HYBRID PROCOAGULANT PROTEINS

Abstract: Improved DNA sequences encoding procoagulant proteins, their production and use are disclosed.

Description: HYBRID PROCOAGUIANT PROTEINS DNA sequences encoding the human coagulation cofactor, factor VIII:C (FVIII) , are known . in the art [see e.g., Toole et al, 1984, Nature 312.:312-317; Wood et al, 1984, Nature 312:330-337? Vehar et al, 1984, Nature 312:337-3421. as are methods for expressing them to produce recombinant FVIII [see e.g. WO 87/04187, WO 88/08035 and WO 88/03558]. Procoagulant-active FVIII analogs and DNA sequences encoding them have also been reported [see e.g. WO 86/06101 and WO 87/07144]. Generally, such analogs are modified such that part or all of the B domain are missing and/or specific amino acid positions are modified, for example, such that normally protease-labile sites are resistant to proteolysis, e.g. by thrombin or activated Protein C. Other analogs include modification at one or more lysine and/or tyrosine residues. For reasons that have still not been determined with certainty, recombinant FVIII expression proceeds at relatively low levels. Various methods have been developed which contribute to enhancing the otherwise low expression level of FVIII and analogs thereof, including expression in the presence of phospholipids or von Willebrand Factor (vWF) or analogs thereof [see e.g., WO 87/04187 and WO 88/08035], expression of FVIII in cells expressing lower levels of an endogenous endoplasmic reticulum protein known as BiP [see e.g., WO 88/03558], and deletion within the B domain of FVIII and analogs thereof [see e.g., WO 86/06101]. Factor V (FV) is another high molecular weight glycoprotein cofactor involved in the coagulation cascade. A human FV gene has been cloned and sequenced [see e.g., Jenny et al, 1987, Proc Natl Acad Sci USA 84:4846-4850]. While different from one another in sequence, FVIII and FV share certain common organizational characteristics, both being expressed by DNAs encoding three "A" domains, a "B" domain and two "C" domains, as depicted schematically below: I A-l l - l A-2 I B ~ 1 A-3 1 - 1 C-l I - I C-2 I Also, highly acidic peptide regions precede A-2 and A-3 in FVIII It had also been surprisingly found that the B domain i dispensible for the procoagulant activity of FVIII, and tha active procoagulant protein can be expressed and secreted b expression of a FVIII-encoding DNA in which the nucleotide regio encoding part or all of the B domain is lacking. Not only i active protein produced and secreted, it accumulates in the medi at higher levels than when expressed by the full-length DNA. The reduced level of active procoagulant FVIII protein in th media has been attributed, at least in part to several factor [see e.g. WO 87/04187, WO 88/08035 and WO 88/03558]. This invention provides new hybrid DNAs encoding hybri procoagulant proteins which may be produced by recombinan techniques in higher yield and/or in more homogeneous form tha recombinant human FVIII. Furthermore, the proteins of thi invention may be more stable procoagulants or may give rise t more stable activated species, and may not be inhibited b antibodies to native FVIII, and thus may be preferred in th treatment of bleeding disorders in patients with such antibodies Hybrid proteins of this invention include procoagulant- or coag ulant-active FVIII proteins which contain - in place of part o all of one or more FVIII domains - corresponding polypeptid portions of FV. DNAs encoding these hybrid proteins include DNA which encode a protein having procoagulant or coagulant activit (as measured by conventional assays such as described below which are capable of hybridizing under stringent conditions to cDNA encoding FVIII, preferably human FVIII, and to a cDNA encod ing FV, preferably human FV. Also encompassed, of course, ar DNAs which would so hybridize but for the inclusion of synonymous codons. The DNA may be further modified, e.g. at one or more sites encoding proteolytic cleavage sites, sulfation sites, etc. as disclosed in WO 87/07144, and/or by deletion within the region encoding the B-domain. The DNA may further be modified as described in WO 87/07144 but at corresponding FV sites. It is contemplated that the hybrid FVIII-FV proteins encoded by these chimeric genes and which contain part or all of the FV B domain in place of the corresponding FVIII region will be capable of expression at levels higher than typically obtained with wild- type FVIII. Indeed, that appears to be true in cases evaluated thus far (see e.g. Table II) . Furthermore, it is contemplated that the proteins produced may be obtainable in one-chain form (e.g. where specific proteolytic cleavage sites are additionally modified) or as two-chain protein. In the latter case, it is contemplated that the protein may be obtained as a complex of subunits of approximately 90 kD and 80 kD upon proteolytic excision the FV or FVIII B-domain or as a complex of an -80 kD light chain subunit with a heavy chain ranging from -90 kD up to about 200 kD. In all cases, it is contemplated that the protein obtained will retain procoagulant activity in conventional coagulation assays, as described in greater detail below. DNA sequences of this invention One aspect of this invention provides a DNA sequence encoding a polypeptide sequence substantially the same as human FVIII or variants thereof modified as is now known in the art or disclosed in various patent applications, except within one or more regions encoding part or all of a FVIII domain. One such domain, for example, encodes the B-domain, spanning a ino acid positions 740 to 1649, with numbering beginning with the mature N-terminus, Ala-l. Within the modified region(s) , the DNA sequence comprises a nucleotide sequence encoding a peptide sequence the same or substantially the same as that of a human FV peptide sequence. preferably corresponding to that modified in FVIII. It should b understood that the remainder of the DNA sequence may encod native human FVIII or may comprise additional modification t delete part of the polypeptide sequence, as disclosed e.g. in W 86/06101 or WO 87/07144, and/or modification at one or mor specific sites as disclosed e.g. in WO 