CA1341174C - Procoagulant proteins derived from factor viii: c - Google Patents
Procoagulant proteins derived from factor viii: cInfo
- Publication number
- CA1341174C CA1341174C CA000506408A CA506408A CA1341174C CA 1341174 C CA1341174 C CA 1341174C CA 000506408 A CA000506408 A CA 000506408A CA 506408 A CA506408 A CA 506408A CA 1341174 C CA1341174 C CA 1341174C
- Authority
- CA
- Canada
- Prior art keywords
- ser
- leu
- thr
- pro
- arg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/948—Microorganisms using viruses or cell lines
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/10—Factor VIII, AHF; related peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/30—Signal or leader sequence
Abstract
Novel procoagulant proteins, derived from human Factor VIII: C, are disclosed which comprise the amino acid sequence:
A-X-B
wherein region A represents the polypeptide sequence Ala-20 through Arg-759 substantially as shown in Table l; region B
represents the polypeptide sequence Ser-1709 through Tyr-2351 substantially as shown in Table 1; and region X represents a polypeptide sequence comprising up to 949 amino acids substantially duplicative of sequences of amino acids within the sequence Ser-760 through Arg-1708 of Table 1, wherein the amino terminus of X is covalently bonded through a peptide bond designated "-" to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B.
Methods of making such proteins and their use in pharmaceutical preparations is also disclosed.
A-X-B
wherein region A represents the polypeptide sequence Ala-20 through Arg-759 substantially as shown in Table l; region B
represents the polypeptide sequence Ser-1709 through Tyr-2351 substantially as shown in Table 1; and region X represents a polypeptide sequence comprising up to 949 amino acids substantially duplicative of sequences of amino acids within the sequence Ser-760 through Arg-1708 of Table 1, wherein the amino terminus of X is covalently bonded through a peptide bond designated "-" to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B.
Methods of making such proteins and their use in pharmaceutical preparations is also disclosed.
Description
x Procoagulant Proteins Derived from Factor VIII:C
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 Application Serial No. 546,650 filed October 28, 1983, now U.S. Patent 4,757,006; and U.S. Patent Application 644,036 filed August 24, 1984, corresponding to International Publication WO 85/01961, published May 9, 1985; 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 anti-genicity. 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 neutrali-zation 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 treat-ment will vary.
y1341 174 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, su ra 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 substant-l0 Tally lower molecular weight. These proteins are schematically depicted by formula (1) as follows:
( 1 ) A-X-B
wherein A represents a polypeptide sequence substantially dupli-cative of 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 2p 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 1 ikewise bonded to the amino terminus of 8.
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 3o 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).
BY waY of example, one compound of this invention contains a region X comprising the amino acid sequence of Ser-760 to Pro-. 1341 174 5' GAATTCCCCAGTGGGTAAGTTCCTTAAATGCTCTGCAAAGAAATTGGGACTTTTCATTAAATCAGAAATT
TTACTTTTTTCCCCTCCTGGGAGCTAAAGATATTTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAG
CAATAAGTC
MET Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe Cys Phe 18 ATG CAA ATA GAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CGA TTC TGC TTT
Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr MET 36 AGT GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTC TCA TGG GAC TAT ATG
Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro 54 CAA AGT GAT CTC GGT GAG CTG CCT GTG GAC GCA AGA TTT CCT CCT AGA GTG 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 GTC GTG TAC AAA AAG ACT CTG TTT GTA GAA
Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp MET Gly Leu 90 TTC ACG GTT CAC CTT TTC AAC ATC GCT AAG CCA AGG CCA CCC TGG ATG GGT CTG
Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys 108 CTA GGT CCT ACC ATC CAG GCT GAG GTT TAT GAT ACA GTG GTC ATT ACA CTT AAG
Asn MET Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys 126 AAC ATG GCT TCC CAT CCT GTC AGT CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA
Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 144 GCT TCT GAG GGA GCT GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu 162 GAT AAA GTC TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG
Asn Gly Pro MET Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His 180 AAT GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT CAT
Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys 198 GTG GAC CTG GTA AAA GAC TTG AAT TCA GGG CTC ATT GGA GCC CTA CTA GTA TGT
Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu 216 AGA GAA GGG AGT CTG GCC AAG GAA AAG ACA CAG ACC TTG CAC AAA TTT ATA CTA
Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 234 CTT TTT GCT GTA TTT GAT GAA GGG AAA AGT TGG CAC TCA GAA ACA AAG AAC TCC
Leu MET Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys MET His Thr 252 TTG ATG CAG GAT AGG GAT GCT GCA TCT GCT CGG GCC TGG CCT AAA ATG CAC ACA
Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys 270 GTC AAT GGT TAT GTA AAC AGG TCT CTG CCA GGT CTC ATT GGA TGC CAC AGG AAA
Ser Val Tyr Trp His Val Ile Gly MET Gly Thr Thr Pro Glu Val His Ser Ile 288 TCA GTC TAT TGG CAT GTG ATT GGA ATG GGC ACC ACT CCT GAA GTC CAC TCA ATA
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu 306 TTC CTC GAA GGT CAC ACA TTT CTT GTG AGG AAC CAT CGC CAG GCG TCC TTG GAA
,q, r c '~
...w.r.~.o,~.". .,"..,....... .~,._W-..,.
......._......m...."~..~,~.,.,..~""....""",~..~...n..,.,.".."..,"w""~."",",..~, ~.,.~,.~" ,.., -..
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 Application Serial No. 546,650 filed October 28, 1983, now U.S. Patent 4,757,006; and U.S. Patent Application 644,036 filed August 24, 1984, corresponding to International Publication WO 85/01961, published May 9, 1985; 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 anti-genicity. 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 neutrali-zation 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 treat-ment will vary.
y1341 174 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, su ra 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 substant-l0 Tally lower molecular weight. These proteins are schematically depicted by formula (1) as follows:
( 1 ) A-X-B
wherein A represents a polypeptide sequence substantially dupli-cative of 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 2p 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 1 ikewise bonded to the amino terminus of 8.
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 3o 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).
BY waY of example, one compound of this invention contains a region X comprising the amino acid sequence of Ser-760 to Pro-. 1341 174 5' GAATTCCCCAGTGGGTAAGTTCCTTAAATGCTCTGCAAAGAAATTGGGACTTTTCATTAAATCAGAAATT
TTACTTTTTTCCCCTCCTGGGAGCTAAAGATATTTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACATTTGTAG
CAATAAGTC
MET Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe Cys Phe 18 ATG CAA ATA GAG CTC TCC ACC TGC TTC TTT CTG TGC CTT TTG CGA TTC TGC TTT
Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr MET 36 AGT GCC ACC AGA AGA TAC TAC CTG GGT GCA GTG GAA CTC TCA TGG GAC TAT ATG
Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro Arg Val Pro 54 CAA AGT GAT CTC GGT GAG CTG CCT GTG GAC GCA AGA TTT CCT CCT AGA GTG 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 GTC GTG TAC AAA AAG ACT CTG TTT GTA GAA
Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp MET Gly Leu 90 TTC ACG GTT CAC CTT TTC AAC ATC GCT AAG CCA AGG CCA CCC TGG ATG GGT CTG
Leu Gly Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys 108 CTA GGT CCT ACC ATC CAG GCT GAG GTT TAT GAT ACA GTG GTC ATT ACA CTT AAG
Asn MET Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys 126 AAC ATG GCT TCC CAT CCT GTC AGT CTT CAT GCT GTT GGT GTA TCC TAC TGG AAA
Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 144 GCT TCT GAG GGA GCT GAA TAT GAT GAT CAG ACC AGT CAA AGG GAG AAA GAA GAT
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu 162 GAT AAA GTC TTC CCT GGT GGA AGC CAT ACA TAT GTC TGG CAG GTC CTG AAA GAG
Asn Gly Pro MET Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser His 180 AAT GGT CCA ATG GCC TCT GAC CCA CTG TGC CTT ACC TAC TCA TAT CTT TCT CAT
Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu Leu Val Cys 198 GTG GAC CTG GTA AAA GAC TTG AAT TCA GGG CTC ATT GGA GCC CTA CTA GTA TGT
Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu 216 AGA GAA GGG AGT CTG GCC AAG GAA AAG ACA CAG ACC TTG CAC AAA TTT ATA CTA
Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser 234 CTT TTT GCT GTA TTT GAT GAA GGG AAA AGT TGG CAC TCA GAA ACA AAG AAC TCC
Leu MET Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys MET His Thr 252 TTG ATG CAG GAT AGG GAT GCT GCA TCT GCT CGG GCC TGG CCT AAA ATG CAC ACA
Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys 270 GTC AAT GGT TAT GTA AAC AGG TCT CTG CCA GGT CTC ATT GGA TGC CAC AGG AAA
Ser Val Tyr Trp His Val Ile Gly MET Gly Thr Thr Pro Glu Val His Ser Ile 288 TCA GTC TAT TGG CAT GTG ATT GGA ATG GGC ACC ACT CCT GAA GTC CAC TCA ATA
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu 306 TTC CTC GAA GGT CAC ACA TTT CTT GTG AGG AAC CAT CGC CAG GCG TCC TTG GAA
,q, r c '~
...w.r.~.o,~.". .,"..,....... .~,._W-..,.
......._......m...."~..~,~.,.,..~""....""",~..~...n..,.,.".."..,"w""~."",",..~, ~.,.~,.~" ,.., -..
