AU682147B2

AU682147B2 – Hybrid human/porcine factor VIII
– Google Patents

AU682147B2 – Hybrid human/porcine factor VIII
– Google Patents
Hybrid human/porcine factor VIII

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Publication number
AU682147B2

AU682147B2
AU42799/93A
AU4279993A
AU682147B2
AU 682147 B2
AU682147 B2
AU 682147B2
AU 42799/93 A
AU42799/93 A
AU 42799/93A
AU 4279993 A
AU4279993 A
AU 4279993A
AU 682147 B2
AU682147 B2
AU 682147B2
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AU
Australia
Prior art keywords
factor viii
human
porcine
ser
leu
Prior art date
1992-04-07
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AU42799/93A
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AU4279993A
(en

Inventor
John S Lollar
Marschall S Runge
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Emory University

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Emory University
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1992-04-07
Filing date
1993-04-07
Publication date
1997-09-25
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1993-04-07
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Emory University

1993-11-08
Publication of AU4279993A
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patent/AU4279993A/en

1997-09-25
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1997-09-25
Publication of AU682147B2
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patent/AU682147B2/en

2013-04-07
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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

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K2319/00—Fusion polypeptide

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

Abstract

The nucleic acid sequence encoding the porcine factor VIII A2 domain is disclosed.The nucleic acid sequence encoding the porcine factor VIII A2 domain is disclosed.