87/07144, which als disclose exemplary oligonucleotides and methods for effectin such additional modification. Variant- DNA in accordance with this invention also includ allelic variations,i.e. variations in sequence due to natura variability from individual to individual, or with other codo substitutions or deletions which still retain Factor VIII:c-typ procoagulant activity. Thus this invention encompasses DNA sequences encoding procoagulant protein which sequences are capable of hybridizin under stringent conditions to the wildtype human FVIII-encodin cDNA insert in pSP64-VIII, discussed below, so long as the DN sequence is modified within the region encoding the B domain a described herein. DNA sequences of this invention are als capable of hybridizing under stringent conditions to a human F cDNA, such as the FV cDNA insert in p T2-V (ATCC No. 40515) . Production of DNA sequences of this invention All variant DNAs of this invention may be prepared by modifyin DNA sequences encoding human FVIII. The variant DNA sequence may be produced by conventional site-directed mutagenesis of DN sequences encoding human Factor VIII:c or analogs thereof and/o by ligation of desired DNA sequences. DNA sequences encoding human Factor VIII:c have been cloned. cDNA encoding the full-length human protein as well as a cDN encoding the deletion analog pDGR-2 have been deposited with th ATCC under accession numbers ATCC 39812 and 53100, respectively Preparation and/or nucleotide sequence of a full-length huma factor VIII:c cDNA has been set forth in detail in U.S. Patent Applications Serial Nos. 546,650 (filed October 28, 1983) and 644,086 (filed August 24, 1984) and in International Patent Application No. PCT/US84/01641, published May 9, 1985 (Publn. No. WO 85/01961) . A pSP64 recombinant clone containing the full- length human FVIII nucleotide sequence, designated as pSP64-VIII, is on deposit at the ATCC under Accession Number ATCC 39812. Preparation and nucleotide sequence of a full-length human FV cDNA has been set forth in Jenny et al, supr . A mammalian expression vetor containing a human FV-encoding nucleotide sequence, as depicted in Jenny et al, supra. has been designated pMT2-V and deposited at the ATCC under Accession No. ATCC 40515. In order to simplify genetic manipulations, unique restriction sites have been engineered into FVIII and FV cDNAs by site- directed mutagenesis. Such techniques have also been used to modify a cDNA at specific sites, whether by replacing or deleting one or more bases. Such methods of mutagenesis include the M13 system of Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982); Methods Enzymol. 100:468-500 (1983); and DNA 2:479-488 (1984) , using single stranded DNA and the method of Morinaga et al.. Bio/ echnology, 636-639 (July 1984), using heteroduplexed DNA. Exemplary oligonucleotides used in accordance with such methods are shown in Table I. It should be understood, of course, that all variant DNAs of this invention may be analogously produced by one skilled in the art by methods such as described herein for exemplary constructs. The convenient exchange of domains between FVIII and FV was accomplished by introducing Mlul sites into cDNAs encoding the respective factors. Because Mlul does not cut within either of the cDNAs nor within the vector backbone, the unique introduced Mlul sites facilitate the ability to exchange domains between the FVIII and FV cDNAs at will. Naturally, if such a site is present in alternative cDNAs or vectors such sites may be altered by site directed mutagenesis, if desired. It should be understood that other unique restriction sites may be similarly introduced instead of Mlul, or naturally occuring restriction sites may be abolished for the sake of convenience. In addition, since Mlul recognizes the DNA sequence [5] » -ACGCGT- [3'], it will encode thr-arg. This allows one to introduce Mlul sites at particular cleavage sites, e.g. thrombin-cleavage sites, in which the N-ter- minal residue is arg. Hopefully this avoids drastic alteration of the conformation at the site and avoids significant interfer¬ ence with biological activity. Results so far are consistent with that goal. The particular Mlul sites introduced to mediat a B-domain exchange between factors V and VIII are at nucleotid positions in our FVIII cDNA corresponding to peptide position 739-740 (changing the encoded sequence -PR- to -TR-) and a positions 1647-1648 (changing -QR- to -TR-) and in our FV cDNA a positions 708-709 (changing -IR- to -TR-) and at 1544-154 (changing -LR- to -TR-) . Partial restriction maps are as shown: 90K FVIII: 5' I I I 3' Mlul9OK A Xhol Mlul Sail 80K FVIII: M1UI80K A Xhol Mlul Sail 94K FV: 5' M1U94K A A sail Mlul BstEII Sail 74K FV: M1U74K Sail BstEII Mlul Sail Thus, to construct the exemplary DNA encoding FVIII but with a FV B domain, we employed the following scheme. First, the FVIII MluI90K expression vector was digested with Mlul and Sail to excise the FVIII B domain and light chain-encoding region. The FVIII MluIδOK expression vector was similarly digested and the light chain-encoding Mlul-Sall fragment was isolated. The FV expression vectors MluI74K and MluI94K were digested with Mlul and BstEII to excise the FV B domain encoding DNA in two pieces. The resultant DNAs were then ligated together as depicted below: 9OK M1UI90K Xhol Mlul Sail 80K FVIII: M1UI80K Mlul Sail A 94K I FV: 3 » M1U94K Mlul BstEII 74K FV: Mlu74K BstEII Mlul The resultant vector containing the FV B-domain-encoding region in the correct orientation within the FVIII-encoding sequence was identified and has been designated pMT2-VIIIB5. It should be noted that the -T(R)- codons at the 5' and 3' B-domain junctions may be engineered back to -P(R)- and -L(R)- codons, respectively, or to any other desired codons by conventional site-directed mutagenesis, if desired. This process may be used to exchange a region other than the B domain-encoding region, or a portion of a region, and may be repeated iteratively to effect multiple exchanges, and if desired, with intervening abolishing (by mutagenesis) of Mlul sites no longer useful. Alternatively, multiple (e.g. 4, 6, 8, etc.) Mlu sites, for example, may be engineered into the