TABLE 1, continued Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu MET Asp Leu Gly Gln 324 ATC TCG CCA ATA ACT TTC CTT ACT GCT CAA ACA CTC TTG ATG GAC CTT GGA CAG
Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly MET Glu Ala Tyr 342 TTT CTA CTG TTT TGT CAT ATC TCT TCC CAC CAA CAT GAT GGC ATG GAA GCT TAT
Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg MET Lys Asn Asn Glu 360 GTC AAA GTA GAC AGC TGT CCA GAG GAA CCC CAA CTA CGA ATG AAA AAT AAT GAA
Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu MET Asp Val Val Arg 378 GAA GCG GAA GAC TAT GAT GAT GAT CTT ACT GAT TCT GAA ATG GAT GTG GTC AGG
Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys 396 TTT GAT GAT GAC AAC TCT CCT TCC TTT ATC CAA ATT CGC TCA GTT GCC AAG AAG
His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr 414 CAT CCT AAA ACT TGG GTA CAT TAC ATT GCT GCT GAA GAG GAG GAC TGG GAC TAT
Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 432 GCT CCC TTA GTC CTC GCC CCC GAT GAC AGA AGT TAT AAA AGT CAA TAT TTG AAC
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe MET Ala Tyr 450 AAT GGG CCT CAG CGG ATT GGT AGG AAG TAC AAA AAA GTC CGA TTT ATG GCA TAC
Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu 468 ACA GAT GAA ACC TTT AAG ACT CGT GAA GCT ATT CAG CAT GAA TCA GGA ATC TTG
Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn 486 GGA CCT TTA CTT TAT GGG GAA GTT GGA GAC ACA CTG TTG ATT ATA TTT AAG AAT
Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro 504 CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT CAC GGA ATC ACT GAT GTC CGT CCT
Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 522 TTG TAT TCA AGG AGA TTA CCA AAA GGT GTA AAA CAT TTG AAG GAT TTT CCA ATT
Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro 540 CTG CCA GGA GAA ATA TTC AAA TAT AAA TGG ACA GTG ACT GTA GAA GAT GGG CCA
Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn MET 558 ACT AAA TCA GAT CCT CGG TGC CTG ACC CGC TAT TAC TCT AGT TTC GTT AAT ATG
Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 576 GAG AGA GAT CTA GCT TCA GGA CTC ATT GGG CCT CTC CTC ATC TGC TAC AAA GAA
Ser Val Asp Gln Arg Gly Asn Gln Ile MET Ser Asp Lys Arg Asn Val Ile Leu 594 TCT GTA GAT CAA AGA GGA AAC CAG ATA ATG TCA GAC AAG AGG AAT GTC ATC CTG
Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg 612 TTT TCT GTA TTT GAT GAG AAC CGA AGC TGG TAC CTC ACA GAG AAT ATA CAA CGC
Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser 630 TTT CTC CCC AAT CCA GCT GGA GTG CAG CTT GAG GAT CCA GAG TTC CAA GCC TCC
Asn Ile MET His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val 648 AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT TTT GAT AGT TTG CAG TTG TCA GTT
X
TABLE 1, continued Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 666 TGT TTG CAT GAG GTG GCA TAC TGG TAC ATT CTA AGC ATT GGA GCA CAG ACT GAC
Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys MET Val Tyr Glu 684 TTC CTT TCT GTC TTC TTC TCT GGA TAT ACC TTC AAA CAC AAA ATG GTC TAT GAA
Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe MET Ser MET Glu 702 GAC ACA CTG ACC CTA TTC CCA TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA
Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 720 AAC CCA GGT CTA TGG ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC
MET Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr 738 ATG ACC GCC TTA CTG AAG GTT TCT AGT TGT GAC AAG AAC ACT GGT GAT TAT TAC
Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile 756 GAG GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA AAC AAT GCC ATT
Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln 774 GAA CCA AGA AGC TTC TCC CAG AAT TCA AGA CAC CCT AGC ACT AGG CAA AAG CAA
Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp Phe 792 TTT AAT GCC ACC ACA ATT CCA GAA AAT GAC ATA GAG AAG ACT GAC CCT TGG TTT
Ala His Arg Thr Pro MET Pro Lys Ile Gln Asn Val Ser Ser Ser Asp Leu Leu 810 GCA CAC AGA ACA CCT ATG CCT AAA ATA CAA AAT GTC TCC TCT AGT GAT TTG TTG
MET Leu Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln 828 ATG CTC TTG CGA CAG AGT CCT ACT CCA CAT GGG CTA TCC TTA TCT GAT CTC CAA
Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser 846 GAA GCC AAA TAT GAG ACT TTT TCT GAT GAT CCA TCA CCT GGA GCA ATA GAC AGT
Asn Asn Ser Leu Ser Glu MET Thr His Phe Arg Pro Gln Leu His His Ser Gly 864 AAT AAC AGC CTG TCT GAA ATG ACA CAC TTC AGG CCA CAG CTC CAT CAC AGT GGG
Asp MET Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu 882 GAC ATG GTA TTT ACC CCT GAG TCA GGC CTC CAA TTA AGA TTA AAT GAG AAA CTG
Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser Thr 900 GGG ACA ACT GCA GCA ACA GAG TTG AAG AAA CTT GAT TTC AAA GTT TCT AGT ACA
Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp 918 TCA AAT AAT CTG ATT TCA ACA ATT CCA TCA GAC AAT TTG GCA GCA GGT ACT GAT
Asn Thr Ser Ser Leu Gly Pro Pro Ser MET Pro Val His Tyr Asp Ser Gln Leu 936 AAT ACA AGT TCG TTA GGA CCC CCA AGT ATG CCA GTT CAT TAT GAT AGT CAA TTA
Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro 954 GAT ACC ACT CTA TTT GGC AAA AAG TCA TCT CCC CTT ACT GAG TCT GGT GGA CCT
Leu Ser Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu MET 972 CTG AGC TTG AGT GAA GAA AAT AAT GAT TCA AAG TTG TTA GAA TCA GGT TTA ATG
Asn Ser Gln Glu Ser 5er Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg 990 AAT AGC CAA GAA AGT TCA TGG GGA AAA AAT GTA TCG TCA ACA GAG AGT GGT AGG
X
...
1341 1 ~4 TABLE 1, continued Leu PheLysGly LysArg HisGlyPro AlaLeuLeuThr LysAspAsn Ala1,008 Ala TTA TTTAAAGGG AAAAGAGCT CATGGACCT GCTTTGTTGACT AAAGATAAT GCC
Leu PheLysVal SerIleSer LeuLeuLys ThrAsnLysThr SerAsnAsn Ser1,026 TTA TTCAAAGTT AGCATCTCT TTGTTAAAG ACAAACAAAACT TCCAATAAT TCA
Ala ThrAsnArg LysThrHis IleAspGly ProSerLeuLeu IleGluAsn Ser1,044 GCA ACTAATAGA AAGACTCAC ATTGATGGG CCATCATTATTA ATTGAGAAT AGT
Pro SerValTrp GlnAsnIle LeuGluSer AspThrGluPhe LysLysVal Thr1,062 CCA TCAGTCTGG CAAAATATA TTAGAAAGT GACACTGAGTTT AAAAAAGTG ACA
Pro LeuIleHis AspArgMET LeuMETAsp LysAsnAlaThr AlaLeuArg Leu1,080 CCT TTGATTCAT GACAGAATG CTTATGGAC AAAAATGCTACA GCTTTGAGG CTA
Asn HisMETSer AsnLysThr ThrSerSer LysAsnMETGlu METValGln Gln1,098 AAT CATATGTCA AATAAAACT ACTTCATCA AAAAACATGGAA ATGGTCCAA CAG
Lys LysGluGly ProIlePro ProAspAla GlnAsnProAsp METSerPhe Phe1,116 AAA AAAGAGGGG CCCATTCCA CCAGATGCA CAAAATCCAGAT ATGTCGTTC TTT
Lys METLeuPhe LeuProGlu SerAlaArg TrpIleGlnArg ThrHisGly Lys1,134 AAG ATGCTATTC TTGCCAGAA TCAGCAAGG TGGATACAAAGG ACTCATGGA AAG
Asn SerLeuAsn SerGlyGln GlyProSer ProLysGlnLeu ValSerLeu Gly1,152 AAC TCTCTGAAC TCTGGGCAA GGCCCCAGT CCAAAGCAATTA GTATCCTTA GGA
Pro GluLysSer ValGluGly GlnAsnPhe LeuSerGluLys AsnLysVal Val1,170 CCA GAAAAATCT GTGGAAGGT CAGAATTTC TTGTCTGAGAAA AACAAAGTG GTA
Val GlyLysGly GluPheThr LysAspVal GlyLeuLysGlu METValPhe Pro1,188 GTA GGAAAGGGT GAATTTACA AAGGACGTA GGACTCAAAGAG ATGGTTTTT CCA
Ser SerArgAsn LeuPheLeu ThrAsnLeu AspAsnLeuHis GluAsnAsn Thr1,206 AGC AGCAGAAAC CTATTTCTT ACTAACTTG GATAATTTACAT GAAAATAAT ACA
His AsnGlnGlu LysLysIle GlnGluGlu IleGluLysLys GluThrLeu Ile1,224 CAC AATCAAGAA AAAAAAATT CAGGAAGAA ATAGAAAAGAAG GAAACATTA ATC
Gln GluAsnVal ValLeuPro GlnIleHis ThrValThrGly ThrLysAsn Phe1,242 CAA GAGAATGTA GTTTTGCCT CAGATACAT ACAGTGACTGGC ACTAAGAAT TTC
MET Lys Asn Leu Phe Leu Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Glu 1,260 ATG AAG AAC CTT TTC TTA CTG AGC ACT AGG CAA AAT GTA GAA GGT TCA TAT GAG
Gly Ala Tyr Ala Pro Leu Val Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn 1,278 GGG GCA TAT GCT CCA GTA CTT CAA GAT TTT AGG TCA TTA AAT GAT TCA ACA AAT
Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu 1,296 AGA ACA AAG AAA CAC ACA GCT CAT TTC TCA AAA AAA GGG GAG GAA GAA AAC TTG
Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr 1,314 GAA GGC TTG GGA AAT CAA ACC AAG CAA ATT GTA GAG AAA TAT GCA TGC ACC ACA
Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys Arg 1,332 AGG ATA TCT CCT AAT ACA AGC CAG CAG AAT TTT GTC ACG CAA CGT AGT AAG AGA
...
TABLE l, continued '7 Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile 1,350 GCT TTG AAA CAA TTC AGA CTC CCA CTA GAA GAA ACA GAA CTT GAA AAA AGG ATA
Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn MET Lys His Leu Thr Pro 1,368 ATT GTG GAT GAC ACC TCA ACC CAG TGG TCC AAA AAC ATG AAA CAT TTG ACC CCG
Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln 1,386 AGC ACC CTC ACA CAG ATA GAC TAC AAT GAG AAG GAG AAA GGG GCC ATT ACT CAG
Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala Asn Arg 1,404 TCT CCC TTA TCA GAT TGC CTT ACG AGG AGT CAT AGC ATC CCT CAA GCA AAT AGA
Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr 1,422 TCT CCA TTA CCC ATT GCA AAG GTA TCA TCA TTT CCA TCT ATT AGA CCT ATA TAT
Leu Thr Arg Val Leu Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr 1,440 CTG ACC AGG GTC CTA TTC CAA GAC AAC TCT TCT CAT CTT CCA GCA GCA TCT TAT
Arg Lys Lys Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys 1,458 AGA AAG AAA GAT TCT GGG GTC CAA GAA AGC AGT CAT TTC TTA CAA GGA GCC AAA
Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu MET Thr Gly Asp Gln Arg 1,476 AAA AAT AAC CTT TCT TTA GCC ATT CTA ACC TTG GAG ATG ACT GGT GAT CAA AGA
Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys Lys Val 1,494 GAG GTT GGC TCC CTG GGG ACA AGT GCC ACA AAT TCA GTC ACA TAC AAG AAA GTT
Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr Ser Gly Lys Val Glu 1,512 GAG AAC ACT GTT CTC CCG AAA CCA GAC TTG CCC AAA ACA TCT GGC AAA GTT GAA
Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser 1,530 TTG CTT CCA AAA GTT CAC ATT TAT CAG AAG GAC CTA TTC CCT ACG GAA ACT AGC
Asn Gly Ser Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr 1,548 AAT GGG TCT CCT GGC CAT CTG GAT CTC GTG GAA GGG AGC CTT CTT CAG GGA ACA
Glu Gly Ala Ile Lys Trp Asn Glu Pro Asn Arg Pro Gly Lys Val Pro Phe Leu 1,566 GAG GGA GCG ATT AAG TGG AAT GAA CCA AAC AGA CCT GGA AAA GTT CCC TTT CTG
Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu 1,584 ATA GTA GCA ACA GAA AGC TCT GCA AAG ACT CCC TCC AAG CTA TTG GAT CCT CTT
Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln 1,602 GCT TGG GAT AAC CAC TAT GGT ACT CAG ATA CCA AAA GAA GAG TGG AAA TCC CAA
Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu 1,520 GAG AAG TCA CCA GAA AAA ACA GCT TTT AAG AAA AAG GAT ACC ATT TTG TCC CTC
Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys 1,638 AAC GCT TGT GAA AGC AAT CAT GCA ATA GCA GCA ATA AAT GAG GGA CAA AAT AAG
Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser 1,656 CCC GAA ATA GAA GTC ACC TGG GCA AAG CAA GGT AGG ACT GAA AGG CTC TGC TCT
Gln Asn Pro Pro Val Leu Lys Arg His Arg Pro Glu Ile Thr Arg Thr Thr Leu 1,674 CAA AAC CCA CCA GTC TTG AAA CGC CAT CAA CGC GAA ATA ACT CGT ACT ACT CTT
iX
1341 174.