Description

GPI DATE 08/11/93 AOJP DATE 13/01/94 APPLN. ID PCT NUMBER 42799/93 PCT/US93/03275 1111111 111111illl ll II AU9342799 tPCT) (51) International Patent Classification 5 C07H 21/00, C12N 15/00, 15/11 C12N 15/12, A61K 37/02
I
(ll) lnternational Publication Number: (43) International Publication Date: WO 93/20093 14 October 1993 (14.10.93) (21) International Application Number: (22) International Filing Date: PCT’US93’03275 7 April 1993 (07.04.93) (81) Designated States: AU. CA, JP, European patent (AT, BE, CH. DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).
Published Ifth international search report.
Priorit data: 864.004 7 April 1992 (07.04.92) (71) Applicant: EMORY UNIVERSITY [US US]; 1380 South Oxford Road, Atlanta, GA 30322 (US).
(72) Inventors: LOLLAR, John. S. 2568 Oak Crossing Drive, Decatur. GA 30345 RUNGE, Marschall, S. 2870 Cedar Canyon Court, Atlanta, GA 30345 (US).
(74) Agents: PABST, Patrea, L, et al.; Kilpatrick Codv, 1100 Peachtree Street, Suite 2800, Atlanta, GA 30309-4530
(US).
fiP:~ 4 7 (54)Title: HYBRID HUMAN’PORCINE FACTOR Vill (57) Abstract A hybrid human/porcine coagulation factor VIlI is produced by isolation and recombination of human and porcine factor VI subunits, or b% genetic engineering of the human and porcine factor VIII genes. Subunits of factor VIIl that have been puri.
fied from human or porcine plasma are isolated, and hybrid human/porcine factor Vlll is produced by mixing either porcine heavy chain subunits with human light chain subunits or by mixing human heavy chain subunits with porcine light chain subuni:s. thereby producing human light chain’porcine heavy chain and human heavy chain ‘porcine light chain hybrid molecules.
These hybrid molecules are isolated by ion exchange chromatography. Alternatively, recombinant DNA methods are used to swap elements of porcine factor VllI for the corresponding elements of human factor Vl11 to produce hybrid human’porcine factor VIII.
I
C_ WO 93/20093 PC//S93/03275 HYBRID HUMAN/PORCINE FACTOR VIII The government has rights in this invention arising from National Institutes of Health Grant No.
HL 40921 that partially funded the research leading to this invention.
Background of the Invention This invention relates generally to a hybrid human/porcine factor VIII and methods of preparation and use thereof.
Blood clotting begins when platelets adhere to the cut wall of an injured blood vessel at a lesion site. Subsequently, in a cascade of enzymatically regulated reactions, soluble fibrinogen molecules are converted by the enzyme thrombin to insoluble strands of fibrin that hold the platelets together in a thrombus. At each step in the cascade, a protein precursor is converted to a protease that cleaves the next protein precursor in the series. Cofactors are required at most of the steps. In its active form, the protein factor VIII is a cofactor that is required for the activation of factor X by the protease, activated factor IX.
Factor VIII or antihemophilic factor was noticed in plasma and named in the 1930s. In the 1940s, a deficiency in factor VIII was associated with the clotting disorder hemophilia A. Factor VIII was found to be X-linked and was hypothesized to be a protein.
Work involving bovine, human, and porcine plasma identified factor VIII as a protein in the 1980s, though its definitive cellular source remains uncertain.
Precisely how factor VIII functions in blood coagulation is unknown. It is known that factor VIII is activated to factor VIIIa proteolytically by thrombin or factor Xa. In combination with calcium and phospholipid, factor VIIIa makes factor IXa a more WO 93/20093 PCr/US9S3!03275 -2efficient activator of factor X by an unknown mechanism.
People deficient in factor VIII or having antibodies against factor VIII who are not treated with factor VIII suffer uncontrolled internal bleeding that may cause a range of serious symptoms, from inflammatory reactions in joints to early death.
Severe hemophiliacs, who number about 10,000 in the United States, can be treated with infusion of factor VIII, which will restore the blood’s normal clotting ability if administered with sufficient frequency and concentration. The classic definition of factor VIII, in fact, is that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A.
Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These include a partially-purified factor VIII derived from the pooled blood of many donors that is heat- and detergent-treated for viruses but contains a significant level of antigenic proteins; a monoclonal antibody-purified factor VIII that has lower levels of antigenic impurities and viral contamination; and recombinant human factor VIII, clinical trials for which are underway. Additionally, a preparation of partially-purified porcine factor VIII is available to treat patients with inhibitors to human factor VIII, those who have circulating antibody molecules that bind and neutralize human factor VIII.
Hemophiliacs require daily replacement of factor VIII to prevent the deforming hemophilic arthropathy that occurs after many years of recurrent hemorrhages into the joints. However, supplies of factor VIII concentrates have never been plentiful enough for treating hemophiliacs adequately because of problems in commercial production and therapeutic use. For
U
WO 93/200933 PCT/US93/03275 -3example, the commonly used plasma-derived is difficult to isolate and purify, is immunogenic, and requires treatment to remove the risk of infectivity from AIDS and hepatitis viruses. Recombinant human factor VIII may lessen the latter two problems. Porcine factor VIII may also present an alternative, since human factor VIII is unstable at physiologic concentrations and pH, is present in blood at an extremely low concentration (0.2 Ag/ml plasma), and its specific clotting activity is low, compared with porcine factor
VIII.
Since many inhibitors of human factor VIII react less strongly with porcine factor VIII, porcine factor VIII is currently used to correct factor VIII deficiency in patients under conditions in which they do not respond to infusions of human factor VIII. A limitation of porcine factor VIII is the development of inhibitory antibodies to it after one or more infusions.
The problems associated with the commonly used, commercially available, plasma-derived factor VIII have stimulated significant interest in the development of a better factor VIII product. There is a need for a more potent factor VIII molecule so that more units of clotting activity can be delivered per molecule; a factor VIII molecule that is stable at a selected pH and physiologic concentration; a factor VIII molecule that is less apt to produce inhibitory antibodies; and a factor VIII molecule that evades immune detection in patients who have already acquired antibodies to human factor VIII.
I is therorez aKbec3t or the present invention to provide a factor VIII that ca ec hemophilia in a patient defici tTfactor VIII or having inhibitors jf tan factor VIII.
It further object of the present invention S’-o to provide methods for treatment of hemophiliacs.
r c Summary of the Invention In a first aspect, the present invention consists in a purified procoagulant hybrid factor VIII molecule comprising porcine and human amino acid sequence.
In a preferred embodiment, at least one sequence of one or more amino acids unique to one species is substituted for the corresponding sequence of one or more amino acids of the other species, wherein the corresponding sequence of one species to be substituted by the sequence of the other species includes a determinant of coagulant activity.
In a second aspect, the present invention consists in the use of the first aspect of the invention in the manufacture of a medicament for use in treating human factor VIII deficiency.
In a third aspect, the present invention consists in a method for preparing purified hybrid human/porcine coagulation factor VIII comprising combining primary amino acid sequence derived from porcine factor VIII with primary amino acid sequence derived from human factor VIII to form a procoagulant hybrid factor VIII molecule.
In a fourth aspect, the present invention consists in an isolated nucleic acid sequence encoding procoagulant hybrid human/porcine factor VIII molecule of the first aspect of the invention.
In a fifth aspect, the present invention consists in an isolated nucleic acid sequence encoding porcine factor VIII A2 domain.
In a sixth aspect, the present invention consists in a purified procoagulant hybrid factor VIII molecule comprising porcine and human amino acid sequence, wherein a factor VIII light or heavy chain of one species, at least one factor VIII domain of one species, or at least one sequence of at least one or more amino acids unique to the factor VIII of one species is substituted for the corresponding heavy or light chain, domain, or amino acid I I 4/1 sequence of the other species, and wherein the hybrid factor VIII molecule lacks the B domain.
In a seventh aspect, the present invention consists in the use of the sixth aspect of the invention in the manufacture of a medicament for treating human factor VIII deficiency or patients having antibodies to factor VIII that inhibit coagulant activity.
I
WO 93/20093 PC/US93/03275 swapping of domains or porcine and human factor VII or creation of hybrid human/porcine factor by replacement of specific amino acid resid of human factor VIII with the homologous ine factor VIII amino acid residues by si irected mutagenesis.
The resultin rid human/porcine factor VIII has specifj activity greater than human factor VIII ane ual to or slightly higher than porcine factor
VIII.
Brief Description of the Drawings Figure 1 (Prior Art) is a diagrammatic representation of factor VIII molecule showing the subunits (heavy and light chains) and the domains.
Detailed Description of the Invention Definitions As used herein, “hybrid human/porcine factor VIII” denotes a functional factor VIII protein molecule with sequence derived from human and porcine factor VIII. This hybrid human/porcine factor VIII has a specific activity equal to or greater than that of porcine factor VIII and has activity in a human factor VIII assay. In some embodiments, this hybrid human/porcine factor VIII is not cross-reactive with all human factor VIII antibodies.
“Specific activity,” as used herein, refers to the activity that will correct the coagulation defect of human factor VIII deficient plasma. Specific activity is measured in units of clotting activity per milligram total factor VIII protein in a standard assay in which the clotting time of human factor VIII deficient plasma is compared to that of normal human plasma. One unit of factor VIII activity is the activity present in one milliliter of normal human plasma. In the assay, the shorter the time for clot formation, the greater the activity of the factor VIII being assayed.
ji
II
L-~L I- WO 93/20093 PCT/US93/03275 -6- A “hybrid factor VIII” or “hybrid protein,” as used herein, is a factor VIII protein in which the amino acid sequence is derived in part from human and in part from porcine origin. This hybrid factor VIII can be made by substitution of isolated, plasmaderived porcine or human subunits (heavy or light chains) for corresponding human or porcine subunits; by substitution of human or porcine domains (Al, A2, A3, B, Cl, and C2) for corresponding porcine or human domains; by substitution of parts of human or porcine domains for parts of porcine or human domains; or by changing one or more amino acid residue(s) in human factor VIII to the residue(s) in the corresponding porcine sequence. A fusion protein is the product of a hybrid gene in which the coding sequence for one protein is extensively altered, for example, by fusing part of it to the coding sequence for a second protein from a different gene to produce a hybrid gene that encodes the fusion protein. As used herein, a fusion protein is a subset of the hybrid protein described in this application.