starting vector to define multiple regions for exchange. In that case, the resultant vectors must be carefully analyzed to ensure that all exchanges have been properly effected without loss or disorientation of any desired domain. The B-domain exchange example illustrates one aspect of this invention, namely, the introduction of convenient restriction sites, such as Mlul sites, at the junctions between different domains within DNAs encoding factors VIII and V, and the use of such DNAs in the construction- of hybrid DNAs encoding FVIII-FV fusion proteins. It should be understood that there is some flexibility in the definition of domain boundaries. For example, the Al domain of FVIII has its 3* boundary between amino acid residues 329 and 336. The 5' border of the A2 domain of FVIII is defined as being between residues 337 and 372. The 3' border of the FVIII A2 domain is defined as being between residues 698 and 740. The 5' border of the FVIII A3 domain is defined as between residues 1689 and 1721. Flexibility in the definition of domain boundaries has been previously discussed. See e.g. Vehar et al, suora; Kane & Davie, 1988, Blood 71:539; Jenny et al, supra. For convenience, the boundaries of the domains are frequently taken to be the sites of cleavage mediated by thrombin, activated protein c or Factor Xa. Of course, this invention also encompasses exchanging portions of one or more domains as well. In the practice of certain specific embodiments of this invention, Mlu sites may be engineered into FVIII and FV encoding DNAs at or within one or more of the following-domain boundaries: AA residues at domain boundaries* Domain 5 » Border 3 ' Border FVIII: Al A2 B A3 Cl C2 Heavy chain acidic domain Light chain acidic domain FV: Al A2 B A3 Cl C2 * The amino acid number is given with respect to the mature FVIII or FV polypeptide Thus, as an example of another domain exchange, the FV A2 domain may be inserted in place of the FVIII A2 domain by expression of a hybrid DNA analogously prepared. For instance, Mlul sites may be engineered into FVIII and FV expression vectors at the 5' and 3' borders of the A2 domains. The Mlul fragments may then be excised from the vectors, and the FV A2-encoding region may be ligated into the FVIII expression vector from which the corresponding A2-encoding region had been excised, with or without retention of the FVIII heavy chain acidic region, depending on the location of the Mlul site introduced into the FVIII cDNA. Other examples of DNA exchange include inserting one or more FV DNA regions into the FVIII DNA in exchange for a non-correspond¬ ing region, e.g. replacing a FVIII Al domain with a FV A2 domain. Of particular interest, aside from exchange of all or part of the B domain, are modifications to particular subregions within the FVIII heavy chain and within the light chain C2 domain. Within the C2 domain, modifications within the most c-terminal 100-200 amino acid residues are of especial interest. Modifications to the heavy chain provide FVIII variants which may be produced in higher yield compared to native sequence FVIII, while modifications in both heavy and light chain regions provide FVIII variants which may be less susceptible to cross-reaction with antibodies to FVIII in certain "inhibitor" patients. More detailed schematic representations of the FVIII heavy and light chains are shown below, with specific domains named, acidic regions indicated by diagonal hatching, and C-terminal amino acid positions of junctions specified: Heavy chain: I < A1 > I / / // i < A2 > i χWΛ i 1 226 336 372 562 698 700 740 Liσht chain: I////I< A3 >I< Cl—>I<—C2 >I 1648 I 1721 2020 2173 2?i2 1689 Thus, DNA and protein species of this invention include embodiments wherein one or more of the Al, A2, A3, Cl and C2 domains are replaced in whole or part with corresponding sequence from FV. The correspondence of domains of FVIII and FV is well known in the art, and is shown in considerable detail e.g. in Kane & Davie, 1988, Blood 71(3) :539-555. For instance, in embodiments wherein part of the heavy chain domains of FVIII are replaced with corresponding FV sequence, the replacement may be in the FVIII region from position 1-225, 226- 372, 336-562 and/or 562-740. In embodiments involving partial domain replacement in the FVIII light chain, the replacement may be within the region from FVIII position 1648-1721, 1689-2020, 2020-2173 and/or 2173-2332. Certain embodiments of this invention are so modified in both heavy and light chain regions. It should be recognized that the regions of FV sequence used to replace native FVIII sequence may be longer or shorter than the corresponding FVIII region. This should be clear, e.g. from Fig. 4 of Kane & Davie, 1988, above. Convenience will usually dictate whether the DNA sequence encoding the desired variant is produced by ligation of Mlul (for example) restriction fragments, site-directed mutagenesis or a combination of both. If the domain exchange process is conducted in vectors other than desired expression vectors, the new DNA sequences encoding the variants of this invention can be excised from the mutagenesis vectors and introduced into appropriate vectors for expression, preferably in mammalian cells. The procoagulant activity produced by the transiently transfected or stably transformed host cells may be measured by using standard assays for blood plasma samples. Table I: Exemplary Oliqonucleotides Name No. Sequence Mutation [FVIII] FVIII 740 1. 5 1 (GTA AAA ACA ATG CCA P 739 R 740-> T 739 R 740 TTG AAA CGC GTA GCT TCT CCC AGA ATT C) 3» . 5«(TTG AAA CGC GTA GCT) 3 - *(1) 5 ' (CCA GTC TTG AAA CGC °-1647 R 1648-> 1647 R 164 CAT ACG CGT GAA ATA ACT CGT ACT ACT C)3' 5• (GCC ATA CGC GTG AAA) 3 • *(3) 5• (GAG GAT GAA AAT p i688 R i689-> T i688 R i68 CAG AGC ACG CGT AGC TTT CAA AAG AAA ACA CG)3 • 6. 5 « (AGA GCA CGC GTA GCT)3 ■ *(5) FVIII 372 7. 5'(GCT TCC TTT ATC CAA I371 R 372-> T 371 R 372 ACG CGT TCA GTT GCC AAG AAG CAT CC) Screen 372 8. 5«(CCA AAC GCG TTC AGT)3' *(7) FVIII 226 9. 