Novel procoagulant proteins

Details Novel procoagulant proteins Novel procoagulant proteins

1986-04-11

1989-09-19

Teskin, Robin

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Enzymatic production of a protein or polypeptide

435 70, 4351723, 4352401, 4352402, 435320, 435948, 43525233, 530383, 536 27, 514 2, 514 8, C12P 2100, C12P 2102, C12N 1500, C07H 1512

Patent

active

048681121

DESCRIPTION:

BRIEF SUMMARY
This invention relates to a novel series of proteins which exhibit procoagulant properties. These proteins have marked structural differences from human factor VIII:C, but have similar procoagulant activity.
Factor VIII:C is the blood plasma protein that is defective or absent in Hemophilia A disease. This disease is a hereditary bleeding disorder affecting approximately one in 20,000 males. The structure of factor VIII:C is described in U.S. Patent Applications Ser. Nos. 546,650 filed Oct. 28, 1983 and 644,036 filed Aug. 24, 1984, which are incorporated herein by reference and in Nature. 312:306, 307, 326 and 342.
One of the problems presently encountered with the use of human factor VIII:C for treatment of hemophilia arises from its antigenicity. A significant percentage of hemophiliacs have developed an immune reaction to the factor VIII:C used for their treatment. Non-hemophiliacs can also develop or acquire hemophilia when their immune systems become sensitized to factor VIII:C and produce circulating antibodies or "inhibitors" to factor VIII:C. In either case, the effect is the neutralization of whatever factor VIII:C is present in the patient, making treatment very difficult. Until now, the method of choice for treating hemophiliacs with this problem has been to administer, in cases of severe bleeding episodes, non-human factor VIII:C, such as treated porcine factor VIII:C. See Kernoff et al., Blood 63:31 (1984). However, the antibodies which neutralize the clotting ability of human factor VIII:C will react to a varying extent with factor VIII:C of other species, and the porcine protein is itself antigenic, thus both the short-term and long-term effectiveness of such treatment will vary.
Additionally, patients frequently display adverse reactions to infusion with the porcine factor VIII:C. The use of porcine factor VIII:C in spite of the risks has been justified because of the lack of reliably effective alternatives. Kernoff, supra at 38. The present invention provides an alternative to the administration of porcine factor VIII:C.
This invention provides for proteins which have procoagulant activity similar to that of factor VIII:C and also have substantially lower molecular weight. These proteins are schematically depicted by formula (1) as follows: the sequence Ala-20 through Arg-759; B represents a polypeptide sequence substantially duplicative of the sequence Ser-1709 through the C-terminal Tyr-2351; and X represents a polypeptide sequence of up to 949 amino acids substantially duplicative of sequences of amino acids within the sequence Ser-760 through Arg-1708. The amino terminus of region X is covalently bonded through a peptide bond (designated "-" in formula 1) to the carboxy terminus of A. The carboxy terminus of region X is likewise bonded to the amino terminus of B. Numbering of amino acids throughout this disclosure is with reference to the numbering of amino acids in Table 1 in which the first amino acid, Met, of the leader sequence is assigned Number 1. Protein domain X may comprise a continuous but shorter sequence selected from the region Ser-760 through Arg-1708. Alternatively X may comprise two or more amino acid sequences selected from that region which are covalently bonded by a peptide bond (maintaining an ascending numerical order of amino acids).


TABLE 1 5' GAATTCCCCACTGGGTAAGTTCCTTAAAGCTCTGGAAAGAAATTCCGACTTTTCATTAAATCAGAAATT TTACTTTTTTCCCCTCCTGGGACCTAAAGATATTTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACAT TTCTAGCAATAAGTC MET Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe Cys Phe 18 ATG CAA ATA CAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CCA TTC TCC TTT Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr MET 36 ACT CCC ACC AGA AGA TAC TAC CTG GGT CCA CTG GAA CTC TCA TGC GAC TAT ATC Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro 54 CAA AGT GAT CTC GGT CAG CTG CCT GTG CAC GCA AGA TTT CCT CCT AGA CTG CCA Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu 72 AAA TCT TTT CCA TTC AAC ACC TCA CTC GTG TAC

REFERENCES:
Wood et al, Nature, vol. 312, 22 Nov. 1984, pp. 330-336, "Expression of Active Human Factor VIII from Recombinant DNA Clones".
Toole et al, Nature, vol. 312, 22 Nov. 1984, pp. 342-347, "Molecular Cloning of a cDNA Encoding Human Antihaemophilic Factor".
Vehar et al, Nature, vol. 312, 22 Nov. 1984, pp. 337-342, "Structure of Human Factor VIII".
Orr et al, Thrombos Haemostasis, vol. 57(1), p. 57, 1985, "Spacer Function Imp for the Heavily Glycosylated Region of Factor VIII".
Toole et al, Proc. Natl. Acad. Sci., vol. 83, pp. 5939-5942, Aug. 1986, "A Large Region (.apprxeq.95 kDa) of Human Factor VIII is Dispensable for in vitro Procoagulant Activity".
Kaufman et al, Proteases in Biological Control and Biotechnol., J. Gen. Biochem. Supp., 10D 275 (1968).

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