TABLE 1, continued g Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu MET Lys 1,692 CAG TCA GAT CAA GAG GAA ATT GAC TAT GAT GAT ACC ATA TCA GTT GAA ATG AAG
Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 1,710 AAG GAA GAT TTT GAC ATT TAT GAT GAG GAT GAA AAT CAG AGC CCC CGC AGC TTT
Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1,728 CAA AAG AAA ACA CGA CAC TAT TTT ATT GCT GCA GTG GAG AGG CTC TGG GAT TAT
Gly MET Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val 1,746 GGG ATG AGT AGC TCC CCA CAT GTT CTA AGA AAC AGG GCT CAG AGT GGC AGT GTC
Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln 1,764 CCT CAG TTC AAG AAA GTT GTT TTC CAG GAA TTT ACT GAT GGC TCC TTT ACT CAG
Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1,782 CCC TTA TAC CGT GGA GAA CTA AAT GAA CAT TTG GGA CTC CTG GGG CCA TAT ATA
Arg Ala Glu Val Glu Asp Asn Ile MET Val Thr Phe Arg Asn Gln Ala Ser Arg 1,800 AGA GCA GAA GTT GAA GAT AAT ATC ATG GTA ACT TTC AGA AAT CAG GCC TCT CGT
Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1,818 CCC TAT TCC TTC TAT TCT AGC CTT ATT TCT TAT GAG GAA GAT CAG AGG CAA GGA
Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp 1,836 GCA GAA CCT AGA AAA AAC TTT GTC AAG CCT AAT GAA ACC AAA ACT TAC TTT TGG
Lys Val Gln His His MET Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 1,854 AAA GTG CAA CAT CAT ATG GCA CCC ACT AAA GAT GAG TTT GAC TGC AAA GCC TGG
Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1,872 GCT TAT TTC TCT GAT GTT GAC CTG GAA AAA GAT GTG CAC TCA GGC CTG ATT GGA
Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val 1,890 CCC CTT CTG GTG TGC CAC ACT AAC ACA CTG AAC CCT GCT CAT GGG AGA CAA GTG
Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1,908 ACA GTA CAG GAA TTT GCT CTG TTT TTC ACC ATC TTT GAT GAG ACC AAA AGC TGG
Tyr Phe Thr Glu Asn MET Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln MET 1,926 TAC TTC ACT GAA AAT ATG GAA AGA AAC TGC AGG GCT CCC TGC AAT ATC CAG ATG
Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile 1,944 GAA GAT CCC ACT TTT AAA GAG AAT TAT CGC TTC CAT GCA ATC AAT GGG TAC ATA
MET Asp Thr Leu Pro Gly Leu Val MET Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1,962 ATG GAT ACA CTA CCT GGC TTA GTA ATG GCT CAG GAT CAA AGG ATT CGA TGG TAT
Leu Leu Ser MET Gly Ser Asn Glu Asn Ili His Ser Ile His Phe Ser Gly His 1,980 CTG CTC AGG ATG GGC AGG AAT GAA AAC ATC CAT TCT ATT CAT TTC AGT GGA CAT
Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys MET Ala Leu Tyr Asn Leu Tyr 1,998 GTG TTC ACT GTA CGA AAA AAA GAG GAG TAT AAA ATG GCA CTG TAC AAT CTC TAT
Pro Gly Val Phe Glu Thr Val Glu MET Leu Pro Ser Lys Ala Gly Ile Trp Arg 2,016 CCA GGT GTT TTT GAG ACA GTG GAA ATG TTA CCA TCC AAA GCT GGA ATT TGG CGG
TABLE 1, continued 9 Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly MET Ser Thr Leu Phe Leu 2,034 GTG GAA TGC CTT ATT GGC GAG CAT CTA CAT GCT GGG ATG AGC ACA CTT TTT CTG
Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly MET Ala Ser Gly His Ile Arg 2,052 GTG TAC AGC AAT AAG TGT CAG ACT CCC CTG GGA ATG GCT TCT GGA CAC ATT AGA
Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala 2,070 GAT TTT CAG ATT ACA GCT TCA GGA CAA TAT GGA CAG TGG GCC CCA AAG CTG GCC
Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 2,088 AGA CTT CAT TAT TCC GGA TCA ATC AAT GCC TGG AGC ACC AAG GAG CCC TTT TCT
Trp Ile Lys Val Asp Leu Leu Ala Pro MET Ile Ile His Gly Ile Lys Thr Gln 2,106 TGG ATC AAG GTG GAT CTG TTG GCA CCA ATG ATT ATT CAC GGC ATC AAG ACC CAG
Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile MET Tyr 2,124 GGT GCC CGT CAG AAG TTC TCC AGC CTC TAC ATC TCT CAG TTT ATC ATC ATG TAT
Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 2,142 AGT CTT GAT GGG AAG AAG TGG CAG ACT TAT CGA GGA AAT TCC ACT GGA ACC TTA
MET Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn 2,160 ATG GTC TTC TTT GGC AAT GTG GAT TCA TCT GGG ATA AAA CAC AAT ATT TTT AAC
Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 2,178 CCT CCA ATT ATT GCT CGA TAC ATC CGT TTG CAC CCA ACT CAT TAT AGC ATT CGC
Ser Thr Leu Arg MET Glu Leu MET Gly Cys Asp Leu Asn Ser Cys Ser MET Pro 2,196 AGC ACT CTT CGC ATG GAG TTG ATG GGC TGT GAT TTA AAT AGT TGC AGC ATG CCA
Leu Gly MET Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr 2,214 TTG GGA ATG GAG AGT AAA GCA ATA TCA GAT GCA CAG ATT ACT GCT TCA TCC TAC
Phe Thr Asn MET Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 2,232 TTT ACC AAT ATG TTT GCC ACC TGG TCT CCT TCA AAA GCT CGA CTT CAC CTC CAA
Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln 2,250 GGG AGG AGT AAT GCC TGG AGA CCT CAG GTC AAT AAT CCA AAA GAG TGG CTC CAA
Val Asp Phe Gln Lys Thr MET Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 2,268 GTG GAC TTC CAG AAG ACA ATG AAA GTC ACA GGA GTA ACT ACT CAG GGA GTA AAA
Ser Leu Leu Thr Ser MET Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 2,286 TCT CTG CTT ACC AGC ATG TAT GTG AAG GAG TTC CTC ATC TCC AGC AGT CAA GAT
Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly 2,304 GGC CAT CAG TGG ACT CTC TTT TTT CAG AAT GGC AAA GTA AAG GTT TTT CAG GGA
Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 2,322 AAT CAA GAC TCC TTC ACA CCT GTG GTG AAC TCT CTA GAC CCA CCG TTA CTG ACT
Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg MET 2,340 CGC TAC CTT CGA ATT CAC CCC CAG AGT TGG GTG CAC CAG ATT GCC CTG AGG ATG
Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr End 2,352 GAG GTT CTG GGC TGC GAG GCA CAG GAC CTC TAC TGA GGGTGGCCACTGCATCCCACCTGCCACTG
CCGTCACCTCTCCCTCCTCAGCTCCAGGGCATGTGTCCCTCCCTGGCTTGCTTCTACCTTTGTGCTAAATCCTAGCAGA
CACTCCCTTG
AAGCCTCCTGAATTAACTATCATCACTCCTGCATTTCTTTGGTGGGGGGCCAGGAGGCTGCATCCATTTTAACTTAACT
CTTACCTATT
TTCTGCAGCTGCTCCCAGA
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to 1000 followed by the amino acid sequence of Asp-1582 to Arg--1708. That compound thus comprises the polypeptide sequence of Ala-20 to Pro-1000 covalently linked by a peptide bond to amino acids Asp-1582 to Tyr-2351. Another exemplary compound contains a region X comprising the amino acid sequence Ser-760 to Thr-778 followed by the sequence Pro-1659 to Arg-1708.
That compound thus comprises the polypeptide sequence Ala-20 to Thr-778 covalently linked by a peptide bond to the sequence Pro-1659 through Tyr-2351. Still another exemplary compound, contains a region X comprising the amino acid sequence Ser-760 to Thr-778 followed by the sequence Glu-1694 to Arg-1708.
That compound thus comprises the polypeptide sequence Ala-20 to Thr-778 covalently linked by a peptide bond to amino acids Glu-1694 through Tyr-2351.
These exemplary compounds are depicted schematically in Table 2.
The amino acid sequence represented by X should be selected so that it does not substantially reduce the procoagulant 2p activity of the molecule, which activity can be conveniently assayed by conventional methods. Compound (2) of Table 2 is a presently preferred embodiment.
The procoagulant protein may be produced by appropriate host cells transformed by factor VIII:C DNA which has been specific-ally altered by use of any of a variety of site-specific muta-genesis techniques which will be familiar to those of ordinary skill in the art of recombinant DNA.
The starting materials may be a DNA sequence which codes for the complete factor VIII:C molecule, e.g., the complete human factor VIII:C as shown in Table 1, a truncated version of that sequence, or it may comprise segments of that DNA sequence, so long as the starting materials contain at least sufficient DNA
to code for the amino acid sequences of the desired polypeptide.
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rd The procoagulent proteins of the present invention, in addition to lacking a substantial amino acid segment of human factor VIII: C, also have fewer potential N-glycosylation sites than human factor VIII. Preferably, at least one N-glycosylation site has been deleted. More preferably, 18 of the 25 potential N-glycosylation sites are not in the molecule. In still more preferred embodiments, up to 19 of the 25 potential N-glycos-ylation sites are removed. While not wishing to be bound by theory, it is presently believed that the antibodies to factor VIII:C which are directed to antigenic determinants contained in the protein segment deleted in accordance with this inven-tion, i.e., in the amino acid segement itself or in the carbo-hydrate portion of the glycosylated protein, will not neutralize the procoagulant proteins of the present invention. Moreover, the fact that the procoagulants of the present invention lack many of the sites for non-human glycosylation by the non-human mammalian or other cells used to produce the proteins is also belived to reduce the antigenicity of that protein, and lessen the likelihood of developing antibodies to the procoagulants.
This may enable facilitating the treatment of patients in need of procoagulant therapy.
I contemplate that my compounds can be produced by recombinant DNA techniques at a much lower cost than is possible for pro-duction of human factor VIII. The host organisms should more - efficiently process and express the substantially simpler molecules of this invention.
The compounds of this invention can be formulated into pharmaceu-tically acceptable preparations with parenterally acceptable vehicles and excipients in accordance with procedures known in the art.
The pharmaceutical preparations of this invention, suitable for parenteral administration, may conveniently comprise a sterile lyophilized preparation of the protein which may be reconsti-tuted by addition of sterile solution to produce solutions preferably isotonic with the blood of the recipient. The prepar-ation may be presented in unit or multi-dose containers, e.g. in sealed ampoules or vials. Their use would be analogous to that of human factor VIII, appropriately adjusted for potency.
One method by which these proteins can be expressed is by use of DNA which is prepared by cutting a full-length factor VIII:C DNA with the appropriate restriction enzymes to remove a portion of the DNA sequence that codes for amino acids 760 to 1708 of human factor VIII: C. The cut DNA is then ligated with an oligonucleotide that resects the cut DNA and maintains the correct translational reading frame.
Preparation of the cDNA has been set forth in detail in U.S.
Patent Applications Serial Nos. 546,650 and 644,036, supra. A
pSP64 recombinant clone containing the nucleotide sequence depicted in Table 1, designated as pSP64-VIII, is on deposit at the American Type Culture Collection under Accession Number ATCC 39812.
Restriction endonucleases are used to obtain cleavage of the human factor VIII:C cDNA, hereinafter the DNA source sequence, at appropriate sites in the nucleotide sequence. Unless otherwise noted, restriction endonucleases are utilized under the conditions and in the manner recommended by their commercial suppliers. The restriction endonucleases selected herein are those which will enable one to excise with substantial speci-ficity sequences that code for the portion of the factor VIII:C molecule desired to be excised. BamHI and SacI are particularly useful endonucleases. However, the skilled artisan will be able to utilize other restriction endonucleases chosen by conventional selection methods. The number of nucleotides deleted may vary but care should be taken to insure that the reading frame of the ultimate cDNA sequence will not be affected.
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The resulting DNA fragments are then purified using conventional techniques such as those set forth in Maniatis et al. , Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory 1982), and Proc. Natl. Acad. Sci. 76:615-619 (1979). The purified DNA is then ligated to form the sequence encoding the polypeptide of the preferred invention. When necessary or desirable, the ligation may be within an oligonucleotide that resects the cut DNA and maintains the correct translational reading frame using standard ligation conditions. Ligation reactions are carried on as described by Maniatis et al., supra at 2453-6 using the buffer described at page 246 thereof and using a DNA
concentration of 1-100 ug/ml, at a temperature of 23°C for blunt ended DNA and 16°C for "sticky ended" DNA. The following double-stranded oligonucleotide is useful when there is BamHI/-SacI deletion such as described infra, 5' P-CATGGACCG-3' 3-TCGAGTACCTGGCCTAG 5';
but other oligonucleotides can be selected by the skilled artisan depending upon the deletions made and reaction conditions.