5»(G GAT AGG GAT GCT GCA A 2 25 R 226-> T 225 R 226 TCT ACG CGT GCC TGG CCT AAA ATG CAC)3 » Screen 226 10.5« (ATC TAC GCG TGC CTG) 3« *(9) FVIII 336 11.5« (GTC CAG AGG AAC CCC L 335 R 336-> T 335 R 336 AA ACG £G£ ATG AAA AAT AAT GAA G)3' Screen 336 12.5* (CCA AAC GCG TAT GAA)3' *(H) FVIII 562 13.5' (CAA AGA ATC TGT AGA T °-561 R 562-> T 561 R 562 ACG CGT GGA AAC CAG ATA ATG TC)3 • Screen 562 14.5' (GTA GAT ACG CGT GGA)3* *(13) (continued >) (Table I, continued) FVIII 698 15.5' (GGG TGC CAC AAC TCA GAC F 697 R 698-> T 697 R 698 ACG CGT AAC AGA GGC ATG ACC G)3• Screen 698 16.5' (TCA GAC ACG CGT AAC)3' *(15) FVIII 700 17.5* (GC CAC AAC TCA GAC TTT N 699 R 700-> T 699 R 700 CGG ACG CGT GGC ATG ACC GCC TAA CTG)3' Screen 700 18.5• (TTT CGG ACG CGT GGC)3• *(17) FVIII 1313 19.5' (GTC ACG CAA CGT AGT K 1312 R 1313-> T 1312 R 1313 ACG CGT GCT TTG AAA CAA TTC)3 « Screen 1313 20.5' (CGT AGT ACG CGT GCT) 3 ' *(19) FVIII 1721 21.5 » (CC CCA CAT GTT CTA AGA N 1720 R 1721— T 1720 R 1721 ACG CGT GCT CAG AGT GGC AGT G)3« Screen 1721 22.5' (TAA GAA CGC GTG CTC)3 ' *(21) FVIII 2020 23.5' (GCA CAC TTT TTC TGG TGT N 2019 K 2020-> T 2019 : 2O20 AC AGC ACG CGT TGT CAG ACT CCC CTG GG)3 » Screen 2020 24.5' (ACA GCA CGC GTT GTC) 3 ' *(23) FVIII 2173 25.5' (GGG CTG TGA TTT A N 2172 S 2173— T 2172 T 2173 ACG CGT TGC AGC ATG CCA TTG GG)3' Screen 2173 26.5'(GAT TTA ACG CGT TGC)3 » *(25) [FACTOR V] NO. SEQUENCE MUTATION FV 709 27. 5» (GGC TGC AGC ATT I 708 R 709— T 708 R 709 AGG AAC GCG TTC ATT CCG AAA CTC ATC ATT G) 3 ■ Sen 709 28. 5' (AGG AAC GCG TTC ATT) 3 • *(27) FV 1545 29. 5' (CAT TGC AGC ATG L 1544 R 1545— T 1544 R 1545 GTA CAC GCG TAG CAA CAA TGG AAA CAG AAG) 3 » (continued >) (Table I, continued) Sen 1545 30. 5• (TAC ACG CGT AGC AA)3• *(29) FV 348 31. CCA GCG AAT ATG GAC AAA AAA γ 347 R 348" >τ 347 R 348 ACG CGT TCT CAG CAT TTG GAT AAT TTC Sen 348 32. AA AAA ACG CGT TCT CAG *(31) FV 506 33. GTA AGA GCA GAT CCC TGG ACA R 505 R 506~ >T 505 R 506 CGC GTG GAA TAC AGA GGG CAG CAG Sen 506 34. GGA CAC GCG TGG AAT *(33) FV 505 35. CTG TAA GAG CAG ATC CCT D 504 R 505- >TR GAC GCG TCG AGG AAT ACA GAG GGC AGC Sen 505 36. CCT GAC GCG TCG AG *(35) FV 1018 37. CAC ACC ATG CTC CTT TAT p 1017 R 1018- >TR CTA CGC GTA CCT TTC ACC CTC TAA GAA GTG SCn 1018 38. TAT CTA CGC GTA CCT *(37) FV 1765 39. GAA AAG AAG TCC CGA AGT W 1764 R 1765- >TR TCT ACG CGT CTC ACA TCC TCA GAA ATG ScN 1765 40, T TCT ACG CGT CTC AC *(39) FV 313 41. CCA GGA ATC TTA AGA AAA T 312 R 313->TR TAA CGC GTG AGC AGA GGC GGC ACA TGA AG Sen 313 42. G AAA ATA ACG CGT GAGC *(41) FV 643 43. GTT AAC TTC CAT GAA TTC p 642 R 643- >TR TAG TAC; g£G £AG CAA AAA GCT GAG GCT G Sen 643 44. CTAGTACGCGTAGCA *(43) FV 1878 45. CCA TTT CTT ATC ATG GAC R 1877 D 1878->TR ACG CGT TGT AGG ATG CCA ATG GGA C (continued >) (Table I, continued) Sen 1878 46. GGACACGCGTTGTAG *(45) FV 2037 47. GAA CTG CAA GGT TGT GAG N 2036 G 2037" >TR GTA ACG C£T TGT TCC ACA CCC CTG GGT ATG Sen 2037 48. GGTAACGCGTTGTTC *(47) * Used for screening mutagenesis event effected with the oligonucleόtide indicated in parentheses. Codons for replacement: amino acids are underlined. As those skilled in this art will appreciate, oligonucleotides can be readily constructed for use in deleting one or more amino acids or for inserting a different (replacement) amino acid at a desired site by deleting one or more codons or substituting the codon for the desired amino acid in the oligonucleotide, respectively. Other mutagenesis oligonucleotides can be designed based on an approximately 20-50) nucleotide sequence spanning the desired site, with replacement or deletion of the original codon(s) one wishes to change. The eukaryotic cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol. Biol.. 159:601-621 (1982); Kaufman, Proc Natl. Acad. Sci. 81:689-693 (1985). Eucaryotic expression vectors useful in producing variants of this invention may also contain inducible promoters or comprise inducible expression systems as are known in the art. Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as haematopoetic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so lon as the selection gene is dominantly acting. The host cells preferably will be established mammalian cell lines. For stable integration of the vector DNA into chromosma DNA, and for subsequent amplification of the integrated vecto DNA, both by conventional methods, CHO (Chinese Hamster Ovary) cells are presently preferred. Alternatively, the vector DNA ma include all or part of the bovine papilloma virus genome (Lusk et al., Cell. 36: 391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Othe usable mammalian cell lines include HeLa, COS-1 monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamste cell lines and the like. Whichever type of expression vector is used, it may be preferable to co-express the variant FVIII DNA with a DNA sequence encodin vWF or an analog thereof or in the presence of phospholipid, e.g. as described in WO 87/06101 and WO 88/08035. It may also be prefered to express the protein in media containing a protease inhibitor such as aprotinin e.g. in an amount from about -0.01- -5%, preferably 0.5-1.0%, (vol/vol) (Aprot., 15-30 Trypsin inhibitor units(TIU)/ml, Sigma) or corresponding amounts of activity units of other protease inhibitors. Stable transformants are screened for expression of the procoagulant product by standard im unological or activity assays. The presence of the DNA encoding the procoagulant proteins may be detected by standard procedures such as Southern blotting. Transient expression of the procoagulant genes during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunologic assay of the proteins in the culture medium. Following the expression of the variant DNA by conventional means, the protein so produced may be recovered, purified, and/or characterized with respect to physioche ical, biochemical and/or clinical parameters, all by known methods. The proteins of this invention should bind to appropriate monoclonal antibodies directed to human Factor VIII:C or FV (i.e., where the epitope recognized is present in the desired protein) and may thus be recovered and/or purified by immunoaffinity chromatography using such antibodies and/or hy conventional FVIII purification methods. Furthermore, these compounds should possess procoagulant activity. The proteins produced in accordance with this invention can be formulated into pharmaceutically acceptable