially isolated !_ clones. This finding suggests that the direction of the reverse transcription is from the oligo dT-hybridizing fragment to the left as denoted in Figure 1. It also suggests the orientation of the reading frame within the cDNA and implies that pDL34 is likely to be a complement of the 3' 20 end of its template mRNA.
• pDL34 is homologous to a large, apparently endothelial cell-specific RNA species.
• RNA was then generated in vitro with SP6 polymerase. This RNA was added to a rabbit reticulocyte lysate containing 35 S-m-thionine.
• 5 1 clonal insert is an untranslated region.
• the predicted amino acid sequence is shown in each of the three reading frames up to the first stop codon encountered.
• the final ten amino acids of the longest open reading frame are underlined and 5 correspond exactly to the decapeptide recently described as comprising the C terminus of plasma vWf (Tatani, e_t al. , 1984, abstract. Circulation 70, 11-210). This finding further corroborates the identification of pDL34 as a Vwf cDNA clone and indicates that the precursor-specific amino 0 acid sequences in pro-vWf extend from the N terminus of the plasma protein.
• the oligo(dT) binding site of pDL34 may be seen as a stretch of 18 dA residues, followed immediately by 12 dCs synthesized during library construction.
• the hexanucleotide 5 AATAAA is located 18 bases upstream of the run of As. This is the characteristic poly(A) ' addition sequence (Proudfoot and Browniee, Nature 263: 211-214, 1976) at an appropriate distance (Fitzgerald and Shenk, Cell 24: 251-260, 1981) for the detected dA tract- to be a portion of the poly(A) tail of o vWf mRNA.
• the (dA), reckons represents a portion of the poly(A) tai.l of vWf mRNA; in this case, the 3' untranslated region would extend for less than 200 bp.
• the original primer for the cDNA library was oligo (dT) lg it is also possible that the (dA), réelle represents an 5 internal complement of the primer located in the 3' untranslated region of the vWf mRNA.
• a partial cDNA clone can be similarly used. It might be expected that this approach would be most useful for the identification of cDNA clones encoding the N terminus of a given protein. Such 5 cDNAs might be expected to yield more readily translated mRNAs because of the presence of the natural initiation codon and, possibly, ribosome binding sites. However, pDL34 contains the C terminus of vWf, as indicated by DNA sequence information ( Figure 5). Thus, the presence of the natural 5' Q end of the mRNA was not a requirement for its j ln vitro translation.
• C terminus may have aided antibody recognition, as protein termini may be generally more antigenic than internal sequences (Walter, et al. , Proc. Nat'l. Acad. Sci. USA, 77: 5197-5200, 1980; Lerner, et al. , Cell 23: 309-10, 1981).
• the anti-vWf monoclonals recognized more than one subset of these translation products, so in the aggregate, their activity appears to be against epitopes from more than one distinct region.
• some of the polypeptides do not contain the authentic C terminus and were immunoprecipitated by antibodies to internal epitopes.
• this method of verification may prove to have general applicability in defining the identity of certain cDNA cloning products in situations where antibodies to a protein are available and protein sequence information is not. Such a situation is not unusual for many large proteins and is particularly common in the study of certain cell-surface antigens.

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