The DNA sequences encoding the novel procoagulant polypeptides - can, in addition to other methods, be derived from the sequence of human factor VIII : C DNA by application of oligonucleotide-med-iated deletion mutagenesis, often referred to as "loopout" muta-genesis, as described for example in Morinaga, Y. et al. Biotech-noloQV,, 2: 636-639 (1984).
The new DNA sequences containing the various deletions can then be introduced into appropriate vectors for expression 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.
-1341 17~
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 5 may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol. Biol., 159: 51-521 (1982); Kaufman, Proc. Natl. Acad. Sci. 82: 689-693 (1985).
Established cell lines, including transformed cell lines, are 10 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 haematopoeitic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so 15 long 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 chromosomal DNA, and for subsequent amplification of the integrated vector DNA, CHO (Chinese hamster ovary) cells are presently preferred.
See U.S. Patent 4,399,216. Alternatively, the vector DNA
could include all or parts of the bovine papilloma virus genome (Lusky et al., Cell, 36: 391-401 (9184) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other 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 hamster cells lines and the like.
Stable transformants then are screened for expression of the procoagulant product by standard immunological or enzymatic 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 enzymatic or immunologic assay of the proteins in the culture medium.
The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention, as described in the claims.
1341 1'74 ug. of the plasmid PACE, a pSP64 (Promega Biotec, Madison, Wis.) derivative, containing nucleotides 562-7269 of human factor VIII:C cDNA (nucleotide 1 is the A of the ATG initiator meth-s ionine codon) was subj ected to partial BamHI digestion in 100u1 containing 50mM Tris.HCl ph 8.0, 50mM MgCl2, and 2.4 units BamHI (New England Biolabs) for 30 minutes at 37°C. The reaction was terminated by the addition of EDTA to 20mM and then extracted once with phenol, once with chloroform , ethanol 10 precipitated and pelleted by centrifugation. DNA was redis-solved, cleaved to completion in 50u1 using 40 units SacI for 1.5 hours at 37°C. DNA was then electrophoresed through a buffered 0.6% agarose gel. An 8.1 kb fragment corresponding to the partial BamHI-SacI fragment of PACE lacking only the sequence corresponding to nucleotides 2992-4774 of the factor VIII:C sequence was purified from the gel using the glass powder technique described in Proc. Nat. Acad. Sci. 76; 615-619 (1979) . Purified DNA was ligated with 100 pmoles of the following double-stranded oligonucleotide 5'P-CATGGACCG-3' 3'-TCGAGTACCTGGCCTAG 5' using standard ligation conditions. The DNA sequence removed represents the deletion of 584 amino acid sequence beginning - 25 with amino acid 998 and continuing through 1581. The oligo-nucleotide inserted, however, encodes amino acids corresponding to 998-1000. Therefore, the polypeptide encoded contains deletion of 581 amino acids.
DNA was then used to transform competent E, coli bacteria, and DNA from several ampicillin resistant transformants was analyzed by restriction mapping to identify a plasmid harboring the desired SacI-BamHI deletion mutant. DNA from this plasmid was digested to completion with KpnI, which cleaves the plasmid uniquely at nucleotide 1816 of the factor VIII:C coding se-quence. This DNA was ligated with a KpnI DNA fragment containing nucleotides 1-1815 of factor VIII:C DNA and a synthetic SalI
site at nucleotides -11 to -5 and then used to transform competent E. coli bacteria.
Plasmid DNA was isolated and oriented by restriction mapping to identify a plasmid, pBSdK, containing the correct 5' to 3' orien-tation of the KpnI insert. SalI digestion, which excises the entire polypeptide coding region from the plasmid,. was performed l0 and the DNA electrophoresed through a buffered 0.6% agarose gel.
The 5.3Kb SalI fragment was purified from the gel as described above. This DNA fragment was ligated with XhoI cut pXMT2 DNA
to give rise to plasmid pDGR-2. pXMT2 is a plasmid capable of expressing heterologous genes when introduced into mammalian cells such as the COS-1 African Green Monkey kidney cell line, and is a derivative of the expression vectors described in Kaufman, supra at 689-93. The expression elements are the same as described for plasmid pQ2 except that it contains a deletion of the adenovirus major late promoter extending from 2p -45 to +156 with respect to the transcription start site of the adenovirus major late promoter. mRNA expression in pXMT
is driven by the SV40 late promoter. The bacterial replicon, however, has been substituted to render bacteria containing the vector resistant to ampicillin rather than tetracycline.
pXMT2 contains a unique Xho I site at a position which allows for expression of inserted cDNA from the SV40 late promoter.
This Xho I site is convenient for inserting factor VIII:C cDNA
constructs since these are flanked by SalI sites.
3o Restriction mapping of transformants identified a plasmid, pDGR-2, containing the correct 5' to 3' orientation of the polypeptide coding sequence relative to the direction of transcription from the SV40 late promoter. pDGR-2 is on deposit at the American Type Culture Collection under Accession number 53100.
Other novel procoagulant proteins may be obtained from constructs produced by oligonucleotide mediated deletion mutagenesis, using for example the "loopout" mutagenesis techniques as described in Morinaga et al. , supra. The deletion mutagenesis is performed using expression plasmid pDGR-2 or any other appropriate plasmid or bacteriophage vector. Other methods for oligonucleotide mediated mutagenesis employing single stranded DNA produced with M13 vectors and the like are also suitable. See Zoller et al., Nucl. Acids Res. 10: 6487-6500 (1982). For example, these deletions can be produced using the oligonucleotides (A) 5' AAAAGCAATTTAATGCCACCCCACCAGTCTTGAAACGCCA
(8) 5' AAAAGCAATTTAATGCCACCGAAGATTTTGACATTTATGA
to cause deletions in factor VIII:C cDNA from nucleotides (A)' 2334 to 4974 or (B) 2334 to 5079. The proteins encoded by these constructs contain deletions of (A) 880 and (B) 915 amino acids relative to Factor VIII: C.
The deleted constructs are tested directly, or after subcloning into appropriate expression vectors, in order to determine if the novel proteins possess procoagulant activity. Procoagulant activity was assayed as described in Examples 3 and 4.
Expression of Procoagulant Molecules in COS Monkey Cells The expression plasmids containing the modified cDNA's prepared as in Examples 1 or 2 and the full-length cDNA, pXMT-VIII, were introduced into COS-1 cells via the DEAE-dextran trans-fection protocol. Sompayrac and Dana 1981, Proc. Natl. Acad.
Sci. 78: 7575-7578. Conditioned media was harvested 48 hours post-transfection and assayed for factor VIII-type activity as described in Toole et. al., 1984, Nature 312:342-347. The results of the experiment are summarized in Table 3. Both plasmids containing the modified cDNAs yielded procoagulant activity and, moreover, the activity was greater than that obtained using wild type cDNA. From these data it was concluded 5 that removal of up to 880 amino acids (95,000 daltons) in a defined domain of human factor VIII does not destroy cofactor activity. Furthermore, these abridged procoagulant proteins retain their ability to be activated by thrombin.
1341 17~r TABLE 3: EXPRESSION OF ABRIDGED FACTOR VIII MOLECULES
amino chromogenic Clotek acids activity activity plasmid deleted (mUml-1) -IIa +IIa ~ fold) No DNA - 0 pXMT-VIII - 15:1 -pDGR-2 581 114 250 5750 (23X) pLA-2 880 162 330 9240 (28X) The plasmids indicated were transfected into COS cells and 48 hr. post-transfection the conditioned media taken for assay by Zp the Kabi Coatest factor VIII:C method (chromogenic activity) and by the one-stage activated partial thromboplastin time (APTT) coagulation assay (Clotek activity) using factor VIII:C
deficient plasma as described (Toole, Nature 1984). For thrombin (IIa) activation, samples were pretreated 1-10 min, with 0.2 units/ml thrombin (IIa) at room temperature. Activation coefficients are provided in parentheses. Activity from media from the wild-type (pXMT-VIII) transfection was too low to directly measure Clotek activity before thrombin activ-ation. From other experiments where the wild type factor VIII activity was concentrated, it was demonstrated to be approximately 30-fold activatable.
Expression of Procoagulant Molecules in CHO Cells A) Expression of pDGR-2 The procoagulant expression vector containing a deletion (relative to the Factor VIII:C cDNA) of 581 amino acids (pDGR-2) was transfected with plasmid pAdD26SV(A)#3 (10 ug pDGR-2:1 ug pAdD26SV(A)#3) by CaP04 coprecipitation into CHO DHFR deficient cells (DUKX-B11) and transformants isolated and grown in increasing concentrations of MTX as described by Kaufman et. al. , (1985). One transformant designated J1 exhibited the following activities as a function of resistance to increasing concen-trations of MTX.
uM MTX mUnits/ml,/day,/106 cells*
0 1.46 0.02 322 0.1 499 B) Expression of pLA-2 The procoagulant expression vector containing a deletion of 880 amino acids (pLA-2) was introduced into CHO DHFR deficient cells (DUKX-B11, Chasin and Urlaub, PNAS 77: 4216-4220, 1980 by protoplast fusion as described (Sandri-Goldin et. al., Mol.
Cell. Biol. l: 743-752). After fusion, fresh medium containing 100 ug/ml of kanamycin, and 10 ug/ml of each of thymidine, adenosine, deoxyadenosine, penicillin, and streptomycin and 10% dialyzed fetal calf serum was added to each plate. The kanamycin was included to prevent the growth of any bacteria which had escaped conversion to protoplasts. Four days later the cells were subcultured 1:15 into alpha-media with 10%
dialyzed fetal calf serum, penicillin, and streptomycin, but lacking the nucleosides. Colonies appeared after 10-12 days after subculturing cells into selective media. A group of B
134 ? ? 7~
transformants were pooled and grown in sequentially increasing concentrations of MTX starting at 0.02 uM with steps to 0.1, 0.2, and 1.0 uM MTX. Results of factor VIII-type activity in cells resistant to increasing concentrations of MTX is shown below.
uM MTX mUnitsfml/day,/106cells*
0.02 530 0 ~ 2 1170 1.0 1890 * Factor VIII activity was determined by the Kabi Coatest factor VIII:C method (chromogenic activity).
Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly MET Glu Ala Tyr 342 TTT CTA CTG TTT TGT CAT ATC TCT TCC CAC CAA CAT GAT GGC ATG GAA GCT TAT
Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg MET Lys Asn Asn Glu 360 GTC AAA GTA GAC AGC TGT CCA GAG GAA CCC CAA CTA CGA ATG AAA AAT AAT GAA
Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu MET Asp Val Val Arg 378 GAA GCG GAA GAC TAT GAT GAT GAT CTT ACT GAT TCT GAA ATG GAT GTG GTC AGG
Phe Asp Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys 396 TTT GAT GAT GAC AAC TCT CCT TCC TTT ATC CAA ATT CGC TCA GTT GCC AAG AAG
His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr 414 CAT CCT AAA ACT TGG GTA CAT TAC ATT GCT GCT GAA GAG GAG GAC TGG GAC TAT
Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 432 GCT CCC TTA GTC CTC GCC CCC GAT GAC AGA AGT TAT AAA AGT CAA TAT TTG AAC
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe MET Ala Tyr 450 AAT GGG CCT CAG CGG ATT GGT AGG AAG TAC AAA AAA GTC CGA TTT ATG GCA TAC
Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile Leu 468 ACA GAT GAA ACC TTT AAG ACT CGT GAA GCT ATT CAG CAT GAA TCA GGA ATC TTG
Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn 486 GGA CCT TTA CTT TAT GGG GAA GTT GGA GAC ACA CTG TTG ATT ATA TTT AAG AAT
Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro 504 CAA GCA AGC AGA CCA TAT AAC ATC TAC CCT CAC GGA ATC ACT GAT GTC CGT CCT
Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile 522 TTG TAT TCA AGG AGA TTA CCA AAA GGT GTA AAA CAT TTG AAG GAT TTT CCA ATT
Leu Pro Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro 540 CTG CCA GGA GAA ATA TTC AAA TAT AAA TGG ACA GTG ACT GTA GAA GAT GGG CCA
Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn MET 558 ACT AAA TCA GAT CCT CGG TGC CTG ACC CGC TAT TAC TCT AGT TTC GTT AAT ATG
Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 576 GAG AGA GAT CTA GCT TCA GGA CTC ATT GGG CCT CTC CTC ATC TGC TAC AAA GAA
Ser Val Asp Gln Arg Gly Asn Gln Ile MET Ser Asp Lys Arg Asn Val Ile Leu 594 TCT GTA GAT CAA AGA GGA AAC CAG ATA ATG TCA GAC AAG AGG AAT GTC ATC CTG
Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln Arg 612 TTT TCT GTA TTT GAT GAG AAC CGA AGC TGG TAC CTC ACA GAG AAT ATA CAA CGC
Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser 630 TTT CTC CCC AAT CCA GCT GGA GTG CAG CTT GAG GAT CCA GAG TTC CAA GCC TCC
Asn Ile MET His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val 648 AAC ATC ATG CAC AGC ATC AAT GGC TAT GTT TTT GAT AGT TTG CAG TTG TCA GTT
X
TABLE 1, continued Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp 666 TGT TTG CAT GAG GTG GCA TAC TGG TAC ATT CTA AGC ATT GGA GCA CAG ACT GAC
Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys MET Val Tyr Glu 684 TTC CTT TCT GTC TTC TTC TCT GGA TAT ACC TTC AAA CAC AAA ATG GTC TAT GAA
Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe MET Ser MET Glu 702 GAC ACA CTG ACC CTA TTC CCA TTC TCA GGA GAA ACT GTC TTC ATG TCG ATG GAA
Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 720 AAC CCA GGT CTA TGG ATT CTG GGG TGC CAC AAC TCA GAC TTT CGG AAC AGA GGC
MET Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr 738 ATG ACC GCC TTA CTG AAG GTT TCT AGT TGT GAC AAG AAC ACT GGT GAT TAT TAC
Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala Ile 756 GAG GAC AGT TAT GAA GAT ATT TCA GCA TAC TTG CTG AGT AAA AAC AAT GCC ATT
Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln 774 GAA CCA AGA AGC TTC TCC CAG AAT TCA AGA CAC CCT AGC ACT AGG CAA AAG CAA
Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp Phe 792 TTT AAT GCC ACC ACA ATT CCA GAA AAT GAC ATA GAG AAG ACT GAC CCT TGG TTT
Ala His Arg Thr Pro MET Pro Lys Ile Gln Asn Val Ser Ser Ser Asp Leu Leu 810 GCA CAC AGA ACA CCT ATG CCT AAA ATA CAA AAT GTC TCC TCT AGT GAT TTG TTG
MET Leu Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln 828 ATG CTC TTG CGA CAG AGT CCT ACT CCA CAT GGG CTA TCC TTA TCT GAT CTC CAA
Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser 846 GAA GCC AAA TAT GAG ACT TTT TCT GAT GAT CCA TCA CCT GGA GCA ATA GAC AGT
Asn Asn Ser Leu Ser Glu MET Thr His Phe Arg Pro Gln Leu His His Ser Gly 864 AAT AAC AGC CTG TCT GAA ATG ACA CAC TTC AGG CCA CAG CTC CAT CAC AGT GGG
Asp MET Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu 882 GAC ATG GTA TTT ACC CCT GAG TCA GGC CTC CAA TTA AGA TTA AAT GAG AAA CTG
Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser Thr 900 GGG ACA ACT GCA GCA ACA GAG TTG AAG AAA CTT GAT TTC AAA GTT TCT AGT ACA
Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp 918 TCA AAT AAT CTG ATT TCA ACA ATT CCA TCA GAC AAT TTG GCA GCA GGT ACT GAT
Asn Thr Ser Ser Leu Gly Pro Pro Ser MET Pro Val His Tyr Asp Ser Gln Leu 936 AAT ACA AGT TCG TTA GGA CCC CCA AGT ATG CCA GTT CAT TAT GAT AGT CAA TTA
Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro 954 GAT ACC ACT CTA TTT GGC AAA AAG TCA TCT CCC CTT ACT GAG TCT GGT GGA CCT
Leu Ser Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu MET 972 CTG AGC TTG AGT GAA GAA AAT AAT GAT TCA AAG TTG TTA GAA TCA GGT TTA ATG
Asn Ser Gln Glu Ser 5er Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg 990 AAT AGC CAA GAA AGT TCA TGG GGA AAA AAT GTA TCG TCA ACA GAG AGT GGT AGG
X
...
1341 1 ~4 TABLE 1, continued Leu PheLysGly LysArg HisGlyPro AlaLeuLeuThr LysAspAsn Ala1,008 Ala TTA TTTAAAGGG AAAAGAGCT CATGGACCT GCTTTGTTGACT AAAGATAAT GCC
Leu PheLysVal SerIleSer LeuLeuLys ThrAsnLysThr SerAsnAsn Ser1,026 TTA TTCAAAGTT AGCATCTCT TTGTTAAAG ACAAACAAAACT TCCAATAAT TCA
Ala ThrAsnArg LysThrHis IleAspGly ProSerLeuLeu IleGluAsn Ser1,044 GCA ACTAATAGA AAGACTCAC ATTGATGGG CCATCATTATTA ATTGAGAAT AGT
Pro SerValTrp GlnAsnIle LeuGluSer AspThrGluPhe LysLysVal Thr1,062 CCA TCAGTCTGG CAAAATATA TTAGAAAGT GACACTGAGTTT AAAAAAGTG ACA
Pro LeuIleHis AspArgMET LeuMETAsp LysAsnAlaThr AlaLeuArg Leu1,080 CCT TTGATTCAT GACAGAATG CTTATGGAC AAAAATGCTACA GCTTTGAGG CTA
Asn HisMETSer AsnLysThr ThrSerSer LysAsnMETGlu METValGln Gln1,098 AAT CATATGTCA AATAAAACT ACTTCATCA AAAAACATGGAA ATGGTCCAA CAG
Lys LysGluGly ProIlePro ProAspAla GlnAsnProAsp METSerPhe Phe1,116 AAA AAAGAGGGG CCCATTCCA CCAGATGCA CAAAATCCAGAT ATGTCGTTC TTT
Lys METLeuPhe LeuProGlu SerAlaArg TrpIleGlnArg ThrHisGly Lys1,134 AAG ATGCTATTC TTGCCAGAA TCAGCAAGG TGGATACAAAGG ACTCATGGA AAG
Asn SerLeuAsn SerGlyGln GlyProSer ProLysGlnLeu ValSerLeu Gly1,152 AAC TCTCTGAAC TCTGGGCAA GGCCCCAGT CCAAAGCAATTA GTATCCTTA GGA
Pro GluLysSer ValGluGly GlnAsnPhe LeuSerGluLys AsnLysVal Val1,170 CCA GAAAAATCT GTGGAAGGT CAGAATTTC TTGTCTGAGAAA AACAAAGTG GTA
Val GlyLysGly GluPheThr LysAspVal GlyLeuLysGlu METValPhe Pro1,188 GTA GGAAAGGGT GAATTTACA AAGGACGTA GGACTCAAAGAG ATGGTTTTT CCA
Ser SerArgAsn LeuPheLeu ThrAsnLeu AspAsnLeuHis GluAsnAsn Thr1,206 AGC AGCAGAAAC CTATTTCTT ACTAACTTG GATAATTTACAT GAAAATAAT ACA
His AsnGlnGlu LysLysIle GlnGluGlu IleGluLysLys GluThrLeu Ile1,224 CAC AATCAAGAA AAAAAAATT CAGGAAGAA ATAGAAAAGAAG GAAACATTA ATC
Gln GluAsnVal ValLeuPro GlnIleHis ThrValThrGly ThrLysAsn Phe1,242 CAA GAGAATGTA GTTTTGCCT CAGATACAT ACAGTGACTGGC ACTAAGAAT TTC
MET Lys Asn Leu Phe Leu Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Glu 1,260 ATG AAG AAC CTT TTC TTA CTG AGC ACT AGG CAA AAT GTA GAA GGT TCA TAT GAG
Gly Ala Tyr Ala Pro Leu Val Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn 1,278 GGG GCA TAT GCT CCA GTA CTT CAA GAT TTT AGG TCA TTA AAT GAT TCA ACA AAT
Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu 1,296 AGA ACA AAG AAA CAC ACA GCT CAT TTC TCA AAA AAA GGG GAG GAA GAA AAC TTG
Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr 1,314 GAA GGC TTG GGA AAT CAA ACC AAG CAA ATT GTA GAG AAA TAT GCA TGC ACC ACA
Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys Arg 1,332 AGG ATA TCT CCT AAT ACA AGC CAG CAG AAT TTT GTC ACG CAA CGT AGT AAG AGA
...
TABLE l, continued '7 Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile 1,350 GCT TTG AAA CAA TTC AGA CTC CCA CTA GAA GAA ACA GAA CTT GAA AAA AGG ATA
Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn MET Lys His Leu Thr Pro 1,368 ATT GTG GAT GAC ACC TCA ACC CAG TGG TCC AAA AAC ATG AAA CAT TTG ACC CCG
Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln 1,386 AGC ACC CTC ACA CAG ATA GAC TAC AAT GAG AAG GAG AAA GGG GCC ATT ACT CAG
Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala Asn Arg 1,404 TCT CCC TTA TCA GAT TGC CTT ACG AGG AGT CAT AGC ATC CCT CAA GCA AAT AGA
Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr 1,422 TCT CCA TTA CCC ATT GCA AAG GTA TCA TCA TTT CCA TCT ATT AGA CCT ATA TAT
Leu Thr Arg Val Leu Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr 1,440 CTG ACC AGG GTC CTA TTC CAA GAC AAC TCT TCT CAT CTT CCA GCA GCA TCT TAT
Arg Lys Lys Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys 1,458 AGA AAG AAA GAT TCT GGG GTC CAA GAA AGC AGT CAT TTC TTA CAA GGA GCC AAA
Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu MET Thr Gly Asp Gln Arg 1,476 AAA AAT AAC CTT TCT TTA GCC ATT CTA ACC TTG GAG ATG ACT GGT GAT CAA AGA
Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys Lys Val 1,494 GAG GTT GGC TCC CTG GGG ACA AGT GCC ACA AAT TCA GTC ACA TAC AAG AAA GTT
Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr Ser Gly Lys Val Glu 1,512 GAG AAC ACT GTT CTC CCG AAA CCA GAC TTG CCC AAA ACA TCT GGC AAA GTT GAA
Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser 1,530 TTG CTT CCA AAA GTT CAC ATT TAT CAG AAG GAC CTA TTC CCT ACG GAA ACT AGC
Asn Gly Ser Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr 1,548 AAT GGG TCT CCT GGC CAT CTG GAT CTC GTG GAA GGG AGC CTT CTT CAG GGA ACA
Glu Gly Ala Ile Lys Trp Asn Glu Pro Asn Arg Pro Gly Lys Val Pro Phe Leu 1,566 GAG GGA GCG ATT AAG TGG AAT GAA CCA AAC AGA CCT GGA AAA GTT CCC TTT CTG
Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu 1,584 ATA GTA GCA ACA GAA AGC TCT GCA AAG ACT CCC TCC AAG CTA TTG GAT CCT CTT
Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln 1,602 GCT TGG GAT AAC CAC TAT GGT ACT CAG ATA CCA AAA GAA GAG TGG AAA TCC CAA
Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu 1,520 GAG AAG TCA CCA GAA AAA ACA GCT TTT AAG AAA AAG GAT ACC ATT TTG TCC CTC
Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys 1,638 AAC GCT TGT GAA AGC AAT CAT GCA ATA GCA GCA ATA AAT GAG GGA CAA AAT AAG
Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser 1,656 CCC GAA ATA GAA GTC ACC TGG GCA AAG CAA GGT AGG ACT GAA AGG CTC TGC TCT
Gln Asn Pro Pro Val Leu Lys Arg His Arg Pro Glu Ile Thr Arg Thr Thr Leu 1,674 CAA AAC CCA CCA GTC TTG AAA CGC CAT CAA CGC GAA ATA ACT CGT ACT ACT CTT
iX
1341 174.