preparations with a pharmaceutically acceptable, e.g. parenterally acceptable, vehicle and/or one or more excipients in accordance with procedures known in the art. Such pharmaceutical preparations suitable for parentera administration may conveniently comprise a sterile lyophilize preparation of the protein which may be reconstituted by additio of sterile solution to produce solutions preferably isotonic wit the blood of the recipient. The preparation may be presented i unit or multi-dose containers, e.g. in sealed ampoules or vials Their use would be analogous to that of human factor VIII (or F in certain cases where one or more FV A and/or C domains ar present) , appropriately adjusted for potency. The invention will be further understood with reference to th following illustrative experimental examples and procedures which are purely exemplary, and should not be taken as limitin the true scope of the present invention. PLASMID DERIVATIONS A. FVIII The mutagenesis of factor VIII cDNAs was performed directly i the expression plasmid in order to minimize inconvenience i shuffling sequences between different vectors. Generally, th approach taken for mutagenesis was derived from the procedure o Morinaga with modifications. This approach is facilitated b the construction of plasmids which have convenient uniqu restriction sites in the factor VIII expression plasmid. Th following depicts the construction of a factor VIII expressio plasmid which has unique Eco RV, Hpal, Cla I and Xba restriction sites. Plasmid pMT2 may be obtained by EcoR digestion of pMT2-VWF, which has been deposited with the America Type Culture Collection under accession number ATCC 67122. EcoR digestion excises the cDNA insert present in pMT2-VWF, yieldin pMT2 in linear form which can be ligated and used to transform E coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DN can be prepared by conventional methods. pMT2VIII was then con structed by digesting pMT2 with Eco RV- and Xbal, treating th digested DNA with Klenow fragment of DNA polymerase I, and ligating Cla linkers (NEBiolabs, CATCGATG) . This removes bases 2171 to 2421 starting from the Hind III site near the SV40 origin of replication and enhancer sequences of pMT2 (the Clal derivative of pMT2) . The factor VIII cDNA was exised from pSP64 VIII with Sail and blunted with T4 DNA polymerase, and EcoRI adapters were added (AATTCCTCGAGAGCT) . The EcoRI-adapted factor VIII cDNA was then ligated into the EcoRI site of the Clal derivative of pMT2. 'The resultant plasmid is called pMT2-VIII. B. FV The mutagenesis of FV cDNAs was performed both in the expression vector and in a cloning shuttle vector. The mutagenesis approach was derived from Morinaga, supra. with modifications. The expression vector used for FV expression was also a pMT2 derivative. Plasmid pMT2 DNA was obtained in linear form as pre¬ viously described from pMT2-VWF (ATCC 67122) . The EcoRI site was then modified by ligation to an adapter containing a Sail site (see below) . The Clal derivative of pMT2 was then constructed by digestion with EcoRV and Xbal, treating the digested DNA with Klenow fragment of DNA polymerase I, and ligating Clal linkers (N. E. Biolabs, CATCGATG). This removes bases 2267-2421, number¬ ing in a clockwise manner from the Hindlll site on the early side of the pMT2 SV40 origin of replication and enhancer sequences. The FV cDNA was assembled from two overlapping Charon 21Aλ recombinants, AV401 and λVl, each containing portions of FV cDNA derived from an oligo (dT)-primed human fetal liver cDNA library (See Jenny et al, supra) . The following strategy was used to assemble the full-length cDNA and remove flanking DNA sequences. The AV401 clone was digested with SacII to remove the FV cDNA, which was ligated to SacII lin- earized Bluescript KS (+) cloning vector (Stratagene) , resulting in the plasmid 401B5-6. λ DNA sequences were removed from 401B5- 6 by digestion with Hhal (nucleotide 41 in the FV cDNA) , treat ment with Klenow fragment of DNA polymerase I, and then digestio with Bgll (nucleotide 4275 in the FV cDNA) . This resulted in 4234 bp blunted Bglll fragment free of flanking λ sequences. This 4234 bp fragment was inserted by ligation into the EcoRV and Ba HI cloning sites of Bluescript KS (+) , generating plasmid Blu401. The FV cDNA in the λVl clone was removed by SacII digestion o λVl. • This SacII fragment was then ligated to the linearize SacII Bluescript KS (+) cloning vector, resulting in the plasmi V1BS10. V1BS10 was then digested with Hpal (nucleotide 6854 i the FV cDNA) and ligated to the following EcoRI modifie adapters: 5' AATTCCGTCGACTCTAGAG 3 « 3' GGCAGCTGAGATCTC 5• resulting in plasmid BluVl-RI. Three restriction sites were important in the construction of th expression vector containing the full-length FV cDNA. A Sai site in the polylinker cloning region of Bluescript KS (+ provides a unique Sail site which is 5' of the FV cDNA sequences There is also a unique BstEII site in the FV cDNA (nucleotid 3666) present in both plasmids Blu401 and BluVl-RI. A uniqu Sail site results from the addition of the EcoRI modifie adapters introduced into the 3• end of the Blu Vl-Rl. The recombinant Blu401 will contribute the 5' end sequences o the FV cDNA, while the Blu Vl-Rl comprises the 3' end of th intact cDNA. Three restriction fragments from the pMT2 deriv ative, Blu401 and Blu Vl-Rl were purified after electrophoresi on a 1% low-melting temperature agarose gel in tris-acetate. Th isolation of the 3625 bp Sall-BstEII fragment was facilitated b also digesting with XMNI to remove a contaminating, co-migratin fragment. The pMT2 expression vector was linearized with Sail digestion and then treated with calf-intestine phosphatase. The three fragments: (i) the 3625 bp Sall/BstEII Blu 401 fragment, (ii) the 3188 bp Sall/BstEII Blu Vl-Rl fragment, and (iii) Sail digested pMT2 were ligated to generate the expression plasmid pMT2-V, which has been deposited with the American Type Culture Collection under Accession No. 40515. A second plasmid containing the full-length FV cDNA was constructed in Bluescript KS (+) . Blu401 was digested with SacII (3' site in Bluescript cloning polylinker) and BstEII (nucleotide 3666 in the