“Factor VIII deficiency,” as used herein, includes deficiency in clotting activity caused by production of a defective factor VIII, by inadequate or no production of factor VIII, or by partial or total inhibition of factor VIII by inhibitors.
Hemophilia A is a type of factor VIII deficiency resulting from a defect in an X-linked gene and the absence or deficiency of the factor VIII protein it encodes.
“Subunits” of human or porcine factor VIII, as used herein, are the heavy and light chains of the protein. The heavy chain of factor VIII contains three “domains,” Al, A2, and B. The light chain of factor VIII also contains three “domains,” A3, CI, and C2.
I WO 93/20093 PCT/US93/03275 -7- General Description of Methods Hybrid human/porcine factor VIII molecules that have greater activity in a standard clotting assay when compared to highly-purified human factor VIII can be constructed as follows.
Four types of hybrid human/porcine factor VIII and the methods for preparing them are disclosed herein: those obtained by substituting a porcine subunit heavy chain or light chain) for the corresponding human subunit; by substituting a porcine domain Al, A2, A3, B, Cl, and C2) for the corresponding human domain; and by substituting part of a porcine domain for the corresponding fragment of the human domain; and by changing one or more amino acid residue(s) in human factor VIII to the residue(s) in the corresponding porcine sequence. The hybrid molecule may contain a greater percentage of human than porcine sequence or vice versa, depending on the origin of the various regions, as described in more detail below.
It is shown below that hybrid human/porcine factor VIII consisting of porcine heavy chain/human light chain and corresponding to the first type of hybrid listed above has greater specific coagulant activity in a standard clotting assay as compared to human factor VIII. The hybrid human/porcine factor VIII can be useful in treating patients with inhibitors, since these inhibitors can react less well with hybrid human/porcine factor VIII than with either.
human or porcine factor VIII.
Hybrid human/porcine factor VIII proteins listed above under group are made by isolation of subunits of plasma-derived factor VIII, followed by reconstitution and purification. Hybrid human/porcine factor VIII proteins described under group above are made by recombinant DNA methods.
.I I I I WO 93/20093 PCT/US93/03275 -8- Preparation of hybrid human/porcine factor VIII molecules from isolated human and porcine factor VIII subunits by reconstitution: Hybrid human/porcine factor VIII molecules are prepared and isolated, and their procoagulant activity is characterized. One method, modified from procedures reported by Fay, et al., 265 J. Biol.
Chem. 6197 (1990), the teachings of which are incorporated herein; and Lollar, et al., 263 J.
Biol. Chem. 10451 (1988), the teachings of which are incorporated herein, involves the isolation of subunits (heavy and light chains) of human and porcine factor VIII, followed by recombination of human heavy chain and porcine light chain or by recombination of human light chain and porcine heavy chain.
Isolation of individual subunits from both species involves dissociation of the light chain/heavy chain dimer by chelation of calcium with ethylenediaminetetraacetic acid (EDTA), followed by Mono S T HPLC (Pharmacia-LKB, Piscataway, NJ). Hybrid human/porcine factor VIII molecules are reconstituted from isolated subunits in the presence of calcium.
Hybrid human light chain/porcine heavy chain or porcine light chain/human heavy chain factor VIII is isolated from unreacted heavy chains by Mono S T
HPLC
by procedures for the isolation of porcine factor VIII, as described by Lollar, et al., 71 Blood 137-143 (1988), the teachings of which are incorporated herein.
These methods, described in detail in the examples below, result in hybrid human light chain/porcine heavy chain molecules with greater than six times the procoagulant activity of human factor
VIII.
M
I
WO 93/20093 PCT/US93/03275 -9- Preparation of human/porcine factor VIII molecules by recombinant engineering of the sequences encoding human and porcine factor VIII subunits: The human factor VIII gene was isolated and expressed in mammalian cells, as reported by Toole, et al., 312 Nature 342-347 (1984) (Genetics Institute); Gitschier, et al., 312 Nature 326-330 (1984) (Genentech); Wood, et al., 312 Nature 330-337 (1984) (Genentech); Vehar, et al., 312 Nature 337-342 (1984) (Genentech)), the teachings of each of which are incorporated herein, and the amino acid sequence was deduced from cDNA. U.S. Patent No.
4,965,199 to Capon et al., discloses a recombinant DNA method for producing factor VIII in mammalian host cells and purification of human factor VIII. Factor VIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells) has been reported.
The cDNA sequence encoding human factor VIII and predicted amino acid sequence are shown below.
Human factor VIII cDNA sequence (SEQ ID NO. 3 on Patentln disk):
CAGTGGGTAA
TTTTACTTTT
TCTCCAGTTG
CTGTGCCTTT
CTGTCATGGG
CCTAGAGTGC
GTAGAATTCA
CTAGGTCCTA
GCTTCCCATC
GCTGAATATG
GGAAGCCATA
CTGTGCCTTA
CTCALTGGAG
TTGCACAAAT
ACAAAGAACT
CACACAGTCA
TCAGTCTATT
GTTCCTTAAA TGCTCTGCAA AGAAATTGGG ACTTTTCATT
TTCC.CCTCCT
AACATTTGTA
TGCGATTCTG
ACTATATGCA
CAAAATCTTT
CGGTTCACCT
CCATCCAGGC
CTGTCAGTCT
ATGATCAGAC
CATATGTCTG
CCTACTCATA
CCCTACTAGT
TTATACTACT
CCTTGATGCA
ATGGTTATGT
GGCATGTGAT
GGGAGCTAAA
GCAATAAGTC
CTTTAGTGCC
AAGTGATCTC
TCCATTCA’AC
TTTCAACATC
TGAGGTTTAT
TCATGCTGTT
CAGTCAAAGG
GCAGGTCCTG
TCTTTCTCAT
ATGTAGAGAA
TTTi:GCTGTA
GGATAGGGAT
AAACAGGTCT
TGGAATGGGC
GATATTTTAG
ATGCAAATAG
ACCAGAAGAT
GGTGAGCTGC
ACCTCAGTCG
GCTAAGCCAA
GATACAGTGG
GGTGTATCCT
GAGAAAGAAG
AAAGAGAATG
GTGGACCTGG
GGGAGTCTGG
TTTGATGAAG
GCTGCATCTG
CTGCCAGGTC
A’eCACTCCTG
AGAAGAATTA
AGCTCTCCAC
ACTACCTGGG
CTGTGGACGC
TGTACAAAAA
GGCCACCCTG
TCATTACACT
ACTGCAAAGC
ATGATAAAGT
GTCCAATGGC
TAAAAGACTT
CCAAGGAAAA
GG.AAAAGTTG
CTCGGGCCTG
TGATTGGATG
AAGTGCACTC
AAATCAGAAA
ACCTTTTGCT
CTGCTTCTT
TGCAGTGGAA
AAGATTTCCT
GACTCTGTTT
GATGGGTCTG
TAAGAACATG
TTCTGAGGGA
CI’TCCCTGGT
CTCTGACCCA
GAATTCAGGC
GACACAGACC
GCACTCAGAA
GCCTAAAATG
CCACAGGAAA
AATATTCCTC
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 GAAGGTCACA CATTCTGT GAGGAACCAT CGCCAGGCGT CCTTGGAAAT CTCGCCAATA
ACTTTCCTTA
ATCTCTTCCC
GAACCCCAAC
GATTCTGAAA
CGCTCAGTTG
GACTGGGACT
TTGAACAATG
ACAGATGAAA
TTACTTTATG
CCATATAACA
CCAAAAGGTG
AAATGGACAG
TATTACTCTA
CTCATCTGCT
AATGTCATCC
CAACGCTTTC
AACATCATGC
CATGAGGTGG
CTGCTCAAAC
ACCAACATGA
TACGAATGAA
TGGATGTGGT
CCAAGAAGCA
ATGCTCCCTT
GCCCTCAGCG
CCTTTAAGAC
GGGAAGTTGG
TCTACCCTCA
TAAAACATTT
TCZACTGTAGA
GTTTCGTTAA
ACAAAGAATC
TGTTTTCTGT
TCCCCAATCC
ACAGCATCAA
C~mACTGGTA
ACTCTTGATG
TGGCATGGAA
AAATAATGAA
CAGGTTTGAT
TCCTAAAACT
AGTCCTCGCC
GATTGGTAGG
TCGTGAAGCT
AGACACACTG
CGGAATCACT
GAAGGATTTT
AGATGGGCCA
TATGGAGAGA
TGTAGATCAA
ATTTGATGAG
AGCTGGAGTG
TGGCTATGTT
CATTCTAAGC
GACCTTGGAC
GCTTATGTCA
GAAGCGGAAG
GATGACAACT
TGGGTACATT
CCCGATGACA
AAGTACAAAA
ATTCAGCATG
TTGATTATAT
GATGTCCGTC
CCAATTCTGC
ACTAAATCAG
GATCTAGCTT
AGAGGAAACC
AACCGAAGCT
CAGCTTGAGG
TTTGATAGTT
ATTGGAGCAC
AGTTTCTACT
AAGTAGACAG
ACTATGATl-G
CTCCTTCCTT
ACATTGCTGC
GAAGTTATAA
AAGTCCGATT
AATCAGGAAT
TTAAGAATCA
CTTTGTATTC
CAGGAGAAAT
ATCCTCGGTG
CAGGACTCAT
AGATAATGTC
GGTACCTCAC
ATCCAGAGTT
TGCAGTTGTC
AGACTGACTT
GTTTTGTCAT
CTGTCCAGAG
TGATCTTACT
TATCCAAATT
TGAAGAGGAG
AAGTCAATAT
TATGGCATAC
CTTGGGACCT
AGCAAGCAGA
AAGGAGATTA
ATTCAAATAT
CCTGACCCGC
TGGCCCTCTC
AGACAAGAGG
AGAGAATATA
CCAAGCCTCC
AGTTTGTTTG
CCTTTCTGTC
1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG AAGACACACT CACCCTATTC
CCATTCTCAG
TGCCACAACT
GACAAGAACA
AGTAAAAACA
AGGCAAAAGC
TGGTTTGCAC
ATGCTCTTGC
AAATATGAGA
TCTGAAATGA
GAGTCAGGCC
AAGAAACTTG
GACAATTTGG
CATTATGATA
TCTGGTGGAC
TTAATGAATA
TTATTTAAAG
AAAGTTAGCA
AAGACTCACA
GAGAAACTGT
CAGACTTTCG
CTGGTGATTA
ATGCCATTGA
AATTTAATGC
ACAGAACACC
GACAGAGTCC
CTTTTTCTGA
CACACTTCAG
TCCAATTAAG
ATTTCAAAGT
CAGCAGGTAC
GTCAATTAGA
CTCTGAGCTT
GCCAAGAAAG
GGAAAAGAGC
TCTCTTTGTT
TTGATGG CCC
CTTCATGTCG
GAACAGAGGC
TTACGAGGAC
ACCAAGAAGC
CACCACAATT
TATGCCTAAA
TACTCCACAT
TGATCCATCA
GCCACAGCTC
ATTAAATGAG
TTCTAGTACA
TGATAATACA
TACCACTCTA
GAGTGAAGAA
TTCATGGGGA
T CATGGCAC CT
AAAGACAAAC
ATCATTATTA
ATGGAAAACC
ATGACCGCCT
AGTTATGAAG
TTCTCCCAGA
CCAGAAAATG
ATACAAAATG
GGGCTATCCT
CCTGGAGCAA
CATCACAGTG
AAACTGGGGA
TCAAATAATC
AGTTCCTTAG
TTTGGCAAAA
AATAATGATT
AAAAATGTAT
GCTTTGTTGA
AAAACTTCCA
ATTGAGAATA
CAGGTCTATG
TACTGAAGGT
ATATTTCAGC
ATTCAAGACA
ACATAGAGAA
TCTCCTCTAG
TATCTGATCT
TAGACAGTAA
GGGACATGGT
CAACTGCAGC
TGATTTCAAC
GACCCCCAAG
AGTCATCTCC
CAAAGTTGTT
CGTCAACAGA
CTAAAGATAA
ATAATTCAGC
GTCCATCAGT
GATTCTGGGG
TTCTAGTTGT
ATACTTGCTG
CCCTAGCACT
GACTGACCCT
TGATTTGTTG
CCAAGAAGCC
TAACAGCCTG
ATTTACCCCT
AACAGAGTTG
AATTCCATCA
TATGCCAGTT
CCTTACTGAG
AGAATCAGGT
GAGTGGTAGG
TGCCTTATTC
AACTAATAGA
CTGGCAAAAT
2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3300 3060 3120 3180 3240 3300 ATATTAGAAA GTGACACTGA ATGGACAAAA ATGCTACAGC AAAAACATGG AAATGGTCCA CCAGATATGT CGTTCTTTAA ACTCATGGAA AGAACTCTCT TTAGGACCAG AAAAATCTGT GTAGGAAAGG GTGAATTTAC AGAAACCTAT TTCTTACTAA AAAAAAATTC AGGAAGAAAT CCTCAGATAC ATACAGTGAC ACTAGGCAAA ATGTAGAAGG AGGTCATTAA ATGATTCAAC GGGGAGGAAG AAAACTTGGA GCATGCACCA CAAGGATATC AAGAGAGCTT TGAAACAATT ATTGTGGATG ACACCTCAAC CTCACACAGA TAGACTACAA GATTGCCTTA CGAGGAGTCA GTTTAAAAAA GTGACACCTT TGATTCATGA CAGAATGCTT TTTGAGGCTA AATCATATGT CAAATAAAAC TACTTCATCA ACAGAAAAAA GAGGGCCCCA TTCCACCAGA TGCACAAAAT GATGCTATTC TTGCCAGAAT CAGCAAGGTG GATACAAAGG GAACTCTGGG CAAGGCCCCA GTCCAAAGCA ATTAGTATCC GGAAGGTCAG AATTTCTTGT CTGAGAAAAA CAAAGTGGTA AAAGGACGTA GGACTCAAAG AGATGGTTTT TCCAAGCAGC CTTGGATAAT TTACATGAAA ATAATACACA CAATCAAGAA AGAAAAGAAG GAAACATTAA TCCAAGAGAA TGTAGTTTTG TGGCACTAAG AATTTCATGA AGAACCTTTT CTTACTGAGC TTCATATGAG GGGGCATATG CTCCAGTACT TCAAGATTTT AAATAGAACA AAGAAACACA CAGCTCATTT CTCAAAAAAA AGGCTTGGGA AATCAAACCA AGCAAATTGT AGAGAAATAT TCCTAATACA AGCCAGCAGA ATTTTGTCAC GCAACGTAGT CAGACTCCCA CTAGAAGAAA CAGAACTTGA AAAAAGGATA CCAGTGGTCC AAAAACATGA AACATTTGAC CCCGAGCACC TGAGAAGGAG AA7AGGGGCCA TTACTCAGTC TCCCTTATCA TAGCATCCCT CAAGCAAATA GATCTCCATT ACCCATTGCA 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380
AAGGTATCAT
AACTCTTCTC
AGTCATTTCT
ATGACTGGTG
TACAAGAAAG
GTTGAATTGC
AATGGGTCTC
GCGATTAAGT
GAAAGCTCTG
GGTACTCAGA
TTTAAGAAAA
GCAATAAATG
ACTGAAAGGC
CGTACTACTC
ATGAAGAAGG
CAAAAGAAAA
AGTAGCTCCC
AAAGTTGTTT
CATTTCCATC
ATCTT’CCAGC
TACAAG GAG C
ATCAAAGAGA
TTGAGAACAC
TTCCAAAAGT
CTGGCCATCT
GGAATGAAGC
CAAAGACTCC
TACCAAAAGA
AGGATAC CAT
AGGGACAAAA
TGTGCTCTCA
TTCAGTCAGA
AAGATTTTGA
CACGACACTA
CACATGTTCT
TCCAGGAATT
TATTAGACCT
AGCATCTTAT
CAAAAAAAAT
GGTTGGCTCC
TGTTCTCCCG
TCACATTTAT
GGATCTCGTG
AAACAGACCT
CTCCAAGCTA
AGAGTGGAAA
TTTGTCCCTG
TAAGCCCGAA
AAACCCACCA
TCAAGAGGAA
CATTTATGAT
TTTTATTGCT
AAGAAACAGG
TACTGATGGC
ATATATCTGA
AGAAAGAAAG
AACCTTTCTT
CTGGGGACAA
AAACCAGACT
CAGAAGGACC
GAZ’GGGAGCC
GGAAAAGTTC
TTGGATCCTC
TCCCAAGAGA
AACGCTTGTG
ATAGAAGTCA
GTCTTGAAAC
ATTGACTATG
GAGGATGAAA
GCAGTGGAGA
GCTCAGAGTG
TCCTTTACTC
CCAGGGTCCT
ATTCTGGGGT
TAGCCATTCT
GTGCCACAAA
TGCCCAAAAC
TATTCCCTAC
TTCTTCAGGG
CCTTTCTGAG
TTGCTTGGGA
AGTCACCAGA
AAAGCAATCA
CCTGGGCAAA
GCCATCAACG
ATGATACCAT
ATCAGAGCCC
GGCTCTGGGA
GCAGTGTCCC
AGCCCTTATA
ATTCCAAGAC
CCAAGAAAGC
AACCTTG GAG
TTCAGTCACA
ATCTGGCAAA
GGAAACTAGC
AACAGAGGGA
AGTAGCAACA
TAACCACTAT