TABLE 1, continued g Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu MET Lys 1,692 CAG TCA GAT CAA GAG GAA ATT GAC TAT GAT GAT ACC ATA TCA GTT GAA ATG AAG
Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 1,710 AAG GAA GAT TTT GAC ATT TAT GAT GAG GAT GAA AAT CAG AGC CCC CGC AGC TTT
Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1,728 CAA AAG AAA ACA CGA CAC TAT TTT ATT GCT GCA GTG GAG AGG CTC TGG GAT TAT
Gly MET Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val 1,746 GGG ATG AGT AGC TCC CCA CAT GTT CTA AGA AAC AGG GCT CAG AGT GGC AGT GTC
Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln 1,764 CCT CAG TTC AAG AAA GTT GTT TTC CAG GAA TTT ACT GAT GGC TCC TTT ACT CAG
Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile 1,782 CCC TTA TAC CGT GGA GAA CTA AAT GAA CAT TTG GGA CTC CTG GGG CCA TAT ATA
Arg Ala Glu Val Glu Asp Asn Ile MET Val Thr Phe Arg Asn Gln Ala Ser Arg 1,800 AGA GCA GAA GTT GAA GAT AAT ATC ATG GTA ACT TTC AGA AAT CAG GCC TCT CGT
Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly 1,818 CCC TAT TCC TTC TAT TCT AGC CTT ATT TCT TAT GAG GAA GAT CAG AGG CAA GGA
Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp 1,836 GCA GAA CCT AGA AAA AAC TTT GTC AAG CCT AAT GAA ACC AAA ACT TAC TTT TGG
Lys Val Gln His His MET Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 1,854 AAA GTG CAA CAT CAT ATG GCA CCC ACT AAA GAT GAG TTT GAC TGC AAA GCC TGG
Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly 1,872 GCT TAT TTC TCT GAT GTT GAC CTG GAA AAA GAT GTG CAC TCA GGC CTG ATT GGA
Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val 1,890 CCC CTT CTG GTG TGC CAC ACT AAC ACA CTG AAC CCT GCT CAT GGG AGA CAA GTG
Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1,908 ACA GTA CAG GAA TTT GCT CTG TTT TTC ACC ATC TTT GAT GAG ACC AAA AGC TGG
Tyr Phe Thr Glu Asn MET Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln MET 1,926 TAC TTC ACT GAA AAT ATG GAA AGA AAC TGC AGG GCT CCC TGC AAT ATC CAG ATG
Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile 1,944 GAA GAT CCC ACT TTT AAA GAG AAT TAT CGC TTC CAT GCA ATC AAT GGG TAC ATA
MET Asp Thr Leu Pro Gly Leu Val MET Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1,962 ATG GAT ACA CTA CCT GGC TTA GTA ATG GCT CAG GAT CAA AGG ATT CGA TGG TAT
Leu Leu Ser MET Gly Ser Asn Glu Asn Ili His Ser Ile His Phe Ser Gly His 1,980 CTG CTC AGG ATG GGC AGG AAT GAA AAC ATC CAT TCT ATT CAT TTC AGT GGA CAT
Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys MET Ala Leu Tyr Asn Leu Tyr 1,998 GTG TTC ACT GTA CGA AAA AAA GAG GAG TAT AAA ATG GCA CTG TAC AAT CTC TAT
Pro Gly Val Phe Glu Thr Val Glu MET Leu Pro Ser Lys Ala Gly Ile Trp Arg 2,016 CCA GGT GTT TTT GAG ACA GTG GAA ATG TTA CCA TCC AAA GCT GGA ATT TGG CGG
TABLE 1, continued 9 Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly MET Ser Thr Leu Phe Leu 2,034 GTG GAA TGC CTT ATT GGC GAG CAT CTA CAT GCT GGG ATG AGC ACA CTT TTT CTG
Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly MET Ala Ser Gly His Ile Arg 2,052 GTG TAC AGC AAT AAG TGT CAG ACT CCC CTG GGA ATG GCT TCT GGA CAC ATT AGA
Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala 2,070 GAT TTT CAG ATT ACA GCT TCA GGA CAA TAT GGA CAG TGG GCC CCA AAG CTG GCC
Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser 2,088 AGA CTT CAT TAT TCC GGA TCA ATC AAT GCC TGG AGC ACC AAG GAG CCC TTT TCT
Trp Ile Lys Val Asp Leu Leu Ala Pro MET Ile Ile His Gly Ile Lys Thr Gln 2,106 TGG ATC AAG GTG GAT CTG TTG GCA CCA ATG ATT ATT CAC GGC ATC AAG ACC CAG
Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile MET Tyr 2,124 GGT GCC CGT CAG AAG TTC TCC AGC CTC TAC ATC TCT CAG TTT ATC ATC ATG TAT
Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 2,142 AGT CTT GAT GGG AAG AAG TGG CAG ACT TAT CGA GGA AAT TCC ACT GGA ACC TTA
MET Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn 2,160 ATG GTC TTC TTT GGC AAT GTG GAT TCA TCT GGG ATA AAA CAC AAT ATT TTT AAC
Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg 2,178 CCT CCA ATT ATT GCT CGA TAC ATC CGT TTG CAC CCA ACT CAT TAT AGC ATT CGC
Ser Thr Leu Arg MET Glu Leu MET Gly Cys Asp Leu Asn Ser Cys Ser MET Pro 2,196 AGC ACT CTT CGC ATG GAG TTG ATG GGC TGT GAT TTA AAT AGT TGC AGC ATG CCA
Leu Gly MET Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr 2,214 TTG GGA ATG GAG AGT AAA GCA ATA TCA GAT GCA CAG ATT ACT GCT TCA TCC TAC
Phe Thr Asn MET Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 2,232 TTT ACC AAT ATG TTT GCC ACC TGG TCT CCT TCA AAA GCT CGA CTT CAC CTC CAA
Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln 2,250 GGG AGG AGT AAT GCC TGG AGA CCT CAG GTC AAT AAT CCA AAA GAG TGG CTC CAA
Val Asp Phe Gln Lys Thr MET Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 2,268 GTG GAC TTC CAG AAG ACA ATG AAA GTC ACA GGA GTA ACT ACT CAG GGA GTA AAA
Ser Leu Leu Thr Ser MET Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp 2,286 TCT CTG CTT ACC AGC ATG TAT GTG AAG GAG TTC CTC ATC TCC AGC AGT CAA GAT
Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly 2,304 GGC CAT CAG TGG ACT CTC TTT TTT CAG AAT GGC AAA GTA AAG GTT TTT CAG GGA
Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 2,322 AAT CAA GAC TCC TTC ACA CCT GTG GTG AAC TCT CTA GAC CCA CCG TTA CTG ACT
Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg MET 2,340 CGC TAC CTT CGA ATT CAC CCC CAG AGT TGG GTG CAC CAG ATT GCC CTG AGG ATG
Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr End 2,352 GAG GTT CTG GGC TGC GAG GCA CAG GAC CTC TAC TGA GGGTGGCCACTGCATCCCACCTGCCACTG
CCGTCACCTCTCCCTCCTCAGCTCCAGGGCATGTGTCCCTCCCTGGCTTGCTTCTACCTTTGTGCTAAATCCTAGCAGA
CACTCCCTTG
AAGCCTCCTGAATTAACTATCATCACTCCTGCATTTCTTTGGTGGGGGGCCAGGAGGCTGCATCCATTTTAACTTAACT
CTTACCTATT
TTCTGCAGCTGCTCCCAGA
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to 1000 followed by the amino acid sequence of Asp-1582 to Arg--1708. That compound thus comprises the polypeptide sequence of Ala-20 to Pro-1000 covalently linked by a peptide bond to amino acids Asp-1582 to Tyr-2351. Another exemplary compound contains a region X comprising the amino acid sequence Ser-760 to Thr-778 followed by the sequence Pro-1659 to Arg-1708.
That compound thus comprises the polypeptide sequence Ala-20 to Thr-778 covalently linked by a peptide bond to the sequence Pro-1659 through Tyr-2351. Still another exemplary compound, contains a region X comprising the amino acid sequence Ser-760 to Thr-778 followed by the sequence Glu-1694 to Arg-1708.
That compound thus comprises the polypeptide sequence Ala-20 to Thr-778 covalently linked by a peptide bond to amino acids Glu-1694 through Tyr-2351.
These exemplary compounds are depicted schematically in Table 2.
The amino acid sequence represented by X should be selected so that it does not substantially reduce the procoagulant 2p activity of the molecule, which activity can be conveniently assayed by conventional methods. Compound (2) of Table 2 is a presently preferred embodiment.
The procoagulant protein may be produced by appropriate host cells transformed by factor VIII:C DNA which has been specific-ally altered by use of any of a variety of site-specific muta-genesis techniques which will be familiar to those of ordinary skill in the art of recombinant DNA.
The starting materials may be a DNA sequence which codes for the complete factor VIII:C molecule, e.g., the complete human factor VIII:C as shown in Table 1, a truncated version of that sequence, or it may comprise segments of that DNA sequence, so long as the starting materials contain at least sufficient DNA
to code for the amino acid sequences of the desired polypeptide.
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rd The procoagulent proteins of the present invention, in addition to lacking a substantial amino acid segment of human factor VIII: C, also have fewer potential N-glycosylation sites than human factor VIII. Preferably, at least one N-glycosylation site has been deleted. More preferably, 18 of the 25 potential N-glycosylation sites are not in the molecule. In still more preferred embodiments, up to 19 of the 25 potential N-glycos-ylation sites are removed. While not wishing to be bound by theory, it is presently believed that the antibodies to factor VIII:C which are directed to antigenic determinants contained in the protein segment deleted in accordance with this inven-tion, i.e., in the amino acid segement itself or in the carbo-hydrate portion of the glycosylated protein, will not neutralize the procoagulant proteins of the present invention. Moreover, the fact that the procoagulants of the present invention lack many of the sites for non-human glycosylation by the non-human mammalian or other cells used to produce the proteins is also belived to reduce the antigenicity of that protein, and lessen the likelihood of developing antibodies to the procoagulants.
This may enable facilitating the treatment of patients in need of procoagulant therapy.
I contemplate that my compounds can be produced by recombinant DNA techniques at a much lower cost than is possible for pro-duction of human factor VIII. The host organisms should more - efficiently process and express the substantially simpler molecules of this invention.
The compounds of this invention can be formulated into pharmaceu-tically acceptable preparations with parenterally acceptable vehicles and excipients in accordance with procedures known in the art.
The pharmaceutical preparations of this invention, suitable for parenteral administration, may conveniently comprise a sterile lyophilized preparation of the protein which may be reconsti-tuted by addition of sterile solution to produce solutions preferably isotonic with the blood of the recipient. The prepar-ation may be presented in unit or multi-dose containers, e.g. in sealed ampoules or vials. Their use would be analogous to that of human factor VIII, appropriately adjusted for potency.
One method by which these proteins can be expressed is by use of DNA which is prepared by cutting a full-length factor VIII:C DNA with the appropriate restriction enzymes to remove a portion of the DNA sequence that codes for amino acids 760 to 1708 of human factor VIII: C. The cut DNA is then ligated with an oligonucleotide that resects the cut DNA and maintains the correct translational reading frame.
Preparation of the cDNA has been set forth in detail in U.S.
Patent Applications Serial Nos. 546,650 and 644,036, supra. A
pSP64 recombinant clone containing the nucleotide sequence depicted in Table 1, designated as pSP64-VIII, is on deposit at the American Type Culture Collection under Accession Number ATCC 39812.