FV cDNA) . This fragment containing the Bluescript vector sequences and the 3625 bp fragment of FV sequences starting at the 5' end of the FV cDNA to the BstEII site was isolated. Blu Vl-RI DNA was digested with BstEII (nucleotide 3666) and Sail (3' of the EcoRI modified adapter), to release a fragment of approximately 3900 bp and which contains the 3' end of the FV cDNA sequences. The exact size of this fragment is unclear because is still contains λ DNa sequences. The BstEII/ SacII Blu401 and BstEII/SacII Blu Vl-RI were ligated together to produce the plasmid BluV. λ DNA sequences were removed by Sail digestion of BluV and the intact full-length FV cDNA (6813 bp) contained on the Sail fragment was isolated. Bluescript KS (+) was linearized by Sail digestion and treated with calf-intestine alkaline phosphatase and ligated to the Sail fragment containing the full-length FV cDNA. The resultant plasmid, designated BluKSVΔλ, contains the full-length FV cDNA devoid of any λ sequences. MUTAGENESIS (1) The mutagenesis was performed with the following DNA preparations. Plasmid pMT2-VIII was linearized with Clal, treated with calf intestinal phosphatase, and separated on a low melting temperature tris-acetate agarose gel. The band of linearized DNA was then extracted by absorption to silica dioxid and eluted in tris-EDTA, or by phenol extraction and ethano precipitation. A second lot of pMT2-VIII was digested with Kpnl Xhol, Kpnl-EcoRV or EcoRV-Xbal and the DNA fragments wer separated by electrophoresis on a low melting temperature agaros gel and extracted as above. Plasmid pMT2-V was linearized wit Nhel, treated with calf intestinal phosphatase, and separated b electrophoresis on a low melting temperature tris-acetate agaros gel. The linearized DNA band was extracted with phenol an precipitated in ethanol. The DNA pellet was resuspended in tris EDTA. A second lot of pMT2-V was digested with Apal and Drall the DNA fragments were separated on low melting temperatur agarose gel and extracted as described above. Plasmid BluKSV wa linearized with SacII, treated with calf intestinal phosphatas and the DNA was separated by electorphoresis on low meltin temperature agarose gel. A second lot of BluKSV was digeste with EcoRV and Sphl and was separated in the same manner. Th DNA fragments were extracted with phenol as described. (2) 1 ug of each of the appropriate plasmids were mixed and th volume was adjusted to 18 ul and 2.0 ul of 2 N NaOH was added. (3) The mixture was denatured at room temperature for 10 min then neutralized with 180 ul of a solution which is 0.02 N HC and 0.1 M Tris-HCl pH 8.0. (4) 20 picomoles of phosphorylated mutageniσ oligonucleotide wa added to 40 ul of the heteroduplex mixture. (5) The mixture was placed in a 68 β C heat block for 90 min After the incubation the mixture was allowed to slowly cool a room temperature. (6) For each mutagenic reaction, 40 ul of the heteroduple oligonucleotide mixture was used. The reactions were made 2 m MgCl , lmM / 9-mercaptoethanol, 400 uM ATP, 100 uM deoxynucleotide triphosphate, 3-4 units/ul of Klenow fragment of E___ coli DNA polymerase I and 400 units/ul of T4 DNA ligase. (7) The reactions were incubated for 10 minutes at room temperature, transferred to 16 β C and incubated overnight. (8) The reaction was terminated by phenol-chloroform extraction and ethanol precipitation, and the resultant pellet was washed with 70% ethanol and resuspended in 10 ul of sterile H 2 0. (9) DNA was then used to transform competent HB101 or DH-5 bacteria. The ampicillin resistant colonies were screened with lxlO 6 cpm/ml of a 32 P-labeled screening oligonucleotide in 5x SSC, 0.1% SDS, Sxdenhardt's reagent, and lOOug/ml denatured salmon sperm DNA. (10) The filters were washed with 5x SSC, 0.1% SDS at a temperature 5 degrees below the calculated melting temperature of the oligonucleotide probe. (11) DNA was prepared from positively hybridizing clones and analyzed initially by digestion with different restriction enzymes and agarose gel electrophoresis. DNA was transferred to nitrocellulose and filters were prepared and hybridized to the screening probes in order to ensure the mutagenic oligonucleotide was introduced into the correct fragment. (12) DNA was then retransformed into Ej. coli and ampicillin resistant colonies were screened for hybridization to the screening oligonucleotide. (13) Final mutations were confirmed by DNA sequencing (e.g., Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 24:5463-5467) . Experimental Strategy and Assay Methods The activity of FVIII-FV hybrid proteins was analyzed by transient expression of the proteins by COS-1 monkey kidney cells transfected with the.hybrid DNAs of this invention. Plasmid DNAs were prepared by banding DNA in CsCl and used to trasnfect COS-1 cells as described (Kaufman, 1985, Proc Natl Acad Sci USA :689). At 60 hr post transfection, conditioned media were taken for FVIII or FV activity assay. FVIII is assayed by the Kabi coatest chromogenic assay method (Kabi) or the ability to clot FVIII- deficient plasma (activated partial thromboplastin time, APTT) before and after thrombin activation. FV is assayed by the ability to clot FV-deficient plasma before and after thrombin activation (APTT) . To analyze the synthesis and secretion of the mutant molecules, and to ensure that introduction of the Mlul restriction sites (Thr-Arg) do not alter cleavage at an mutagenized cleavage sites, the transfected cells were labele with 35 S-methionine for 6 hrs at 60 hr post-transfeetion, and th conditioned media and cell extracts were prepared and analyzed b immunoprecipitation with anti-FVIII or FV-specific antibodies an electrophoresis of the precipitates on SDS-polyacrylamide gels. EXAMPLES Example 1: Alteration of the 90 kDa FVIII cleavage site Mutagenesis of the 90 kDa cleavage site in Factor VIII wa performed by the gapped heteroduplex method as describe (Morinaga et al. Biotechnology 8 :636-639) using the Kpn-EcoR 10.1 kb fragment from pMT2-VIII and the Clal linearized pMT2-VII DNA to produce gapped heteroduplexes. The utagenic oligonucleo tide was No. 1 in Table I and the screening oligonucleotide wa the 15-mer No. 2 in Table I. The resultant mutant