AAAAACAGCT
TGCAATAGCA
GCAAGGTAGG
GGAAATAACT
ATCAGTTGAA
CCGCAGCTTT
TTATGGGATG
TCAGTTCAAG
CCGTGGAGAA
4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 CTAAATGAAC ATTTGGGACT CCTGGGGCCA TATATAAGAG CAGAAGTTGA AGATAATATC
ATGGTAACTT
TATGAGGAAG
ACCAAAACTT
TGCAAAGCCT
ATTGGACCCC
ACAGTACAGG
ACTGAAAATA
TTTAAAGAGA
TTAGTAATZGG
AACATCCATT
AAAATGGCAC
AAAGCTCGAA
ACACTTTTTC
ATTAGAGATT
AGACTTCATT
AAGGTGGATC
AAGTTCTCCA
TGGCAGACTT
TCAGAAATCA
ATCAGAGGCA
ACTTTTGGAA
GGGCTTATTT
TTCTGGTCTG
AATTTGCTCT
TGGAAAGAAA
ATTATCGCTT
CTCAGGATCA
CTATTCATTT
TGTACAATCT
TTTGGCGGGT
TGGTGTACAG
TTCAGATTAC
ATTCCGGATC
TGTTGGCACC
GCCTCTACAT
ATCGAGGAAA
GGCCTCTCGT
AGGAGCAGAA
AGTGCAACAT
CTCTGATGTT
CCACACTAAC
GTTTTTCACC
CTGCAGGGCT
CCATGCAATC
AAGGATTCGA
CAGTGGACAT
CTATCCAGGT
GGAATGCCTT
CAATAAGTGT
AGCTTCAGGA
AATCAATGCC
AATGATTATT
CTCTCAGTTT
TTCCACTGGA
CCCTATTCCT
CCTAGAAAAA
CATATGGCAC
GACCTGGAAA
ACACTGAACC
ATCTTTGATG
CCCTGCAATA
AATGGCTACA
TGGTATCTGC
GTGTTCACTG
GTTTTTGAGA
ATTGGCGAGC
CAGACTCCCC
CAATATGGAC
TGGAGCACCA
CACGGCATCA
ATCATCATGT
ACCTTAATGG
TCTATTCTAG
ACTTTGTCAA
CCACTAAAGA
AAGATGTCCA
CTGCTCATGG
AGACCAAAAG
TCCAGATGGA
TAATGGATAC
TCAGCATGGG
TACGAAAAAA
CAGTGGAAAT
ATCTACATGC
TGGGAATGGC
AGTGGGCCCC
AGGAGCCCTT
AGACCCAGGG
ATAGTCTTGA
TCTTCTTTGG
CCTTATTTCT
GCCTAATGAA
TGAGTTTGAC
CTCAGGCCTG
GAGACAAGTG
CTGGTACTTC
AGATCCCACT
ACTACCTGGC
CAGCAATGAA
AGAGGAGTAT
GTTACCATCC
TGGGATGAGC
TTCTGGACAC
AAAGCTGGCC
TTCTTGGATC
TGCCCGTCAG
TGGGAAGAAG
CAATGTGGAT
5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600
TCATCTGGGA
CACCCAACTC
AATAGTTGCA
GCTTCATCCT
CTCCAAGGGA
GTGGACTTCC
CTTACCAGCA
ACTCTCTTTT
CCTGTGGTGA
AGTTGGGTGC
TACTGAGGGT
GGCAGTGTCC
TGAAGCCTCC
TGCATCCAAT
CTTCCAATAT
GAAAAGTTAG
CTTTGAAAAA
AACTCTCAGT
TAAAACACAA
ATTATAGCAT
GCATGCCATT
ACTTTACCAA
GGAGTAATGC
AGAAiGACAAT
TGTATGTGAA
TTCAGAATGG
ACTCTCTAGA
ACCAGATTGC
GGCCACTGCA
CTCCCTGGCT
TGAATTAACT
TTAACTTAA
AACTAGGCAA
GCCTCTCAGA
GATATTTATG
TGTTTATTAT
TATTTTTAAC
TCGCAGCACT
GGGAATGGAG
TAVGTTTGCC
CTGGAGACCT
GAAAGTCACA
GGAGTTCCTC
CAAAGTAAAG
CCCACCGTTA
CCTGAGGATG
GCACCTGCCA
TGCCTTCTAC
ATCATCAGTC
TCTTACCTAT
AAAGAAGTGA
GTCZA’CCACTT
ATGTTAACAT
CCTGATCAAG
CCTCCAATTA
CTTCGCATGG
AGTAAAGCAA
ACCTGGTCTC
CAGGTGAATA
GGAGTAACTA
ATCTCCAGCA
GTTTTTCAGG
CTGACTCGCT
GAGGTTCTGG
CTGCCGTCAC
CTTTGTGCTA
CTGCATTTCT
TTTCTGCAGC
GGAGAAACCT
CCTCTGTTGT
TTCAGGTTAA
CATGGAACAA
TTGCTCGATA
AGTTGATGGG
TATCAGATGC
CTTCAAAACGC
ATCCAAAAGA
CTCAGGGAGT
GTCAAGATGG
GAAATCAAGA
ACCTTCGAAT
GCTGCGAGGC
CTCTCCCTCC
AATCCTAGCA
TTGGTGGGGG
TGCTCCCAGA
GCATGAAAGC
AGAAAAACTA
GCCTCATACG
AGCATGTTTC
CATCCGTTTG
CTGTGATTTA
ACAGATTACT
TCGACTTCAC
GTGGCTGCAA
AAAATCTCTG
CCATCAG IGG
CTCC-TTCACA
TCACCCCCAG
ACAGGACCTC
TCAGCTCCAG
GACACTGCCT
GCCAGGAGGG
TTACTCCTTC
ATTCTTCCCT
TGTGATGAAA
TTTAAAATAA
AGGATCAGAT
6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7330 7440 7500 7560 7620 7680 7740 CAATACAATC TTGGAGTCAA AAGGCAAATC ATTTGGACAA TCTGCAAAAT GGAGAGAATA
CAATAACTAC
TTAGTCATTA
TAGATGGGGT
CAAATGTGCA
CAATAAAAAA
CTTCTTGAAA
CATACATTTA
TACAAACTTT
CTGAAA.ATAA
GTCCTACTTA
GAAAAAACAC
CCCCATAAGA
CTACACAGAA
TGGAGGAAGC
AAAGAAAAAT
GAGTATTTTC
CTGTCACAGT
AAGTTCTTAA
TACAGTAAAG
TGAGGGGCAC
TCAAGAATCC
TTTTTCTGAC
ATAAGTCAGG
TTTGTGATGG
ATATTTCTGT
GTAATTCTAA
CACAACAAAA
CATAGTTGAA
TCCAGTCTGC
TTGTGAAGGG
CTCTCCTGAT
AT CCAAAG AC
GGATCCCAAT
TAATAATCCT
ATAGTCACAA
AGTTTAGAGG
TCTGTTTCTG
ATTCTTATCT
CTAAGTCCCC
GAGTGTCCAT
AGGATGCAAT
CCAAGAAAGA
GGAAATATGA
TAATGCACT”C
ATGTAACAGG
ATATCAAGGA
CATATCACCA
TTTACTGCTC
AGTAAAGGGG
TGCAACCCAG
CTGAGAAAAG
GCTTGACCCT
TCCACAAATG
CTAACTTACA
CTTCCTTACA
CCAAAACTAG
TGAAATTATA
AGATATAAAG
TGTTGAAAGC
AAATGATGAT
GGAAAATCCA
AGTTTACTCT
GGAAATTATA
GGTCAGAAGA
CACAATAGGA
CTTCCATCTG
GCTGGAGGCA
GGCAAATGGA
GCAAAACAAT
TATCTG–ACCT
ATGCAGGTGC
GAAATGAATA
CATAGATATA
CATTCT’TAAA
TAAGGCATTC
CCATTGGTCT
TTTGAAATAA
GACATTAGGC
TGGTTATCTG
CTCCCTCTAC
TACCGTGACT
AAATTGGACT
TCCCCCTTCT
CCTGCACCCC
AGGATAAGTT
AAACAGGAGA
GGCTACTTTT
CTTTGGAAAC
AAATGGTTTA
AGTTGTTTTG
ATTATGTTAT
CTGAGAATTA
TGTATAAATG
TAATTCTGAC
AATAACATGT
TTCTAAAGGA
AGATAGGAGA
TAATTTCCTG
GAAAACTAGA
GGTGAAAACA
TGCCCTCCAC
TTCACTATGA
ATAGAGCAGT
TCCTAATATG
TTCTATGCTG
TATAACATAG
TAGCCCTGTG
TTTTATAGCC
7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 CGGTAGAGGA GTTAACCCCA AAGGTGATAT GGTTTTATTT CCTGTTATGT TTAACTTGAT AATCTTATTT TG–GCATTCTT TTCCCATTGA CTATATACAT CTCTATTTCT CAAATGTTCA TGGAACTAGC TCTTTTATTT TCCTGCTGGT TTCTTCAGTA ATGAGTTAAA TAAAACATTG
ACACATACA
8880 8940 9000 9009 Predicted human factor VIII ATRRYYLGAV ELSWDYMQSD LGELPVDARF IAKPRPPWMG LLGPTIQAEV YDTVVITLKN REKEDDKVFP GGSHTYVWQV LKENGPMASD EGSLAKEKTQ TLH-KFILLFA VFDEGKSWHS SLPGLIGCHR KSVYWHVIGM GTTPEVHSIF MDLGQFLLFC HISSHQHDGM EAYVKVDSCP DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE RKYKKVRFMA YTDETFKTRE AIQHESGILG TDVRPLYSRR LPKGVKHLKD FPILPGETFK RDLASGLIGP LLICYKESVD QRGNQIMSDK VQLEDPEFQA SNIMHSINGY VFDSLQLSVC KMVYEDTLTL FPFSGETVFM SMENPGLWIL DSYEDISAYL LSKNNAIEPR SFSQNSRHPS amino acid sequencs (SEQ ID NO. 4 PPRVPKSFPF NTSVVYKKTL FVEFTVHLFN MASHPVSLHA VGVSYWKASE GAEYDDQTSQ PLCLTYSYLS HVDLVKDLHS GLIGALLVCR ETKNSLMQDR DAASARAWPK MHTVNGYVNR LEGHTFLVRN HRQASLEISP ITFLTAQTLL EEPQLRMKNN EEAEDYDDDL TDSEMDVVRF EDWDYAPLVL APDDRSYKSQ YLNNGPQRIG PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RNVILFSVFD ENRSWYLTEN IQRFLPNPAG LHEVAYWYIL SIGAQTDFLS VFFSGYTFKH GCHNSDFRNR GMTALLKVSS CDKNTGDYYE TRQKQFNAr2T IPENDIEKTD PWFAHRTP4P on Patent In disk): 120 180 240 300 360 420 480 540 600 660 720 780 KIQNVSSSDL LMLLRQSPTP LHHSGDMVFT PESGLQLRLN TSSLGPPSMP VHYDSQLDTT GKNVSSTESG RLFKGKRAHG LIENSPSVWQ NILESDTEFK KEGPIPPDAQ NPDMSFFKML QNFLSEKNKV VVGKGEFTKD KETLIQENVV LPQIHTVTGT TKKHTAHFSK KGEEENLEGL PLEETELEKR IIVDDTSTQW PQANRSPLPI AKVSSFPSIR NNLSLAILTL EMTGDQREVG YQKDLFPTET SNGSPGHLDL HGLSLSDLQE AKYETFSDDP SPGAIDSI4NS EKLGTTAATE LKKLDFKVSS TSNNLISTIP LFGKKSSPLT ESGGPLSLSE ENNDSKLLES PALLTKDNAL FKVSISLLKT NKTSNNSATN KVTPLIHDRM LMDKNATALR FLPESARWIQ RTHGKNSLNS VGLKEMVFPS SRNLFLTNLD KNFMKNLFLL STRQNVEGSY GNQTKQIVEK YAC.TTRI SPN SKNMKHLTPS TLTQIDYNEK PIYLTRVLFQ DNSSHLPAAS SLGTSATNSV TYKKVENTVL VEGSLLQGTE GAIKWNEANR
LNHMSNKTTS
GQGPSPKQLV
NLH-ENNTHNQ
EGAYAPVLQD
TSQQNFVTQR
EKGAITQSPL
YRKKDSGVQE
PKPDLPKTSG
PGKVPFLRVA
LNACESHAI
EIDYDDTISV
RAQSGSVPQF
RPYSFYSSLI
VDLEKDVHSG
LSEMTHFRPQ
SDNLAAGTDN
GLMRSQESSW
RKTHIDGPSL
SKNMEMVQQK
SLGPEKSVEG
EKKIQEEIEK
FRSLNDSTNR
SKRALKQFRL
SDCLTRSHSI
SSH-FLQGAKK
KVELLPKVH I
TESSAKTPSK
AAINEGQNKP
EMKKEDFDIY
KKVVFQEFTD
SYEEDQRQGA
LIGPLLVCHT
840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 LLDPLAWDNH YGTQIPKEEW KSQEKSPEKT AFKKKDTILS EIEVTWAKQG RTERLCSQNP PVLKRHQREI TRTTLQSDQE DEDENQSPRS FQKKTRHYFI AAVERLWDYG MSSSPHVLRN GSFTQPLYRG ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS EPRKNFVKPN ETKTYFWKVQ HHMAPT-KDEF DCKAWAYFSD NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR APCNIQMEDP TFKENYRFH-A INGY IMDTLP
GVFETVEMLP
GQYGQWAPKL
FIIMYSLDGK
TLRI4ELMGCD
PQVNNPKEWL
KVFQGNQDSF
GLVMAQDQRI
SKAGIWRVEC
ARLHYSGSIN
KWQTYRGNST
LN SCSMPLG4 QVD FQKTMKV
TPVVNSLDPP
RWYLLSMGSN
LIGEHLHAGM
AWSTKEPFSW
GTLMVFFGNV
ESKAISDAQ-I
TGVTTQGVKS
LLTR\.LRIHP
ENIHSIHFSG
STLFLVYSNK
IKVDLLAPMI
DSSGIKHNIF
TASSYFTNMF
LLTSMYVKEF
QSWVHQIALR
HVFTVRKKEE YKMALYNLYP CQTPLGMASG HIRDFQITAS IHGIKTQGAR QKFSSLYISQ NPPIIARYIR LHPTH-YSIRS ATWSPSKARL HLQGRSNAWR LISSSQDGHQ WTLFFQNGKV
MEVLGCEAQDLY
1980 2040 2100 2160 2220 2280
_I
WO 93/20093 PCT/US93/03275 -21- Recombinant hybrid human/porcine factor VIII is prepared starting with human cDNA (Biogen, Inc.) encoding the factor VIII sequence corresponding to domains A1-A2-A3-Cl-C2. This cDNA lacks the entire B domain and corresponds to residues 1-740 and 1649-2332 of single chain human factor VIII, according to the numbering system of Wood et al., 312 Nature 330-337 (1984), the teachings of which are incorporated herein. The B domain is deleted, since it does not appear to be necessary for biological function.
Porcine factor VIII has been isolated and purified from plasma (Fass, et al., 59 Blood 594 (1982). The amino acid sequence of the B and part of the A2 domains of porcine factor VIII, as reported by Toole, et al., 83 Proc. Nat’l. Acad. Sci. U.S.A.
5939-5942 (1986), the teachings of which are incorporated herein, and the corresponding genomic DNA sequence are shown below. The coding region in the nucleotide sequence begins at position 675 (GGT CTC TGG which corresponds to amino acids (Gly-Leu- Tyr), the NH2 terminal amino acids.
Genomic DNA sequence of the B and part of the A2 domains of porcine factor VIII (SEQ ID NO. 5 on Patentln disk: GAATTCTTCA CTCAGATTCT rCTGTTCACA GTAGAAATTC AGTATTGTTA GCACTCTTTT AGTTACCTGT ATCCTAAACC TCAGAGTATG TGTTTGG CAT CTCATTTACA AATTAGAAAA CAGCTGGTAA ATTGTATAGC CACTATGTTG CATAGCATAA TATCATCACC AAAGAGTCCG CTCCATTTTT ATCCCAATAT AGGTACACTA GAGCCATGGT GATATAATGG TACCGACTAG AGAGAACCTC TAACACAGAT CTTCCTTATC TCCAGGTCTC GGATGACAGC CTTACTGAAG ACACTTATGA AGATATTCCA GCTTTGCCCA GAATTCAAGA GTCCAGAAGA TGACGTGGAG AACTAAGTGT CCCCTCTGGT TAAGTCCTGC TCCCTTATAC TTACTCATCC TACAAATTGG TATGTTATGT GATTTGAATG CATTATCAGA TACTACTAGT CTGGAGCTCA GAGAGTTCCT TGGACTTGCT TAAAGCAACA TAGGATTCGA ACCGAGGCAA TCGTACTCTA GAACCCATGC TAGCCCGCCT ATATAAACTT GGCTGAATTA AGTCACGATC TGTGACTAAG AGTCTCAACT ATTGTATGTC AATTATATTT ATATTCATTT AAATCACAGC CCTTTCTTGT GGTCACAAAC TGGGCTGCAG TCCATGGTGT ACATTTAACC CAACGACCTC TGTTT GTTT TTGTTTTTGT TTCATTTTTC TGGGAATAGA CTTGCTTGGG ACCTGGGCTG TGAGTAACCA GAGTTTTATT TGGGTCCTAG GGTGCCACAA CTCAGACTTG CGGAACAGAG GTGTATAGTT GTGACAGGGA CACTGGTGAT TATTATGACA GGCTTCTTGC TGAGT 3AAA GAATGTCATT GAACCCAGAA CCCCC’TAGTG CGAGCCAAXAA GCAATTCCAA ACCATCACAA CTTGACCCGC AGTCTGGAGA GAGAACCCAA GCACTGGAAG GATGGGTCGA TGCTCTTGGG ACAGAATCCT GCTCCACATG 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 GCTCATCCTC ATCTGATCTT CAAGAAGCCA GGAATGAGGC TGATGATTAT TTACCTGGAG 1080 CAAGAGAAAG AAACACGGCC CCATCCGCAG CGGCACGTCT CAGACCAGAG CTGCATCACA 1140 GTGCCGAAAG AGTACTTACT CCTGAGCCAG AGAAAGAGTT GAAGAAACTT GATTCTTAAA 1200 TGTCTAGTTC ATCAGACCTT CTAAAGACTT -‘CCAACAAT TCCATCAGAC ACGTTGTCAG 1260 Predicted amino acid sequence of the B and part of the A2 domains of porcine factor VIII, based on genomic DNA sequence above (SEQ ID NO. 6 in Patentln disk): (Amino acid residues labeled “Xaa”l represent deletions in porcine B domain relative to human B domain.) Gly Leu Trp Val Leu Giy Cys His Met Ser Asp Leu Arg Asn Arg Gly 1 5 10 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Thr Gly Asp L.j 25 Tyr Tyr Asp Asn Thr Tyr Glu Asp Leu Pro Gly Phe Leu Leu Ser Giy 40 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gin Asn Ser Arg Pro Pro 55 Ser Ala Ser Gin Lys Gin Phe Gin Thr Ile Thr Ser Pro Glu Asp Asp 70 75 Val Glu Leu Xaa Asp Pro Gin Ser Gin Glu Arg Thr Gin Ala Leu Giu 90 Glu Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gin Asn 100 105 Pro Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gin Glu Ala Arg Asn 115 120 125 Glu Ala Xaa Xaa Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Asn Thr 130 135 140 Ala Pro Ser Ala Ala Ala Arg Leu Arg Pro G.Lu Leu His His Ser Ala 145 150 155 160 Glu Arg Val Leu Thr Pro Glu Pro Glu Lys Xaa Xaa Xaa, Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Giu Leu Lys Lys Leu Asp Ser Lys 180 185 190 Met Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser 195 200 205 Asp Thr Leu Ser Ala Giu Thr Glu Arg Thr His Ser Leu Gly Pro Pro 210 23,5 220 His Pro Gin Val Asn Phe Arg Ser Gin Leu Gly Ala Ile Val Lou Gly 225 230 235 240 Lys Asn Ser Ser His Phe Ile Gly Ala Gly Val Pro Leu Gly Ser Thr 245 250 255 Glu Phe Asp His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270 Xaa Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser Asp Gly 275 280 285 Ile Phe Glu Lys Giu Arg Ala His Gly Pro Ala Ser Leu Thr Lys Asp 290 295 300 Asp Val Leu Phe 305 Arg Val Tyr Leu Leu Leu Thr Giu 340 Thr Ala Ser Gly 355 Xaa Xaa Xaa Xaa 370 Xaa Xaa Xaa Xaa 385 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 