Restriction endonucleases are used to obtain cleavage of the human factor VIII:C cDNA, hereinafter the DNA source sequence, at appropriate sites in the nucleotide sequence. Unless otherwise noted, restriction endonucleases are utilized under the conditions and in the manner recommended by their commercial suppliers. The restriction endonucleases selected herein are those which will enable one to excise with substantial speci-ficity sequences that code for the portion of the factor VIII:C molecule desired to be excised. BamHI and SacI are particularly useful endonucleases. However, the skilled artisan will be able to utilize other restriction endonucleases chosen by conventional selection methods. The number of nucleotides deleted may vary but care should be taken to insure that the reading frame of the ultimate cDNA sequence will not be affected.
Y
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The resulting DNA fragments are then purified using conventional techniques such as those set forth in Maniatis et al. , Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory 1982), and Proc. Natl. Acad. Sci. 76:615-619 (1979). The purified DNA is then ligated to form the sequence encoding the polypeptide of the preferred invention. When necessary or desirable, the ligation may be within an oligonucleotide that resects the cut DNA and maintains the correct translational reading frame using standard ligation conditions. Ligation reactions are carried on as described by Maniatis et al., supra at 2453-6 using the buffer described at page 246 thereof and using a DNA
concentration of 1-100 ug/ml, at a temperature of 23°C for blunt ended DNA and 16°C for "sticky ended" DNA. The following double-stranded oligonucleotide is useful when there is BamHI/-SacI deletion such as described infra, 5' P-CATGGACCG-3' 3-TCGAGTACCTGGCCTAG 5';
but other oligonucleotides can be selected by the skilled artisan depending upon the deletions made and reaction conditions.
The DNA sequences encoding the novel procoagulant polypeptides - can, in addition to other methods, be derived from the sequence of human factor VIII : C DNA by application of oligonucleotide-med-iated deletion mutagenesis, often referred to as "loopout" muta-genesis, as described for example in Morinaga, Y. et al. Biotech-noloQV,, 2: 636-639 (1984).
The new DNA sequences containing the various deletions can then be introduced into appropriate vectors for expression 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.
-1341 17~
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 5 may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol. Biol., 159: 51-521 (1982); Kaufman, Proc. Natl. Acad. Sci. 82: 689-693 (1985).
Established cell lines, including transformed cell lines, are 10 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 haematopoeitic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so 15 long 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 chromosomal DNA, and for subsequent amplification of the integrated vector DNA, CHO (Chinese hamster ovary) cells are presently preferred.
See U.S. Patent 4,399,216. Alternatively, the vector DNA
could include all or parts of the bovine papilloma virus genome (Lusky et al., Cell, 36: 391-401 (9184) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other 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 hamster cells lines and the like.
Stable transformants then are screened for expression of the procoagulant product by standard immunological or enzymatic 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 enzymatic or immunologic assay of the proteins in the culture medium.
The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary, and should not be taken as limiting the true scope of the present invention, as described in the claims.
1341 1'74 ug. of the plasmid PACE, a pSP64 (Promega Biotec, Madison, Wis.) derivative, containing nucleotides 562-7269 of human factor VIII:C cDNA (nucleotide 1 is the A of the ATG initiator meth-s ionine codon) was subj ected to partial BamHI digestion in 100u1 containing 50mM Tris.HCl ph 8.0, 50mM MgCl2, and 2.4 units BamHI (New England Biolabs) for 30 minutes at 37°C. The reaction was terminated by the addition of EDTA to 20mM and then extracted once with phenol, once with chloroform , ethanol 10 precipitated and pelleted by centrifugation. DNA was redis-solved, cleaved to completion in 50u1 using 40 units SacI for 1.5 hours at 37°C. DNA was then electrophoresed through a buffered 0.6% agarose gel. An 8.1 kb fragment corresponding to the partial BamHI-SacI fragment of PACE lacking only the sequence corresponding to nucleotides 2992-4774 of the factor VIII:C sequence was purified from the gel using the glass powder technique described in Proc. Nat. Acad. Sci. 76; 615-619 (1979) . Purified DNA was ligated with 100 pmoles of the following double-stranded oligonucleotide 5'P-CATGGACCG-3' 3'-TCGAGTACCTGGCCTAG 5' using standard ligation conditions. The DNA sequence removed represents the deletion of 584 amino acid sequence beginning - 25 with amino acid 998 and continuing through 1581. The oligo-nucleotide inserted, however, encodes amino acids corresponding to 998-1000. Therefore, the polypeptide encoded contains deletion of 581 amino acids.
DNA was then used to transform competent E, coli bacteria, and DNA from several ampicillin resistant transformants was analyzed by restriction mapping to identify a plasmid harboring the desired SacI-BamHI deletion mutant. DNA from this plasmid was digested to completion with KpnI, which cleaves the plasmid uniquely at nucleotide 1816 of the factor VIII:C coding se-quence. This DNA was ligated with a KpnI DNA fragment containing nucleotides 1-1815 of factor VIII:C DNA and a synthetic SalI
site at nucleotides -11 to -5 and then used to transform competent E. coli bacteria.
Plasmid DNA was isolated and oriented by restriction mapping to identify a plasmid, pBSdK, containing the correct 5' to 3' orien-tation of the KpnI insert. SalI digestion, which excises the entire polypeptide coding region from the plasmid,. was performed l0 and the DNA electrophoresed through a buffered 0.6% agarose gel.
The 5.3Kb SalI fragment was purified from the gel as described above. This DNA fragment was ligated with XhoI cut pXMT2 DNA
to give rise to plasmid pDGR-2. pXMT2 is a plasmid capable of expressing heterologous genes when introduced into mammalian cells such as the COS-1 African Green Monkey kidney cell line, and is a derivative of the expression vectors described in Kaufman, supra at 689-93. The expression elements are the same as described for plasmid pQ2 except that it contains a deletion of the adenovirus major late promoter extending from 2p -45 to +156 with respect to the transcription start site of the adenovirus major late promoter. mRNA expression in pXMT
is driven by the SV40 late promoter. The bacterial replicon, however, has been substituted to render bacteria containing the vector resistant to ampicillin rather than tetracycline.
pXMT2 contains a unique Xho I site at a position which allows for expression of inserted cDNA from the SV40 late promoter.
This Xho I site is convenient for inserting factor VIII:C cDNA
constructs since these are flanked by SalI sites.
3o Restriction mapping of transformants identified a plasmid, pDGR-2, containing the correct 5' to 3' orientation of the polypeptide coding sequence relative to the direction of transcription from the SV40 late promoter. pDGR-2 is on deposit at the American Type Culture Collection under Accession number 53100.
Other novel procoagulant proteins may be obtained from constructs produced by oligonucleotide mediated deletion mutagenesis, using for example the "loopout" mutagenesis techniques as described in Morinaga et al. , supra. The deletion mutagenesis is performed using expression plasmid pDGR-2 or any other appropriate plasmid or bacteriophage vector. Other methods for oligonucleotide mediated mutagenesis employing single stranded DNA produced with M13 vectors and the like are also suitable. See Zoller et al., Nucl. Acids Res. 10: 6487-6500 (1982). For example, these deletions can be produced using the oligonucleotides (A) 5' AAAAGCAATTTAATGCCACCCCACCAGTCTTGAAACGCCA
(8) 5' AAAAGCAATTTAATGCCACCGAAGATTTTGACATTTATGA
to cause deletions in factor VIII:C cDNA from nucleotides (A)' 2334 to 4974 or (B) 2334 to 5079. The proteins encoded by these constructs contain deletions of (A) 880 and (B) 915 amino acids relative to Factor VIII: C.
The deleted constructs are tested directly, or after subcloning into appropriate expression vectors, in order to determine if the novel proteins possess procoagulant activity. Procoagulant activity was assayed as described in Examples 3 and 4.
Expression of Procoagulant Molecules in COS Monkey Cells The expression plasmids containing the modified cDNA's prepared as in Examples 1 or 2 and the full-length cDNA, pXMT-VIII, were introduced into COS-1 cells via the DEAE-dextran trans-fection protocol. Sompayrac and Dana 1981, Proc. Natl. Acad.
Sci. 78: 7575-7578. Conditioned media was harvested 48 hours post-transfection and assayed for factor VIII-type activity as described in Toole et. al., 1984, Nature 312:342-347. The results of the experiment are summarized in Table 3. Both plasmids containing the modified cDNAs yielded procoagulant activity and, moreover, the activity was greater than that obtained using wild type cDNA. From these data it was concluded 5 that removal of up to 880 amino acids (95,000 daltons) in a defined domain of human factor VIII does not destroy cofactor activity. Furthermore, these abridged procoagulant proteins retain their ability to be activated by thrombin.
1341 17~r TABLE 3: EXPRESSION OF ABRIDGED FACTOR VIII MOLECULES
amino chromogenic Clotek acids activity activity plasmid deleted (mUml-1) -IIa +IIa ~ fold) No DNA - 0 pXMT-VIII - 15:1 -pDGR-2 581 114 250 5750 (23X) pLA-2 880 162 330 9240 (28X) The plasmids indicated were transfected into COS cells and 48 hr. post-transfection the conditioned media taken for assay by Zp the Kabi Coatest factor VIII:C method (chromogenic activity) and by the one-stage activated partial thromboplastin time (APTT) coagulation assay (Clotek activity) using factor VIII:C
deficient plasma as described (Toole, Nature 1984). For thrombin (IIa) activation, samples were pretreated 1-10 min, with 0.2 units/ml thrombin (IIa) at room temperature. Activation coefficients are provided in parentheses. Activity from media from the wild-type (pXMT-VIII) transfection was too low to directly measure Clotek activity before thrombin activ-ation. From other experiments where the wild type factor VIII activity was concentrated, it was demonstrated to be approximately 30-fold activatable.
Expression of Procoagulant Molecules in CHO Cells A) Expression of pDGR-2 The procoagulant expression vector containing a deletion (relative to the Factor VIII:C cDNA) of 581 amino acids (pDGR-2) was transfected with plasmid pAdD26SV(A)#3 (10 ug pDGR-2:1 ug pAdD26SV(A)#3) by CaP04 coprecipitation into CHO DHFR deficient cells (DUKX-B11) and transformants isolated and grown in increasing concentrations of MTX as described by Kaufman et. al. , (1985). One transformant designated J1 exhibited the following activities as a function of resistance to increasing concen-trations of MTX.
uM MTX mUnits/ml,/day,/106 cells*
0 1.46 0.02 322 0.1 499 B) Expression of pLA-2 The procoagulant expression vector containing a deletion of 880 amino acids (pLA-2) was introduced into CHO DHFR deficient cells (DUKX-B11, Chasin and Urlaub, PNAS 77: 4216-4220, 1980 by protoplast fusion as described (Sandri-Goldin et. al., Mol.
Cell. Biol. l: 743-752). After fusion, fresh medium containing 100 ug/ml of kanamycin, and 10 ug/ml of each of thymidine, adenosine, deoxyadenosine, penicillin, and streptomycin and 10% dialyzed fetal calf serum was added to each plate. The kanamycin was included to prevent the growth of any bacteria which had escaped conversion to protoplasts. Four days later the cells were subcultured 1:15 into alpha-media with 10%
dialyzed fetal calf serum, penicillin, and streptomycin, but lacking the nucleosides. Colonies appeared after 10-12 days after subculturing cells into selective media. A group of B
134 ? ? 7~
transformants were pooled and grown in sequentially increasing concentrations of MTX starting at 0.02 uM with steps to 0.1, 0.2, and 1.0 uM MTX. Results of factor VIII-type activity in cells resistant to increasing concentrations of MTX is shown below.
uM MTX mUnitsfml/day,/106cells*
0.02 530 0 ~ 2 1170 1.0 1890 * Factor VIII activity was determined by the Kabi Coatest factor VIII:C method (chromogenic activity).