was verifie to be correct by DNA sequencing (Sanger et al 1977 PNAS 74: 5463). The resultant DNA (MluI90K) was prepared by banding t equilibrium in CsCl and transfected into COS-1 cells as describe above. Analysis of 35 S-methionine labeled cell extracts an conditioned medium by immunoprecipitation and gel electrophoresis showed that the factor VIII synthesized was similar in size and amount to wildtype factor VIII and was cleaved appropriately by thrombin. The mutation had no effect on factor VIII activity as measured by the Kabi-coatest activity assay (Table II) . Example II: Alteration of the 80 kDa factor VIII cleavage site: Mutagenesis of the 80 kDa cleavage site was performed by preparing a gapped heteroduplex using Cla linearized pMT2-VIII DNA and the EcoRV-Xbal 9.2 kb fragment of pMT2-VIII. The mutagenic oligonucleotide was No. 3 in Table I and the screening oligonucleotide was No. 4 in Table I. The resultant mutant was verified to be correct by DNA sequencing as above. The resultant DNA (MluI80K) was prepared and transfected into COS-1 cells as described above. Analysis of secreted FVIII activity by the Kabi-coatest method and analysis of 35 S-methionine labeled cell extracts and conditioned medium by immunoprecipitation and gel electrophoresis demonstrated the factor VIII was appropriately secreted, cleaved by thrombin, and not altered in its cofactor activity (Table II) . Example III: Mutagenesis of the 73 kDa cleavage site in FVIII The mutagenesis was performed as described in Example II utilizing the 44-mer oligonucleotide No. 5 in Table I and the screening oligonucleotide No. 6, Table I. The resultant DNA (MluI73k) was verified to be correct by DNA sequencing. The resultant DNA was transfected into COS 1 cells as described above. The protein synthesized was similar to wildtype FVIII in both factor VIII activity and expression level (Table II) . Example IV: Mutagenesis of the 94 kDa factor V cleavage site Mutagenesis of the 94 kDA cleavage site of factor V was performed using a gapped heteroduplex prepared with the Nhel/Apal 10.2 kb fragment of pMT2V and Drall 11.8 kb linearized pMT2V. The mutagenic oligonucleotide was a 43-mer No. 27 Table I and the 15-mer screening oligonucleotide No. 28, Table I. The resultan DNA (MluI94K) was confirmed to be correct by DNA sequencing an was transfected into COS-1 cells as described above. Th resultant secreted protein was similar in amount to wildtyp factor V, as determined by factor V clotting assays before an after thrombin activation and by analysis of the synthesis an secretion of factor V by 35 S-methionine labeling of cells an analysis of cell extracts and conditioned medium by SDS-poly acrylamide gel electrophoresis of i munoprecipitated factor V. Example V: Mutagenesis of the 74 kDa factor V cleavage site Mutagenesis was performed as described above by preparin gapped heteroduplexes with the Eco RV-Sphl 9.0 kb fragment o BluKSVΔλ and Sad 9.8 kb linearized BluKSVAλ and using the mutagenic oligonucleotide (No. 29, Table I) and the 15-me screening oligonucleotide (No. 30, Table I) . The resultant DN (MluI74K) was confirmed to be correct by DNA sequencing. Example VI: Preparation of DNA encoding FVIII with a FV B domain A hybrid factor VHI/factor V was assembled utilizing th following approach. The factor VIII expression plasmid MluI90 was digested with Sail and Mlul and the large 7.2 kb fragment wa isolated by electrophoresis on a low melting temperature agaros gel. The factor VIII expression plasmid MluIδOK was digeste with Mlul and Sail and the small 2.3 kb fragment was isolated b electrophoresis on a low melting temperature gel. The factor V domain was assembled from two fragments: 1) plasmid MluI74K wa digested with Mlul and BstEII to yield a 1.1 kb 3' end of th factor V B domain, and 2) plasmid MluI94K was digested with Mlu and BstEII to yield a 1.4 kb 5' end of the factor" V B domain These frage ents were isolated after electrophoresis on a lo melting temperature agarose gel. The hybrid was assembled by a 4-way ligation of the abov fragments in low melting agarose. The hybrid was verified to b properly assembled by extensive restriction endonucleas digestion and gel electrophoresis analysis and by sequencing across the factor V-factor VIII junctions. The resultant DNA, PMT2-VIIIB5, was transfected into COS-1 cells and analyzed as described below. Factor VIII activity was measured by the Kabi-coatest assay method (Table II) . In control experiments, wildtype Factor VIII is expressed and secreted as a two chain molecule. Factor V is secreted as a single chain polypeptide and is expressed at a 10- 40- fold greater level than FVIII. The hybrid exhibited 2-4 fold higher activity than the wildtype FVIII protein, perhaps as a consequence of more efficient secretion from the cell. Analysis of the 35 S-methionine labeled conditioned medium revealed that the hybrid protein was secreted as a single chain polypeptide which migrates with an apparent molecular weight (approximately 300 kDa) slightly less than factor V. The appropriate cleavage products were obtained by thrombin digestion prior to electrophoresis. In addition, the hybrid was immunoprecipitated by a monoclonal antibody specific to the heavy chain of factor VIII and by a monoclonal antibody specific to the B-domain of factor V, suggesting that the protein is properly folded. Example VII: Replacement of the FVIII light chain with the FV light chain Plasmid MluI90K is digested with Sail and EcoRV and the 8.9 kb factor VIII heavy chain encoding fragment isolated. The factor VIII B domain is isolated from MluI80K or MluI73K by digestion with Mlul and EcoRV and the fragments of 1178 and 1054 bp, respectively, are isolated. The 2.0 kb Mlul-Sall factor V light chain encoding fragment is isolated from MluI74K. The fragments are assembled with a 3-way ligation by standard techniques. This will yield a hybrid that contains the acidic region of the light chain of factor VIII if one uses the 1178 bp fragment or a hybrid that lacks the acidic region by using the 1054 bp fragment. Example VIII: Replacement of the heaw chain of FVIII with the FV heav y chain The -9.9 kb Xho -Mlul fragment encoding the B domain & light chain of FVIII