Asn Pro Leu Ser 435 Ser Gly Ser Gly 450 Ile Arq Val Ala 465 Lys Glu Met Met Lys Val Asn Ile Ser Leu Val Lys 310 315 Lys Thr Asn Arg Lys Ile His Ile 325 330 Asn Arg Ala Ser Ala Thr Phe Met 345 Leu Asn His Val Ser Asn Xaa Xaa 360 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 390 395 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 405 410 Xaa Xaa Xaa Xaa Trp Ile Lys Gly 425 Ser Giu Arg Gly Pro Ser Pro Glu 440 Lys Ser Val Lys Gly Gin Ser Ser 455 460 Val Giu Glu Giu Glu Leu Ser Lys 470 475 Thr Abn Lys Asp Asp Ala 335 Asp Lys Asn 350 Xaa Xaa Xaa 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 415 Pro Leu Gly 430 Leu Leu Thr 445 Gly Gin Gly Gly Xaa Xaa Ala 320 Ala Thr Xaa Xaa Xaa 400 Xaa Lys Ser Arg Xaa 480 Leu Pro 7isn Ser Glu Leu Thr Phe Leu Thr Asn Ser 485 490 495 Ala Giu Pro Phe 545 His Arg Pro Va2l Xaa 625 Glu Trp Xaa Asp G iu Gin 530 His Ala Ala Leu Pro 610 Xaa Ile Ser Xaa Val Gin Gly Asn Asp Thr 500 Met Glu Arg Arg G a Xaa 515 520 Val Tyr Thr Ala Thr Giy 535 Gin Ser Thr Giu Pro Ser 550 Pro Val Pro Gin Asp Ser 565 Giu Thr His Ile Ala His 580 Giu Ala Pro Gly Asn Phe 595 600 Arg Arg Val Lys Gin Ser 615 Xaa Xaa Xaa Xaa Xaa Leu 630 Lys Pro Glu Arg Gly Val 645 Xaa Xaa Xaa Xaa Xaa Xaa 660 Xaa Xaa Xaa Xaa Xaa Xaa 675 680 His Ser Gin Gly Lys 505 Leu Val Gin Glu Lys 525 Thr Lys Asn Phe Leu 540 Val Giu Gly Phe Asp 555 Arg Ser Leu Asn Asp 570 Phe Ser Ala Ile Arg 585 Thr Gly Pro Gly Pro 605 Xaa Xaa Xaa Xaa Xaa 620 Lys Gin Ile Arg Leu 635 Val Leu Asn Ala Thr 650 Xaa Xaa Xaa Xaa Xaa 665 Xaa Xaa Xaa Xaa Xaa 685 Lys Ser 510 Vai Asp Arg Asn Gly Gly Ser Ala 575 Giu Giu 590 Arg Ser Xaa Xaa Pro Leu Ser Thr 655 Xaa Xaa 670 Xaa Xaa Arg Leu Ile Ser 560 G iu Ala Aia Xaa Glu 640 Arg Xaa Xaa Xaa Xaa 690 Xaa Xaa 705 Xaa Xaa Xaa Xaa Gin Gly Met Ala 770 Gly Pro 785 Gly Leu Val His Ala His Pro Val 850 Xaa Xaa 865 Xaa Xaa Xaa Xaa Ala 755 Gly Leu Ser Arg Gly 835 Asn Xaa Xaa Xaa Xaa Xaa 740 Lys Gly Ala G iu Glu 820 Asp Leu Xa a Xaa Xaa Xaa 725 Xaa Arg Gin Ser Ala 805 Asp Ile Asn Xaa Xaa Xaa 695 Xaa Xaa 710 Xaa Xaa Xaa Xaa Asn Asn Gly Lys 775 Gly Lys 790 Ser Gly Leu Leu Giy Gin Lys Val1 855 Xaa Xaa 870 Xaa Xaa Xaa Xaa Leu 760 Ile Leu Lys Pro G lu 840 Asn Xaa Xaa Xaa Xaa Xaa 745 Ser Ser Giu Ala Gin 825 Ile Arg Xaa Xaa Xaa Xaa 700 Xaa Xaa Xaa 715 Xaa Xaa Xaa 730 Xaa Giu Ser Leu Pro Phe Ala Leu Gly 780 Lys Ala Vai 795 Giu Phe Leu 810 Lys Thr Ser Phe Leu Gin Pro Giy Arg 860 Thr Pro Ser 875 Xaa Xaa Xaa Xaa Xaa Xaa Ser Pro 750 Leu Thr 765 Lys Ser Leu Ser Pro Lys Asn Val 830 Lys Thr 845 Xaa Xaa Lys Leu Xaa Xaa Xaa 735 Ile Leu Ala Ser Val 815 Ser Arg Xaa Leu Xaa Xaa 720 Xaa Leu Giu Aia Ala 800 Arg Cys Gly Xaa Gly 880 Pro Trp Asp Ala Thr 945 Leu Glu Glu Phe Pro Met Pro Glu Ser Leu 900 Ile Leu Ser 915 Ala Lys Asn 930 Lys Gin Gly Arg Arg His Asp Lys Met 980 Asp Phe Asp 995 Gin Lys Arg 1010 Lys Xaa Xaa 885 Glu Lys Ser Leu Pro Lea Glu Gly Gin 935 Gly Pro Gly 950 Gin Arg Asp 965 Asp Tyr Asp Ile Tyr Gly Thr Arg His Xaa Xaa Xaa Xaa Xaa Xaa Xaa 890 Pro Lys Ser Thr Ala Leu Arg 905 910 Asp Arg His Glu Ser Asn His 920 925 Ala Phe Thr Gin Arg Glu Ala 940 Arg Leu Cys Pla Pro Lys Pro 955 Ile Ser Leu Pro Thr Pro Gin 970 Asp Ile Phe Ser Thr Glu Thr 985 990 Glu Asp Glu Asn Gin Asp Pro 1000 1005 Tyr Phe Ile Ala Ala Val Glu Xaa Glu 895 Thr Lys Ser Ile Ala Trp Pro Val 960 Pro Glu 975 Lys Gly Arg Ser Gin Leu 1015 1020 Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg Asn Arg Ala 1025 1030 1035 1040 Gin Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe Arg Glu Phe 1045 1050 1055 Ala Asp Gly Ser Phe Thr Asn Pro Ser Tyr Arg Gly Glu Leu Asn Lys 1060 1065 1070 His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn 1075 1080 1085 0 Ile Met 1090
L.J
WO 93/20093 PCT/US93/03275 Both porcine and human factor VIII are isolated from plasma as a two subunit protein. FIG. 1 (prior art) illustrates diagrammatically the subunit structure of the molecule. The subunits, known as the heavy chain and light chain, are held together by a non-covalent bond that requires calcium or other divalent metal ions. The heavy chain of factor VIII contains three domains, Al, A2, and B, which are linked covalently. The light chain of factor VIII also contains three domains designated A3, Cl, and C2.
The B domain has no known function and can be removed from the molecule proteolytically or by recombinant DNA technology methods without significant alteration in any measurable parameter of factor VIII. Human recombinant factor VIII has a similar structure and function to plasma-derived factor VIII, though it is not glycosylated unless expressed in mammalian cells.
Both human and porcine activated factor VIII (factor VIIIa) have three subunits due to cleavage of the heavy chain between the Al and A2 domains. This structure is designated A1/A2/A3-C1-C2. Human factor VIIIa is not stable under the conditions that stabilize porcine factor Villa. This is because of the weaker association of the A2 subunit of human factor VIIIa. Dissociation of the A2 subunit of human and porcine factor VIIIa is associated with loss of activity.
Since the nucleotide sequence of the porcine B domain is known, full length hybrids can be constructed. Individual domains of porcine factor VIII cDNA can be cloned and substituted for the corresponding human domains by established mutagenesis techniques. These factor VIII cDNA molecules can be cloned into expression vectors for ultimate expression of active hybrid human/porcine factor VIII protein molecules.
r I WO 93/20093 PCT/US93/03275 -31- The complete A2 domain of porcine factor VIII, homologous to residues 372-740 in mature human factor VIII, was sequenced and the amino acid sequence was predicted. These sequences are shown below.
Sense Strand Nucleotide Sequence of A2 Domain of Factor VIII (SEQ ID NO. 1 on Patent In disk):
TAAGCACCCT
CCCCG CGGTC
TCAGCGAATT
TAAGACTCGT
AGTTGGAGAC
CCCTCATGGA
ACATTTGAAA
TGTGGAAGAT
CATTAATCTA
AGAATCTGTA
TTCTGTATTC
CAATCCGGAT
CATCAATGGC
CTGGTACATT
CACCTTCAAA
AACGGTCTTC
AAGACGTGGG
CCCAGCCCCA
GGTAGGAAAT
AAAGCTATTC
ACACTTTL’CA
ATCACTGATG
GACATGCCAA
GGGCCAACCA
GAGAAAGATC
GACCAAAGAG
GATGAGAATC
GGATTACAGC
TATGTTTTTG
CTAAGTGTTG
CACAAAATGG
ATGTCAATGG
TGCACTACAT
GTGACAGAAG
ACAAAAAAGC
CGTATGAATC
TTATATTTAA
TCAGCGCTTT
TTCTGCCAGG
AGTCCGATCC
TGGCTTCGGG
GAAACCAGAT
AAAG CTGGTA
CCCAGGATCC
ATAGCTTG CA
GAGCACAGAC
TCTATGAAGA
AAAACCCAGG
CTCTGCAGAG
TTATAAAAGT
TCGATTCGTC
AGGAATCCTG
GAATAAAGCG
GCACCCAGGG
AGAGACTTTC
TCGGTGCCTG
ACTCATTGGC
GATGTCAGAC
CCTCGCAGAG
AGAGTTCCAA
GCTGTCGGTT
GGACTTCCTC
CACACTCACC
TCTCTGGGTC
GAGGAGGACT
CTCTACTTGA
GCTTACACGG
GGACCTTTAC
AGCCGACCAT
AGACTTCTAA
AAGTIT.TAAAT
ACCCGCTACT
CCTCTCCTCA
AAGAGAAACG
AATATTCAGC
GCTTCTAACA
TGTTTGCACG
TCCGTCTTCT
CTGTTCCCCT
CTAGGGTGCC
GGGACTACGC
ACAGTGGTCC
ATGTAACATT
TTTATGGAGA
ATAACATCTA
AAGGTTGGAA
GGACAGTGAC
ACTCGAGCTC
TCTGCTACAA
TCATCCTGTT
GCTTCCTCCC
TCATGCL~CAG
AGGTGGCATA
TCTCTGGCTA
TCTCAGGAGA
ACAACTCAGA
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 CTTGCGGAAC AGAGGGATGA CAGCCTTACT GAAGGTGTAT AGTTGTGACA GGGACATTGG TGATTATTAT GACAACACTT ATGAAGATAT TCCAGGCTTC TTGCTGAGTG GAAAGAATGT 1080 :f 0 CATTGAACCC AGAAGCTTTG CCCAGAATTC AAGACCCCCT AGTGCGAGCA 1130 Predicted Amino Acid Sequence of A2 domain of Porcine Factor VIII (Defined as C residues homologous to human factor VIII sequence 373-740, derived from cDNA sequence except as noted) (Underlined residues are from known porcine amino acid sequence) (SEQ ID No.2 on Patentln disk): VAKKHPKTWV HYISAEEEDW DYAPAVPSPS DRSYKSLYLN SGPQRIGRKY KKARFVAYTD VTFKTRKAIP YESGILGPlL LYGEVGDTLL IIFKNKASRP YNIYPHGITD VSALHPGRLL lzo KGWKHLKDMP ILPGETFKYK WTVTVEDGPT SDPRCLTRYY SSSINLEKDL ASGLIGPLLI 180 CYKESVDQRG NQMI4SDKRNV ILFSVFDENQ SWYLAENIQR FLPNPDGLQP QDPEFQASNI 240 MHSINGYVFD LQLSVCLHEV AYWYILSVGA QTDFLSVFFS GYTFKHKMVY EDTLTLFPFS 300 GETVFMSMEN PGLWVLGCHN SDLRNRGMTA LLKVYSCDRD IGDYYDNTYE DIPGFLLSGK 360 NV IEPR
CA
C_ I WO 93/20093 PMTUS93/03275 -34- Pharmaceutical Compositions Pharmaceutical compositions containing hybrid human/porcine factor VIII, alone or in combination with appropriate pharmaceutical stabilization compounds, delivery vehicles, and/or carrier vehicles, are prepared according to known methods, as described in Remington’s Pharmaceutical Sciences by E.W. Martin, the teachings of which are incorporated herein.
In one preferred embodiment, the preferred carriers or delivery vehicles for intravenous infusion are physiological saline or phosphate buffered saline.
In another preferred embodiment, suitable stabilization compounds, delivery vehicles, and carrier vehicles include but are not limited to other human or porcine proteins such as albumin.
Phospholipid vesicles or liposomal suspensions are also preferred as pharmaceutically acceptable carriers or delivery vehicles. These can be prepared according to methods known to those skilled in the art and can contain, for example, phosphatidylserine/phosphatidylcholine or other compositions of phospholipids or detergents that together impart a negative charge to the surface, since factor VIII binds to negatively charged phospholipid membranes.
Liposomes may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the hybrid human/porcine factor VIII is then introduced into the container. The Scontainer is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
I
WO 93/20093 PCT/US93/03275 The hybrid human/porcine factor VIII can be combined with other suitable stabilization compounds, delivery vehicles, and/or carrier vehicles, including vitamin K dependent clotting factors, tissue factor, and von Willebrand factor (vWf) or a fragment of vWf that contains the factor VIII binding site, and polysaccharides such as sucrose.
Hybrid human/porcine factor VIII can also be delivered by gene therapy in the same way that human factor VIII can be delivered, using delivery means such as retroviral vectors. This method consists of incorporation of factor VIII cDNA into human cells that are transplanted directly into a factor VIII deficient patient or that are placed in an implantable device, permeable to the factor VIII molecules but impermeable to cells, that is then transplanted. The preferred method will be retroviral-mediated gene transfer. In this method, an exogenous gene a factor VIII cDNA) is cloned into the genome of a modified retrovirus. The gene is inserted into the genome of the host cell by viral machinery where it will be expressed by the cell. The retroviral vector is modified so that it will not produce virus, preventing viral infection of the host. The general principles for this type of therapy are known to those skilled in the art and have been reviewed in the literature Kohn, and P.W. Kantoff, 29 Transfusion 812-820, 1989).
Hybrid human/porcine factor VIII can be stored bound to vWf to increase the half-life and shelf-life of the hybrid molecule. Additionally, lyophilization of factor VIII can improve the yields of active molecules in the presence of vWf. Current methods for storage of human and porcine factor VIII used by commercial suppliers can be employed for storage of hybrid human/porcine factor VIII. These methods include: lyophilization of factor VIII in a II I WO 93/20093 PCT/US93/03275 -36partially-purified state (as a factor VIII “concentrate” that is infused without further purification); immunoaffinity-purification of factor VIII by the Zimmerman method and lyophilization in the presence of aloumin, which stabilizes the factor VIII; lyophilization of recombinant factor VIII in the presence of albumin.
Additionally, hybrid hum’in/porcine factor VIII has been indefinitely stable at 40 C in 0.6 M NaCl, mM MES, and 5mM CaCI 2 at pH 6.0 and also can be stored frozen in these buffers and thawed with minimal loss of activity.