Claims (20)
1. A protein exhibiting procoagulant activity having the amino acid sequence:
A-X-B
wherein region A represents the polypeptide sequence Ala 20 through Arg-759 as shown below:
Claim 1 - (Cont'd) region B represents the polypeptide sequence Ser-1709 through Tyr 2351 as shown below:
and region X represents a polypeptide sequence comprising less than 949 amino acids selected from one or more sequences of amino acids within the sequence Ser-760 through Arg-1708 as shown below, and maintaining an ascending numerical order of amino acids as shown below, wherein X is selected so that it does not substantially reduce the procoagulant activity of the molecule:
34~
wherein the amino acid terminus of X is covalently bonded through a peptide bond to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B.
A-X-B
wherein region A represents the polypeptide sequence Ala 20 through Arg-759 as shown below:
Claim 1 - (Cont'd) region B represents the polypeptide sequence Ser-1709 through Tyr 2351 as shown below:
and region X represents a polypeptide sequence comprising less than 949 amino acids selected from one or more sequences of amino acids within the sequence Ser-760 through Arg-1708 as shown below, and maintaining an ascending numerical order of amino acids as shown below, wherein X is selected so that it does not substantially reduce the procoagulant activity of the molecule:
34~
wherein the amino acid terminus of X is covalently bonded through a peptide bond to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B.
2. A protein of claim 1 comprising the amino acid sequence Ala-20 through Pro-1000 followed by Asp-1582 through Tyr-2351 as shown below:
Claim 2 - (Cont'd) Claim 2 - (Cont'd) Claim 2 - (Cont'd) Claim 2 - (Cont'd) Claim 2 - (Cont'd) wherein Pro 1000 is covalently bonded by a peptide bond to Asp-1582.
Claim 2 - (Cont'd) Claim 2 - (Cont'd) Claim 2 - (Cont'd) Claim 2 - (Cont'd) Claim 2 - (Cont'd) wherein Pro 1000 is covalently bonded by a peptide bond to Asp-1582.
3. A protein of claim 1 comprising the amino acid sequence Ala-20 through Thr-778 followed by Pro-1659 through Tyr-2351 as shown below:
Claim 3 - (Cont'd) Claim 3 - (Cont'd) Claim 3 - (Cont'd) wherein Thr-778 is covalently bonded by a peptide bond to Pro-1659.
Claim 3 - (Cont'd) Claim 3 - (Cont'd) Claim 3 - (Cont'd) wherein Thr-778 is covalently bonded by a peptide bond to Pro-1659.
4. A protein of claim 1 comprising the amino acid sequence Ala-20 through Thr-778 followed by Glu-1694 through Tyr-2351 as shown below:
wherein Thr-778 is covalently bonded by a peptide bond to Glu-1694.
wherein Thr-778 is covalently bonded by a peptide bond to Glu-1694.
5. A DNA molecule encoding the protein of claim 1 as depicted in claim 1.
6. A DNA molecule encoding the protein of claim 2 as depicted in claim 2.
7. A DNA molecule encoding the protein of claim 3 as depicted in claim 3.
8. A DNA molecule encoding the protein of claim 4 as depicted in claim 4.
9. A genetically engineered host cell containing, and capable of expressing, a DNA molecule encoding the protein of claim 1.
10. A genetically engineered host cell of claim 9 wherein the host cell is a mammalian, yeast or bacterial cell.
11. A method for producing a protein exhibiting procoagulant properties which comprises culturing a genetically engineered cell of claim 9 under suitable conditions permitting expression of the protein.
12. A pharmaceutical preparation useful for therapeutic treatment of Hemophilia A comprising a sterile preparation of a protein of claim 1 in admixture with a pharmaceutically accepted carrier.
13. A pharmaceutical preparation useful for therapeutic treatment of Hemophilia A comprising a sterile preparation of a protein of claim 2 in admixture with a pharmaceutically accepted carrier.
14. A pharmaceutical preparation useful for therapeutic treatment of Hemophilia A comprising a sterile preparation of a protein of claim 3 in admixture with a pharmaceutically accepted carrier.
15. A pharmaceutical preparation useful for therapeutic treatment of Hemophilia A comprising a sterile preparation of a protein of claim 4 in admixture with a pharmaceutically accepted carrier.
16. A use of an effective dose of the preparation of claim 12 for treating Hemophilia A.
17. A use of an effective dose of the preparation of claim 13 for treating Hemophilia A.
18. A use of an effective dose of the preparation of claim 14 for treating Hemophilia A.
19. A use of an effective dose of the preparation of claim 15 for treating Hemophilia A.
20. A protein exhibiting procoagulant activity having the amino acid sequence:
A-X-B
wherein region A represents the polypeptide sequence Ala-20 through Arg-759 as shown below:
region B represents the polypeptide sequence Ser-1709 through Tyr 2351 as shown below:
60~
and region X represents a polypeptide sequence comprising less than 949 amino acids, wherein said polypeptide sequence comprises an amino acid sequence selected from the group consisting of:
(a) a continuous but shorter amino acid sequence selected from the sequence Ser-760 through Arg-1708 as shown below;
(b) the amino acid sequence from Ser-760 through Pro-1000 followed by the amino acid sequence from Asp-1582 through Arg-1708;
(c) the amino acid sequence from Ser-760 through Thr-778 followed by the amino acid sequence from Pro-1659 through Arg-1708;
(d) the amino acid sequence from Ser-760 through Thr-778 followed by the amino acid sequence from Glu-1694 through Arg-1708;
wherein the amino acid terminus of X is covalently bonded through a peptide bond to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B.
A-X-B
wherein region A represents the polypeptide sequence Ala-20 through Arg-759 as shown below:
region B represents the polypeptide sequence Ser-1709 through Tyr 2351 as shown below:
60~
and region X represents a polypeptide sequence comprising less than 949 amino acids, wherein said polypeptide sequence comprises an amino acid sequence selected from the group consisting of:
(a) a continuous but shorter amino acid sequence selected from the sequence Ser-760 through Arg-1708 as shown below;
(b) the amino acid sequence from Ser-760 through Pro-1000 followed by the amino acid sequence from Asp-1582 through Arg-1708;
(c) the amino acid sequence from Ser-760 through Thr-778 followed by the amino acid sequence from Pro-1659 through Arg-1708;
(d) the amino acid sequence from Ser-760 through Thr-778 followed by the amino acid sequence from Glu-1694 through Arg-1708;
wherein the amino acid terminus of X is covalently bonded through a peptide bond to the carboxy terminus of A, and the carboxy terminus of X is likewise bonded to the amino terminus of B.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US72535085A | 1985-04-12 | 1985-04-12 | |
| US10,085 | 1986-04-11 | ||
| US07/010,085 US4868112A (en) | 1985-04-12 | 1986-04-11 | Novel procoagulant proteins |
| US725,350 | 1991-07-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1341174C true CA1341174C (en) | 2001-01-30 |
Family
ID=24914180
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000506408A Expired - Lifetime CA1341174C (en) | 1985-04-12 | 1986-04-11 | Procoagulant proteins derived from factor viii: c |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4868112A (en) |
| EP (1) | EP0218712B1 (en) |
| JP (1) | JPH0788399B2 (en) |
| AT (1) | ATE72838T1 (en) |
| AU (2) | AU5772886A (en) |
| CA (1) | CA1341174C (en) |
| DE (1) | DE3683980D1 (en) |
| DK (1) | DK166457B1 (en) |
| ES (1) | ES8801674A1 (en) |
| LU (1) | LU90458I2 (en) |
| MX (1) | MX9203440A (en) |
| WO (1) | WO1986006101A1 (en) |
Families Citing this family (138)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4965199A (en) * | 1984-04-20 | 1990-10-23 | Genentech, Inc. | Preparation of functional human factor VIII in mammalian cells using methotrexate based selection |
| KR910006424B1 (en) * | 1985-08-21 | 1991-08-24 | 인코텍스 비.브이 | Method of manufacturing knitted briefs |
| US5198349A (en) * | 1986-01-03 | 1993-03-30 | Genetics Institute, Inc. | Method for producing factor VIII:C and analogs |
| FI98829C (en) * | 1986-01-27 | 1997-08-25 | Chiron Corp | Process for Preparation of Recombinant Protein Complex with Human Factor VIII: C Activity |
| US5595886A (en) * | 1986-01-27 | 1997-01-21 | Chiron Corporation | Protein complexes having Factor VIII:C activity and production thereof |
| US5422260A (en) * | 1986-05-29 | 1995-06-06 | Genetics Institute, Inc. -Legal Affairs | Human factor VIII:c muteins |
| US5451521A (en) * | 1986-05-29 | 1995-09-19 | Genetics Institute, Inc. | Procoagulant proteins |
| IL84168A0 (en) * | 1986-10-15 | 1988-03-31 | Rorer Int Overseas | Human factor viii-c analogs,process for the preparation thereof and pharmaceutical compositions containing the same |
| US6060447A (en) * | 1987-05-19 | 2000-05-09 | Chiron Corporation | Protein complexes having Factor VIII:C activity and production thereof |
| EP0690126B1 (en) * | 1987-06-12 | 2001-11-28 | Baxter Aktiengesellschaft | Novel proteins with factor VIII activitiy: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them |
| AU627150B2 (en) * | 1987-06-12 | 1992-08-20 | Immuno Ag | Novel proteins with factor viii activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them |
| US6346513B1 (en) | 1987-06-12 | 2002-02-12 | Baxter Trading Gmbh | Proteins with factor VIII activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10399009I1 (en) * | 1984-01-12 | 2003-10-23 | Chiron Corp | DNA sequences encoding factor VIIIc and related DNA constructions |
| ZA852099B (en) * | 1984-03-26 | 1985-12-24 | Meloy Lab | Recombinant factor viii-c. |
| IL74909A (en) * | 1984-04-20 | 1992-01-15 | Genentech Inc | Preparation of functional human factor viii and dna sequences,expression vectors,transformed microorganisms and cell lines used therein |
-
1986
- 1986-04-11 CA CA000506408A patent/CA1341174C/en not_active Expired - Lifetime
- 1986-04-11 AT AT86902972T patent/ATE72838T1/en active
- 1986-04-11 MX MX9203440A patent/MX9203440A/en unknown
- 1986-04-11 EP EP86902972A patent/EP0218712B1/en not_active Expired - Lifetime
- 1986-04-11 JP JP61502448A patent/JPH0788399B2/en not_active Expired - Lifetime
- 1986-04-11 DE DE8686902972T patent/DE3683980D1/en not_active Expired - Lifetime
- 1986-04-11 US US07/010,085 patent/US4868112A/en not_active Expired - Lifetime
- 1986-04-11 WO PCT/US1986/000774 patent/WO1986006101A1/en active IP Right Grant
- 1986-04-11 ES ES553935A patent/ES8801674A1/en not_active Expired
- 1986-04-11 AU AU57728/86A patent/AU5772886A/en not_active Abandoned
- 1986-12-12 DK DK601786A patent/DK166457B1/en not_active IP Right Cessation
-
1990
- 1990-06-18 AU AU57534/90A patent/AU629649B2/en not_active Ceased
-
1999
- 1999-10-12 LU LU90458C patent/LU90458I2/en unknown
Also Published As
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|---|---|
| WO1986006101A1 (en) | 1986-10-23 |
| AU629649B2 (en) | 1992-10-08 |
| DK601786D0 (en) | 1986-12-12 |
| ES553935A0 (en) | 1988-02-16 |
| JPS62502941A (en) | 1987-11-26 |
| LU90458I2 (en) | 2001-04-13 |
| AU5753490A (en) | 1991-02-07 |
| ATE72838T1 (en) | 1992-03-15 |
| EP0218712A1 (en) | 1987-04-22 |
| AU5772886A (en) | 1986-11-05 |
| DK601786A (en) | 1986-12-12 |
| DK166457B1 (en) | 1993-05-24 |
| EP0218712B1 (en) | 1992-02-26 |
| DE3683980D1 (en) | 1992-04-02 |
| MX9203440A (en) | 1992-07-01 |
| ES8801674A1 (en) | 1988-02-16 |
| EP0218712A4 (en) | 1987-10-27 |
| US4868112A (en) | 1989-09-19 |
| JPH0788399B2 (en) | 1995-09-27 |
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