is isolated from MluI90K, and the -2.3 kb Sall- Mlul FV heavy chain encoding fragment is isolated from MluI94K. These were ligated together to produce plasmid VIIIHCV, which was transfected into COS-1 cells and analyzed by immune precipitation of 35 S-methionine labeled conditioned medium. The hybrid is secreted as both a single chain polypeptide and as a two chain polypeptide species at -20-50-fold higher levels than wildtype FVIII similarly produced by COS cells. (ND = not determined) Example IX: Replacing the FVIII light chain with the FV light chain Plasmid MluI73K is digested with Mlul and Sail to excise the -10.1 kb FVIII light chain encoding fragment. Plasmid MluI74K is digested with Mlul and Sail to excise the -2045 bp FV light chain which is then isolated and ligated to the MluI/Sall-digested MluI73K plasmid DNA. The resultant plasmid encodes a hybrid protein comprising the FVIII heavy chain, FVIII B domain, FVIII light chain acidic region and the FV light chain. If MluI80K is used in place of MluI80K, a -9.95 kb fragment encoding the FVIII light chain with the acidic region is excised. Accordingly, the hybrid protein produced will lack that acidic region. Example X: Replacing the FVIII light chain and B domain with corresponding elements from FV A set of overlapping synthetic oliognucleotides are prepared, kinased and ligated to produce the following synthetic DNA: CGCGTGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGACTATGAT- ACTTTATTGAGCATGATGAGAAGTCAGTCTAGTTCTCCTTTAACTGATACTA- GATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGAA- CTATGGTATAGTCAACTTTACTTCTTCCTTCTAAAACTGTAAATACTACTCCTACTT- AATCAGAGCA TTAGTCTCGTGCGC That synthetic DNA represents a Mlul cassette encoding the acidic region present between the 80 and 73 kD cleavage sites of FVIII. Alternatively, Mlul sites may be introduced into the FVIII ex- pression vector at the 80 and 73 kD sites using oligonucleotides #3 and #5 from Table I, as separately described in Examples II and III. The corresponding cassette may then simply be excised from the vector with Mlul. The cassette is then ligated to the MluI/BstEII fragment isolated from MluI74K in Example VI, and the resulting Mlul fragment containing the FVIII acidic region- encoding fragment ligated in the correct orientation to the partial FV B domain-encoding fragment is isolated. That fragment is then ligated by the procedure of Example VI to the Sall/Mlul fragment of MluI90K, the MluI/BstEII fragment of MluI94K (both obtained as in Example VI) , and to the Mlul/Sall fragment of MluI74K encoding the FV light chain. The hybrid is verified to be properly assembled as in Example VI. The resultant DNA, pMT2-VIIIB5/LC5 encodes a hybrid protein comprising, in N- terminal to C-terminal order, the FVIII heavy chain with its acidic region, the FV B domain, the acidic region from the FVIII light chain, and the FV light chain. Example XI: Deleting the FVIII B domain and replacing the light chain with the FV light chain Example X is repeated with the following modifications. The Mlul cassette encoding the FVIII light chain acidic region is ligated directly to the Mlul/Sall digested MluI90K FVIII plasmid and the Mlul/Sall fragment of MluI74K which encodes the FV light chain. The properly assembled hybrid cDNA encodes a protein comprising the FVIII heavy chain linked to the FV light chain by the acidic region derived from the FVIII light chain. Example XII: Replacing the A2. B. A3. Cl and C2 FVIII domains A. The Mlul cassette encoding the FVIII light chain acidic region is ligated to the BstEII/MluI FV fragment encoding the C- terminal part of the FV B domain. The correctly oriented BstEII/MluI reaction product encoding the C-terminal part of the FV B domain linked to the FVIII light chain acidic region thus obtained is ligated to the Mlul/Sall fragment from MluI74 which encodes the FV light chain. The correctly oriented BstEII/Sall fragment (fragment "A") is identified and gel purified. B. A FV vector is prepared with a Mlul site introduced just 5" of the region encoding the A2 domain at peptide positions 312-313 using oligonucleotide #41. The MluI/BstEII fragment encoding the FV A2 and part of the FV B domain is excised therefrom and gel purified (fragment "B") . C. A FVIII vector is prepared with a Mlul site introduced just 5' to the FVIII A2 region at peptide position 372 using oligon c- leotide #7. That vector is then digested with Mlul and Sail to excise most of the FVIII coding region leaving only the Al and heavy chain acidic region. The larger Mlul/Sall fragment is then gel purified and ligated to fragments A and B. The correctly oriented resulting vector contains the following coding region: I A-l I-I A-2 I B 1-I A-3 I-I C-l 1-1 C-2 1 j< Fv >I l< F V > I |<FVIII>| I j A FVIII

Claims: What is claimed is: 1. A DNA encoding a protein with procoagulant activity a measured by a standard clotting assay, which DNA is capable o - hybridizing under stringent conditions to a cDNA sequenc encoding human FVIII and to a cDNA sequence encoding human FV, respectively. 2. A DNA of claim 1 which encodes a polypeptide sequence o human " FVIII but containing the B domain of human FV in place o the FVIII B-domain. 3. An expression vector containing a DNA of claim 1. 4. An expression vector containing a DNA of claim 2. 5. A host cell containing and capable of expressing a DNA o claim 1. 6. A host cell containing and capable of expressing a DNA o claim 2. 7. A method for producing a procoagulant protein whic comprises culturing a host cell genetically engineered to contai and be capable of expressing a DNA of claim 1 or 2 under suitabl conditions permitting expression of the DNA and production of th protein so produced. 8. A procoagulant protein encoded by the DNA of claim 1 or 2. 9. A pharmaceutical composition containing a protein of claim in admixture with a pharmaceutically acceptable vehicle. 10. A pharmaceutical composition containing a protein produce by the method of claim 7.

Inventors: KAUFMAN RANDAL J, PITTMAN DEBRA D, KAUFMAN, RANDAL, J., PITTMAN, DEBRA, D.
Applicants: GENETICS INST, GENETICS INSTITUTE, INC.
Application year: 1989
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N5, C12N15, C12P21, C12R1