Method of Treatment Hybrid human/porcine factor VIII is used to treat uncontrolled bleeding due to factor VIII deficiency intraarticular, intracranial, or gastrointestinal hemorrhage) in hemophiliacs with and without inhibitory antibodies and in patients with acquired factor VIII deficiency due to the development of inhibitory antibodies. The active materials are preferably administered intravenously.
Additionally, hybrid human/porcine factor VIII can be administered by transplant of cells genetically engineered to produce the hybrid or by implantaticn of a device containing such cells, as described anove.
In a preferred embodiment, pharmaceutical compositions of hybrid human/porcine factor VIII alone or in combination with stabilizers, delivery vehicles, and/or carriers are infused into patients intravenously according to the same procedure that is used for infusion of human or porcine factor VIII.
The treatment dosages of hybrid human/porcine factor VIII composition that must be administered to a patient in need of such treatment will vary depending on the severity of the factor VIII deficiency.
Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and
I
WO 93/20093 PCI/US93/03275 -37duration of each patient’s bleeding episode.
Accordingly, the hybrid human/porcine factor VIII is included in the pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the hybrid to stop bleeding, as measured by standard clotting assays.
Usually, the desired plasma factor VIII level to be achieved in the patient through administration of the hybrid human/porcine factor VIII is in the range of 30-100% of normal. In a preferred mode of administration of the L brid human/porcine factor VIII, the composition is given intravenously at a preferred dosage in the range from about 20 to units/kg body weight; the interval frequency is in the range from about 8 to 24 hours (in severely affected hemophiliacs); and the duration of treatment in days is in the range from 1 to 10 days or until the bleeding episode is resolved. See, Roberts, and M.R. Jones, “Hemophilia and Related Conditions Congenital Deficiencies of Prothrombin (Factor II, Factor V, and Factors VII to XII),” Ch.
153, 1453-1474, 1460, in Hematoloy, Williams, W. J., et al., ed., 1990. Patients with inhibitors may require more hybrid human/porcine factor VIII, or patients may require less hybrid human/porcine factor VIII because of its higher specific activity than human factor VIII. As in treatment with human or porcine factor VIII, the amount of factor VIII infused is defined by the one-stage factor VIII coagulation assay and, in selected instances, in vivo recovery is determined by measuring the factor VIII in the patient’s plasma after infusion. It is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the WO< 93/200933 PCr/US9310327S -38administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Treatment '.ke the form of a single intravenous admai:.'station of the composition or periodic or continuous administration over an extended period of time, as required. Alternatively, hybrid human/porcine factor VIII can be administered subcutaneously or orally with liposomes in one or several doses at varying intervals of time. The hybrid human/porcine factor VIII molecule and the methods for isolation, characterization, making, and using it generally described above will be further understood with reference to the following nonlimiting examples. Example 1: Assay of porcine factor VIII and hybrid human/porcine factor VIII Porcine factor VIII has more coagulant activity than human factor VIII, based on specific activity of the molecule. These results are shown in Table II in Example 4. This conclusion is based on the use of appropriate standard curves that allow human and porcine factor VIII to be fairly compared. Coagulation assays are based on the ability of factor VIII to shorten the clotting time of plasma derived from a patient with hemophilia A. Two types of assays were employed: the one-stage and the two-stage assay. In the one-stage assay, 0.1 ml hemophilia A plasma (George King Biomedical, Inc.) was incubated with 0.1 ml activated partial thromboplastin reagent (APTT) (Organon Teknika) and 0.01 ml sample or standard, consisting of diluted, citrated normal human plasma, for 5 min at 37 0 C in a water bath. Incubation was followed by addition of 0.1 ml 20 mM CaCl 2 and the time for development of a fibrin clot was determined by visual inspection. L WO 93/20093 3CT/US93/03275 -39- A unit of factor VIII is defined as the amount present in 1 ml of citrated normal human plasma. With human plasma as the standard, porcine and human factor VIII activity were compared directly. Dilutions of the plasma standard or purified proteins were made into 0.15 M NaCl, 0.02 M HEFES, pH 7.4. The standard curve was constructed based cn 3 or 4 dilutions of plasma, the highest dilution being 1/50, and on logl, clotting time plotted against log 0 plasma concentration, which results in a linear plot. The units of factor VIII in an unknown sample were determined by interpolation from the standard curve. The one-stage assay relies on endogenous activation of factor VIII by activators formed in the hemophilia A plasma, whereas the two-stage assay measures the procoagulant activity of preactivated factor VIII. In the two-stage assay, samples containing factor VIII that had been reacted with thrombin were added to a mixture of activated partial thromboplastin and human hemophilia A plasma that had been preincubated for 5 min at 37°C. The resulting clotting times were then converted to units/ml, based on the same human standard curve described above. The relative activity in the two-stage assay was higher than in the one-stage assay because the factor VIII had been preactivated. Example 2: Characterization of the functional difference between human and porcine factor VIII. The isolation of porcine and human plasma-derived factor VIII and iuman recombinant factor VIII have been described in the literature. Fulcher, C. and T. S. Zimmerman, 79 Proc. Nat'l. Acad. Sci. U.S.A. 1648-1652 (1982); Toole, et al., 312 Nature 342- 347 (1984) (Genetics Institute); Gitschier, et al., 312 Nature 326-330 (1984) (Gener ech); Wood, et al., 312 Nature 330-337 (Genentech); I I I I I WO 93/20093 PCT/US93/03275 Vehar, et al., 312 Nature 337-342 (1984) (Genentech); Fass, et al., 59 Blood 594 (1982); Toole, et al., 83 Proc. Nat'l. Acad. Sci. U.S.A. 5939-5942 (1986). This can be accomplished in several ways. All these preparations are similar in subunit composition, although this is the first description of the functional difference between human and porcine factor VIII, not noted previously in part due to the lack of use of a common standard by which to compare them. For comparison of human recombinant and porcine factor VIII, preparations of highly-purified human recombinant factor VIII (Cutter Laboratories, Berkeley, CA) and porcine factor VIII (immunopurified as described in Fass, et al., 59 BloLd 594 (1982)) were subjected to high-pressure liquid chromatography (HPLC) over a Mono Q (Pharmacia-LKB, Piscataway, NJ) anion-exchange column (Pharmacia, Inc.). The purposes of the Mono Q" HPLC step were. elimination ot minor impurities and exchange of human and porcine factor VIII into a common buffer for comparative purposes. Vials containing 1000-2000 units of factor VIII were reconstituted with 5 ml H,0. Hepes (2 M at pH 7.4) was then added to a final concentration of 0.02 M. Factor VIII was applied to a Mono QM HR 5/5 column equilibrated in 0.15 M NaCl, 0.02 M Hepes, 5 mM CaC12, at pH 7.4 (Buffer A plus 0.15 M NaCl); washed with 10 ml Buffer A 0.15 M NaCl; and eluted with a 20 ml linear gradient, 0.15 M to 0.90 M NaCl in Buffer A at a flow rate of 1 ml/min. For comparison of human factor VIII (derived from plasma and purified by Mono Q HPLC) and porcine factor VIII, immunoaffinity-purified, plasma-derived porcine factor VIII was diluted 1:4 with 0.04 M Hepes, mM CaCl 2 0.01% Tween-80, at pH 7.4, and subjected to Mono Qm HPLC under the same conditions described in the previous paragraph for human factor VIII. These WO 93/20093 PCT/US93/03275 -41procedures for the isolation of human and porcine factor VIII are standard for those skilled in the art. Column fractions were assayed for factor VIII activity by a one-stage coagulation assay. The average results of the assays, expressed in units of activity per A 280 of material, are given in Table I, and indicate that porcine factor VIII has at least six times greater activity than human factor VIII when the one-stage assay is used. TABLE I: COMPARISON OF HUMAN AND PORCINE FACTOR VIII COAGULANT ACTIVITY Activity (U/Ao 0 Porcine 21,300 Human plasma-derived 3,600 Human recombinant 2,400 Example 3: Comparison of the stability of human and porcine factor Villa The results of the one-stage assay for factor VIII reflect activation of factor VIII to factor VilIa in the sample and possibly loss of formed factor VIIIa activity. A direct comparison of the stability of human and porcine factor VIII was made. Samples from Mono Q HPLC were diluted to the same concentration and buffer composition and reacted with thrombin. At various times, samples were removed for two-stage coagulation assay. Typically, peak activity (at 2 min) was 10-fold greater for porcine than human factor VIIIa, and the activities of both porcine and human factor VIIIa subsequently decreased, with human factor VIIIa activity decreasing more rapidly. Generally, attempts to isolate stable human factor VIIIa are not successful even when conditions that produce stable porcine factor VIIIa are used. To demonstrate this, Mono Q HPLC-purified human factor VIII was activated with thrombin and subjected to Mono S" cation-exchange (Pharmacia, Inc.) HPLC under conditions that produce stable porcine factor VIIIa WO 93/20093 PCT/US93/03275 -42- (Lollar, and Parker, 28 Biochemistry 666, 1989, the teachings of which are incorporated herein). Human factor VIII, 43 gg/ml (0.2 pM) in 0.2 M NaCI, 0.01 M Hepes, 2.5 mM CaCl 2 at pH 7.4, in 10 ml total volume, was reacted with thrombin (0.036 pM) for min, at which time FPR-CH 2 Cl D-phenyl-prolylarginyl-chloromethyl ketone was added to a concentration of 0.2 AM for irreversible inactivation of thrombin. The mixture then was diluted 1:1 with mM 2-(N-morpholino)ethane sulfonic acid (MES), 5 mM CaCl 2 at pH 6.0, and loaded at 2 ml/min onto a Mono S M HR 5/5 HPLC column equilibrated in 5 mM MES, 5 mM CaC1 2 at pH 6.0 (Buffer B) plus 0.1 M NaCI. Factor VIIIa was eluted without column washing with a 20 ml gradient from 0.1 M NaCI to 0.9 M NaC1 in Buffer B at 1 ml/min. The fraction with coagulant activity in the twostage assay eluted as a single peak under these conditions. The specific activity of the peak fraction was approximately 7,500 U/A 28 o. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the Mono S" factor VIIIa peak, followed by silver staining of the protein, revealed two bands corresponding to a heterodimeric (A3-C1-C2/A1) derivative of factor VIII. Although the A2 fragment was not identified by silver staining under these conditions because of its low concentration, it was identified as a trace constituent by 'SI-labeling. In contrast to the results with human factor VIII, porcine factor VIIIa isolated by Mono S T HPLC under the same conditions had a specific activity 1.6 x 106 U/A 280 Analysis of porcine factor VIIIa by SDS- PAGE revealed 3 fragments corresponding to Al, A2, and A3-C1-C2 subunits, demonstrating that porcine factor VIIIa possesses three subunits. WO 93/20093 PCT/US93/03275 -43- The results of Mono S T HPLC of human thrombinactivated factor VIII preparations at pH 6.0 indicate that human factor Villa is labile under conditions that yield stable porcine factor VIIIa. However, although trace amounts of A2 fragment were identified in the peak fraction, determination of whether the coagulant activity resulted from small amounts of heterotrimeric factor Villa or from heterodimeric factor VIIIa that has a low specific activity was not possible from this method alone. A way to isolate human factor Villa before it loses its A2 subunit is desirable to resolve this question. To this isolation was accomplished in a procedure that involves reduction of the pH of the Mono S T buffers to pH 5. Mono Q"-purified human factor VIII (0.5 mg) was diluted with H 2 0 to give a final composition of 0.25 mg/ml (1 pM) factor VIII in 0.25 M NaC1, 0.01 M Hepes, 2.5 mM CaCI,, 0.005% Tweenat pH 7.4 (total volume 7.0 ml). Thrombin was added to a final concentration of 0.072 MM and allowed to react for 3 min. Thrombin was then inactivated with FPR-CH 2 Cl (0.2 IM). The mixture then was diluted 1:1 with 40 mM sodium acetate, 5 mM CaCd 2 0.01% Tweenat pH 5.0, and loaded at 2 ml/min onto a Mono S T HR 5/5 HPLC column equilibrated in 0.01 M sodium acetate, 5 mM CaC1 2 0.01% Tween-80, at pH 5.0, plus 0.1 M NaCl. Factor Villa was eluted without column washing with a 20 ml gradient from 0.1 M NaCl to 1.0 M NaCl in the same buffer at 1 ml/min. This resulted in recovery of coagulant activity in a peak that contained detectable amounts of the A2 fragment as shown by SDS-PAGE and silver staining. The specific activity of the peak fraction was ten-fold greated' than that recovered at pH 6.0 (75,000 U/Ao 2 vs. 7,500 U/A 280 However, in contrast to porcine factor Villa isolated at pH 6.0, which is indefinitely stable at WO 93/20093 PC/US93/03275 -44- 4 0 C, human factor Villa activity decreased steadily over a period of several hours after elution from Mono S T Additionally, the specific activity of factor VIIIa purified at pH 5.0 and assayed immediately is only 5% that of porcine factor Villa, indicating that substantial dissociation occurred prior to assay. These results demonstrate that both human and porcine factor Villa are composed of three subunits (Al, A2, and A3-C1-C2). Dissociation of the A2 subunit is responsible for the loss of activity of both human and porcine factor Villa under certain conditions, such as physiological ionic strength, pH, and concentration. The relative stability of porcine factor VIIIa under certain conditions is because of stronger association of the A2 subunit. Example 4: Preparation of hybrid human/porcine factor VIII. Porcine factor VIII light chains and factor VIII heavy chains were isolated as follows. A 0.5 M solution of EDTA at pH 7.4 was added to Mono Qpurified porcine factor VIII to a final concentration of 0.05 M and was allowed to stand at room temperature for 18-24 h. An equal volume of 10 mM histidine-Cl, mM EDTA, 0.02% v/v Tween 80, at pH 6.0 (Buffer B), was added, and the solution was applied at 1 ml/min to a Mono S M HR 5/5 column previously equilibrated in Buffer A plus 0.25 M NaCl. Factor VIII heavy chains did not bind the resin, as judged by SDS-PAGE. Factor VIII light chain was eluted with a linear, 20 ml, 0.1- 0.7 M NaCl gradient in Buffer A at 1 ml/min and was homogeneous by SDS-PAGE. Factor VIII heavy chains were isolated by mono Q T HPLC in the following way. Factor VIII heavy chains do not adsorb to mono S T during the purification of factor VIII light chains. The fall-through material that contained factor VIII heavy chains was adjusted to pH 7.2 by addition of M Hepes buffer, pH 7.4, and applied to a mono Q -r WO 93/20093 PCT/US93/03275 HPLC column equilibrated in 0.1 M NaCI, 0.02 M Hepes, 0.01% Tween-80, ph 7.4. The co. .mn was washed with mL of this buffer, and factor VIII heavy chains were eluted with a 20 mL 0.1-1.0 M NaCl gradient in this buffer. Human light chains and heavy chains were isolated in the same manner. Human and porcine light and heavy chains were reconstituted according to the following steps. Ten il human or porcine factor VIII light chain, 100 ig/ml, was mixed in 1 M NaCI, 0.02 M Hepes, 5 mM CaCI 2 0.01% Tween-80, pH 7.4, with 25 tl heterologous heavy chain, 60 pg/ml, in the same buffer; 10 Ml 0.02 M Hepes, 0.01% Tween-80, pH 7.4; 5 41 0.6 M CaCI 2 for 14 hr at room temperature. The mixture was diluted 1/4 with 0.02 M MES, 0.01% Tween-80, 5 mM CaCI 2 pH 6, and applied to Mono S T Hr5/5 equilibrated in 0.1 M NaC1, 0.02 M MES, 0.01% Tween-80, 5mM CaCI 2 pH 6.0. A 20 ml gradient was run from 0.1 1.0 M NaCl in the same buffer at 1 ml/min, and 0.5 ml fractions were collected. Absorbance was read at 280 nm of fractions, and fractions were assayed with absorbance for factor VIII activity by the one-stage clotting assay. Heavy chains were present in excess, because free light chain (not associated with heavy chain) also binds Mono S; excess heavy chains ensure that free light chains are not part of the preparation. Reconstitution experiments followed by Mono S T HPLC purification were performed with all four possible combinations of chains: human light chain/human heavy chain, human light chain/porcine heavy chain, porcine light chain/porcine heavy chain, porcine light chain/human heavy chain. Table II shows that human light chain/porcine heavy chain factor VIII has activity comparable to native porcine factor VIII (Table indicating that structural elements in the porcine heavy chain are responsible WO 93/20093 PCT/US93/03275 -46for the increased coagulant activity of porcine factor VIII compared to human factor VIII. TABLE II: COMP7YTSON OF HYBRID HUMAN/PORCINE FACTOR VIII COAGULANT ACTIVITY WITH HUMAN AND PORCINE FACTOR VIII Activity (U/A28o) Porcine light chain/porcine heavy chain 30,600 Human light chain/porcine heavy chain 44,100 Porcine light chain/human heavy chain 1,100 Human light chain/human heavy chain 1,000 Example 5: Isolation and sequencing of the A2 domain of porcine factor VIII. Only the B domain and part of the A2 domain of porcine factor VIII have been sequenced previously (Toole, et al., 83 Proc. Nat'l. Acad. Sci. U.S.A. 5939-5942 (1986)). The genomic DNA sequence of the porcine factor VIII B domain and the cDNA sequence for the entire porcine factor VIII A2 domain are disclosed herein. The porcine factor VIII A2 domain was cloned by reverse transcription of porcine spleen total RNA and PCR amplification; degenerate primers based on the known human factor VIII cDNA sequence and an exact porcine primer based on a part of the porcine factor VIII sequence were used. A 1 kb PCR product was isolated and amplified by insertion into a Bluescript" (Stratagene) phagemid vector. The porcine A2 domain was completely sequenced by dideoxy sequencing. The sequence is as described above. Example 6: Preparation of recombinant hybrid human/porcine factor VIII The sequence of human factor VIII has been described in the literature (Toole, et al., 312 Nature 342-347 (1984) (Genetics Institute); Gitschier, WO 93/20093 PCT/US93/03275 -47et al., 312 Nature 326-330 (1984) (Genentech); Wood, et al., 312 Nature 330-337 (1984) (Genentech); Vehar, et al., 312 Nature 337-342 (1984) (Genentech)). The sequence is as described above. Making recombinant hybrid human/porcine factor VIII requires that a region of human factor VIII cDNA (Biogen Corp.) be removed and the homologous porcine cDNA sequence inserted. Subsequently, the hybrid cDNA is expressed in an appropriate expression system. In these experiments, for example, the entire cDNA sequence corresponding to the A2 domain of human factor VIII is removed by oligonucleotide-mediated mutagenesis, a method commonly known to those skilled in the art (see, Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Chapter 15, Cold Spring Harbor Press, Cold Spring Harbor, 1989). The steps were as follows: E. coli CJ236 cells were transformed with Bluescript T phage containing the human factor VIII cDNA insert. Single-stranded Bluescript"/human factor VIII circular DNA was produced with M13K07 helper phage and then purified by standard methods (Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Chapter 4, Cold Spring Harbor Press, Cold Spring Harbor, 1989). A mutagenic oligonucleotide wps synthesized corresponding to the 3' end of the Al domain and the 5' end of the A3 domain: CCTTCCTTTATCCAAATACGTAGATCAAGAGGAAATTGAC 3'. Additionally this oligonucleotide provides a SnaB1 restriction site that can be used to insert the porcine A2 domain. On hybridization of this oligonucleotide to single strand Bluescript"/human factor VIII, the region between the Al and A3 domains, i.e, the A2 domain, was "looped out." The resulting heteroduplex was extended to circular, double-stranded ~L~h L I g II WO 93/20093 PCr/US93/03275 -48- DNA by use of T7 polymerase, ligated, and used to transform E. coli XLl-blue M (Stratagene) cells. Transformants were screened by isolation of phagemid DNA from several colonies, Xhol digestion, and examination of the size of phagemid DNA by agarose gel electrophoresis. Three clones were identified that were shorter than human factor VIII/Bluescript T by 1 kb, as expected for deletion of the 1 kb A2 domain. The results were confirmed by sequencing across the boundaries of the Al and A3 domains. The porcine A2 domain has been inserted between the Al and A3 domains of the human factor VIII cDNA by PCR amplification of the porcine A2 domain; (2) gel purification of the PCR product (agarose gel electrophoresis of the PCR product producing a band visualized by ethidium bromide staining, followed by excision of the band and purification of the DNA to remove agarose and other contaminants); and (3) ligation by using T4 DNA ligase of the porcine A2 cDNA to the human A2-domainless cDNA linearized by using the SnaB1 restriction site. The primers used for PCR amplification of the porcine A2 were as follows: primer: 5' GTAGCGTTGCCAAGAAGCACCCTAAGACG 3' 3' primer: 5' GAAGAGTAGTACGAGTTATTTCTCTGG GTTCAATGAC 3'. The 3' primer contains nucleotides corresponding to residues 736-740 of the porcine factor VIII protein sequence (at the C-terminus of the A2 domain), and residues 1649-1656 of the human factor VIII sequence (at the N-terminus of the A3 domain). The A3 sequence residues were included because the looping out procedure removed these residues. The ligated product was used to transform XL1-Blue cells, producing several co3onies that contained the desired porcine A2 insert when analyzed by PCR. The product contains an unwanted thymine at the Al-A2 junction as a result of the PCR amplification of the porcine A2 domain. This r WO 93/20093 PCT/US93/03275 -49single base can be looped out by use of the mutagenic oligonucleotide CCTTTATCCAAATACGTAGCGTTTGCCAAGAAG 3' and the product can be cloned exactly as described above (Under Example 6, paragraph 3) for the production of human A2-deficient cDNA. Cloning of the porcine Al, A3, Cl, and C2 domains is feasible with the same strategy that was used for cloning the porcine A2 domain. Fragments of these domains can be cloned by the looping out mutagenesis technique. Excision of the corresponding domains in human factor VIII and any fragments thereof, including single amino acid eliminations, is feasible by looping out mutagenesis as described above. All possible domain replacements, fragments of domain replacements, or single amino acid residue replacements are possible by this approach. The biological activity of recombinant hybrid human/porcine factor VIII can be evaluated initially by use of a COS-cell mammalian transient expression system. Hybrid human/porcine cDNA can be transfected into COS cells, and supernatants can be analyzed for factor VIII activity by use of one-stage and two-stage coagulation assays as described above in Example 1. Additionally, factor VIII activity can be measured by use of a chromogenic substrate assay, which is more sensitive and allows analysis of larger numbers of samples. This assay has been described (Lollar, P., G.J. Knutson, and D.N. Fass, 24 Biochemistry 8056- 8064, 1985). Similar assays are standard in the assay of factor VIII activity (Wood, et al., 312 Nature 330-337, 1984; Toole, et al., 312 Nature 342-347, 1984). Expression of recombinant factor VIII in COS cells is a standard procedure (Toole, et al., 312 Nature 342-347, 1984; Pittman, and R.J. Kaufman, 85 Proc. Nat'l. Acad. Sci. USA 2429-2433, 1988). The human factor VIII cDNA used as starting WO 93/20093 PCI'/US93/03275 material for the recombinant molecules described herein has been expressed in COS cells yielding a product with biological activity. This material will be used a standard to compare hybrid human/porcine factor VIII molecules. The activity in the assays is converted to a specific activity for proper comparison of the hybrid molecules. For this, a measurement of the mass of factor VIII produced by the cells is necessary and can be done by immunoassay with purified human and/or porcine factor VIII as standards. Immunoassays for factor VIII are routine for those skilled in the art (See, Lollar, et al., 7i Blood 137-143, 1988). Sequences of human and porcine factor VIII likely to be involved as epitopes as recognition sites for inhibitory antibodies) can be determined through use of commercially available predictive computer programs, such as MacVector (IBI Corp., New Haven, CT). Sequences of porcine factor VIII that are not antigenic compared to corresponding (homologous) antigenic human sequences will be identified, and substitutions will be made to insert porcine sequences and delete human sequences according to standard recombinant DNA methods. It is already known that porcine factor VIII reacts less than human factor VIII with some inhibitory antibodies; this provides a basis for current therapy for patients with inhibitors. After the recombinant hybrids are made, they will be tested in vitro for reactivity with the Bethesda inhibitor assay. Those constructs that are less reactive than native human factor VIII and native porcine factor VIII will be candidates for replacement therapy. SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Lollar, John S. Runge, Marschall S. (ii) TITLE OF INVENTION: Hybrid Human/Porcine Factor VIII (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Kilpatrick Cody STREET: 1100 Peachtree Street CITY: Atlanta STATE: Georgia COUNTRY: US ZIP: 30309 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.0 sion #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 07/864,004 FILING DATE: 07 APRIL 1992 CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: NAME: Pratt, John S. REGISTRATION NUMBER: 29,476 REFERENCE/DOCKET NUMBER: EMU106AU i (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 404-815-6500 TELEFAX: 404-815-6555 INFORMATION FOR SEQ ID NO:1: i)SEQUENCE CHARACTERISTICS: LENGTH: 1130 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: cD11A ,iii) 'HYPOTHETICAL: No (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminaL (vi) ORIGINAL SOURCE: ORGANISM: Porcine (F1 TISSUE TYPE: Blood (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TAAGCACCCT AAGACGTGGG TGCACTACAT CTCTGCAGAG GAGGAGGACT GGGACTACGC CCCCGCGGTC CCCAGCCCCA GTGACAGAAG TTATAAAAGT CTCTACTTGA ACAGTGGTCC 120 TCAGCGAATT GGTAGGAAAT ACAAAAAAGC TCGATTCGTC, GCTTACACGG ATGTAACATT 180 TAAGACTCGT AA.AGCTATTC CGTATGAATC AGGAATCCTG GGACCTTTAC TTTATGGAGA 240 AGTTGrAGAC ACACTTTTGA TTATATTTAA GAATAAAGCG AGCCGACCAT ATAACATCTA 300 CCCTCATGGA ATCACTGATG TCAGCGCTTT GCACCCAGGG AGACTTCTAA AAGGTTGGAA 360 ACATTTGAAA TGTGGAAGAT CATTAATCTA AGAATCTGTA TTCTGTATTC CAATCCGGAT CATCAATGGC CTGGTACA2T CACCTTCAAA AACGGTCTTC CTTGCGGAAC TGATTATTAT GACATGCCAA GGGCCAACCA GAGAAAGATC GACCAAAGAG GATGAGAATC GGATTACAGC TATGTTTTTG CTAAGTGTTG CACAAAATGG ATGTCAATGG AGAGGGATGA GACAACACTT TTCTGCCAGG AGTCCGATCC TGGCTTCGGG GAAACCAGAT AAAGCTGGTA CCCAGC7.TCC ATAGCTTGCA GAGCACAGAC TCTATGAAGA AAAACCCAGG CAGCCTTACT ATGAAGATAT CCCAGAATTC AGAGACTTTC TCGGTGCCTG ACTCATTGGC GATGTCAGAC CCTCGCAGAG AGAGTTCCAA GCTGTCGGTT GGACTTCCTC CACACTCACC jGTC GAAGGTGTAT TCCAGGCTTC AAGACCCCCT AAGTATAAAT ACCCGCTACT CCTCTCCTCA AAGAGAAACG AATATTCAGC GCTTCTAACA TGTTTGCACG TCCGTCTTCT CTGTTCCCCT CTAGGGTGCC AGTTGTGACA TTGCTGAGTG AGTGCGAGCA GGACAGTGAC ACTCGAGCTC TCTGCTACAA TCATCCTGTT GCTTCCTCCC TCATGCACAG AGGTGGCATA TCTCTGGCTA. TCTCAGGAGA ACAACTCAGA. GGGACATTGG GAAAGAATGT 420 480 540 600 660 720 780 840 900 960 1020 1080 1130 CATTGAACCC AGAAGCTTTG INFORMATION FOR SEQ ID NQ:2: ()SEQUENCE CHARACTERISTICS: LENGTH: 367 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Porcine TISSUE TYPE: Spleen (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr 1 5 10 Glu Glu Asp Trp Asp Tyr Ala Pro Ala Val Pro Ser 25 Ser Tyr Lys Ser Leu Tyr Leu Asn Ser Gly Pro Gln 40 Lys Tyr Lys Lys Ala Arg Phe Val Ala Tyr Thr Asp 55 Thr Arg Lys Ala Ile Pro Tyr Glu Ser Gly Ile Leu 70 75 Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe 90 Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr 100 105 Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys His 115 120 Ile Pro Arg Val Gly Lys Asp Leu 125 Ser Ala Glu Ser Asp Arg Ile Gly Arg Thr Phe Lys Pro Leu Leu Asn Lys Ala Val Ser Ala 110 Lys Asp Met I Pro Ile Leu Pro Gly Glu Thr Phe Lys Tyr Lys 130 135 Trp Thr Val Thr Val 140 Glu Asp Gly Pro Thr 145 Ser Pro Met Asn Pro 225 Met Cys Thr Met Val 305 Ser Ser Leu Leu Met Ser 195 Gin Ser 210 Asp Gly His Ser Leu His Asp Phe 275 Val Tyr 290 Ile Asn 165 Ile Cys 180 Asp Lys Trp Tyr Leu Gin Ile Asn 245 Glu Val 260 Leu Ser Glu Asp Lys Ser Asp 150 Leu Glu Lys Tyr Lys Glu Arg Asn Val 200 Leu Ala Glu 215 Pro Gin Asp 230 Gly Tyr Val Ala Tyr Trp Val Phe Phe 280 Thr Leu Thr Pro Asp Ser 185 Ile Asn Pro Phe Tyr 265 Ser Leu Arg Cys 155 Leu Ala 170 Val Asp Leu Phe Ile Gin Glu Phe 235 Asp Ser 250 Ile Leu Gly Tyr Phe Pro Leu Thr Ser Gly Gin Arg Ser Val 205 Arg Phe 220 Gin Ala Leu Gin Ser Val Thr Phe 285 Phe Ser Arg Tyr Tyr 160 Leu Ile Gly 175 Gly Asn Gin 190 Phe Asp Glu Leu Pro Asn Ser Asn Ile 240 Leu Ser Val 255 Gly Ala Gin 270 Lys His Lys Gly Glu Thr 295 300 Phe Met Ser Met Glu Asn Pro Gly 310 Leu Trp Val Leu Gly Cys His 315 320 I O i I IY Asn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Tyr 325 330 335 Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp Asn Thr Tyr Glu Asp 340 345 350 Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val Ile Glu Pro Arg 355 360 365 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 9009 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA tn (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapien TISSUE TYPE: Liver (ix) FEATURE: NAME/KEY: miscfeature (Domain Structure) LOCATION: 5001 7053 OTHER INFORMATION: /note= "Equivalent to the A3-C1-C2 domain" 1111-- I I- I p I I I (ix) FEATURE: NAME/KEY: misc -feature (Domain Structure) LOCATION: 1 2277 OTHER INFORMATION: /note= "Equivalent to the A1-A2 domain" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CAGTGGGTAA GTTCCTTAAA TGCTCTGCAA. AGAAATTGGG ACTTTTCATT AAATCAGAAA TTTTACTTTT TTCCCCTCCT GGGAGCTAAA GATATTTTAG AGAAGAATTA ACCTTTTGCT TCTCCAGTTG AACATTTGTA CTGTGCCTTT TGCGATTCTG CTGTCATGGG ACTATATGCA CCTAGAGTGC CAAAATCTTT GTAGAATTCA CGGTTCACCT CTAGGTCCTA CCATCCAGGC GCTTCCCATC CTGTCAGTCT GCTGAATATG ATGATCAGAC GGAAGCCATA CATATGTCTG CTGTGCCTTA CCTACTCATA CTCATTGGAG CCCTACTAGT TTGCACAAAT TTATACTACT ACAAAGAACT CCTTGATGCA GCAATAAGTC ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA AAGTGATCTC GGTGAGCTGC CTGTGGACGC, AAGATTTCCT TCCATTCAAC ACCTCAGTCG TGTACAAAAA GACTCTGTTT TTTCAACATC GCTAAGCCAA GGCCACCCTG GATGGGTCTG TGAGGTTTAT GATACAGTGG TCATTACACT TAAGAACATG TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT GCAGGTCCTG AAAGAGAATG GTCCAATGGC CTCTGACCCA TCTTTCTCAT GTGGACCTGG TAAAAGACTT GAATTCAGGC ATGTAGAGAA GGGAGTCTGG CCA-AGGAAAA GACACAGACC TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG 120 180 240 300 360 420 480 540 600 660 720 780 840 900 CACACAGTCA ATGGTTATGT TCAGTCTATT GAAGGTCACA ACTTTCCTTA ATCTCTTCCC GAACCCCAAC GATTCTGAAA CGCTCAGTTG GACTGGGACT TTGAACLATG ACAGATGAAA TTACTTTATG CCATATAACA CCAAAAGGTG AAATGGACAG TATTACTCTA CTCATCTGCT AATGTCATCC GGCATGTGAT CATTTCTTGT CTGCTCAAAC ACCAACATGA TACGAkATGAA TGGATGTGGT CCAAGAAGCA ATGCTCCCTT GCCCTCAGCG CCTTTAAGAC GGGAAGTTGG TCTACCCTCA TAAAACATTT TGACTGTAGA GTTTCGTTAA ACAAAGAATC TGTTTTCTGT AAACAGGTCT TGGAATGGGC GAGGAACCAT ACTCTTGATG TGGCATGGAA AAATAATGAA CAGGTTTGAT TCCTAAAACT AGTCCTCGCC GATTGGTAGG TCGTGAAGCT AGACACACTG CGGAATCACT GAAGGATTTT AGATGGGCCA TATGGAGAGA TGTAGATCAA ATTTGATGAG CTGCCAGGTC ACCACTCCTG CGCCAGGCGT GACCTTGGAC GCTTATGTCA GAAGCGGAAG GATGACAACT TGGGTACATT CCCGATGACA AAGTACAAAA ATTCAGCATG TTGATTATAT GATGTCCGTC CCAATTCTG C ACTAAATCAG GATCTAGCTT AGAGGAAACC AACCGAAGCT TGATTGGATG AAGTGCACTC CCTTGGAAAT AGTTTCTACT AAGTAGACAG ACTATGATGA CTCCTTCCTT ACATTGCTGC GAAGTTATAA AAGTCCGATT AATCAGGAAT TTAAGAATCA CTTTGTATTC CAGGAGAAAT ATCCTCGGTG CAGGACTCAT AGATAATGTC GGTACCTCAC CCACAGGAAA AATATTCCTC CTCGCCAATA GTTTTGT CAT CTGTCCAGAG TGATCTTACT TATCCAAATT TGAAGAGGAG AAC-TCAATAT TATGGCATAC CTTGGGACCT AGCAAGCAGA AAGGAGATTA ATTCAAATAT CCTGACCCGC TGG~CCCTCTC AGACAAGAGG AGAGAATATA 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 I CAACGCTTTC AACATCATGC CATGAGGTGG TTCTTCTCTG CCATTCTCAG TGCCACAACT GACAAGAACA AGTAAAAACA AGGCAAAAGC TGGTTTGCAC ATGCTCTTGC AAATATGAGA TCTGAAATGA GAGTCAGGCC AAGAAACTTG GACAATTTGG CATTATGATA TCTGGTGGAC TCCCCAATCC ACAGCATCAA CATACTGGTA GATATACCTT GAGAAACTGT CAGACTTTCG CTGGTGATTA ATGCCATTGA AATTTAATGC ACAGAACACC GACAGAGTCC CTTTTTCTGA CACACTTCAG TCCAATTAAG ATTTCAAAGT CAGCAGGTAC GTCAATTAGA CTCTGAGCTT AGCTGGAGTG TGGCTATGTT CATTCTAAGC CAAACACAAA CTTCATGTCG GAACAGAGGC TTACGAGGAC ACCAAGAAGC CACCACAATT TATGCCTAAA TACTCCACAT TGATCCATCA GCCACAGCTC ATTAAATGAG TTCTAGTACA TGATAATACA TACCACTCTA GAGTGAAGAA CAGCTTGAGG TTTGATAGTT ATTGGAGCAC ATGGTCTATG ATGGAAAACC ATGACCGCCT AGTTATGAAG TTCTCCCAGA CCAGAAAATG ATACAAAATG GGGCTATCCT CCTGGAGCAA CATCACAGTG AAACTGGGGA TCAAATAATC AGTTCCTTAG TTTGGCAAAA AATAATGATT ATCCAGAGTT TGCAGTTGTC AGACTGACTT AAGACACACT CAGGTCTATG'- TACTGAAGGT ATATTTCAGC ATTCAAGACA ACATAGAGAA TCTCCTCTAG TATCTGATCT TAGAC)AGTAA GGGACATGGT CAACTGCAGC TGATTTCAAC GACCCCCAAG AGTCATCTCC CAAAGTTGTT CCAAGCCTCC AGTTTGTTTG CCTTTCTGTC CACCCTATTC GATTCTGGGG TTCTAGTTGT ATACTTGCTG CCCTAGCACT GACTGACCCT TGATTTGTTG CCAAGAAGCC TAACAGCCTG ATTTACCCCT AACAGAGTTG AATTCCATCA TATGCCAGTT CCTTACTGAG AGAATCAGGT 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 TTAATGAATA GCCAAGAAAG TTCATGGGGA AAAAATGTAT CGTCAACAGA GAGTGGTAGG I TTATTTAAAG A-AAGTTAGCA AAGACTCACA ATATTAGAAA ATGGACAAAA AAAAACATGG CCAGATATGT ACTCATGGAA TTAGGACCAG GTAGGAAAGG AGAAACCTAT AAAAAAATTC CCTCAGATAC ACTAGGCAAA AGGTCATTAA GGGGAGGAAG GCATGCACCA GGAAAAGAGC TCTCTTTGTT TTGATGGCCC GTGACACTGA ATGCTACAGC AAATGGTCCA CGTr"CTTTAA AGAACTCTCT AAAAATCTGT GTGAATTTAC TTCTTACTAA AGGAAGAAAT ATACAGTGAC ATGTAGAAGG ATGATTCAAC AAAACTTGGA CAAGGATATC TCATGGACCT AAAGACAAAC ATCATTATTA GTTTAAAAAA TTTGAGGCTA ACAGAAAAAA GATGCTATTC GAACTCTGGG GGAAGGTCAG AAAGGACGTA CTTGGATAAT AGAAAAGAAG TGGCACTAAG TTCATATGAG AAATAGAACA AGGCTTGGGA TCCTAATACA GCTTTGTTGA AAAACTTCCA ATTGAGAATA GTGACACCTT AATCATATGT GAGGGCCCCA TTGCCAGAAT CAAGGCCCCA AATTTCTTGT GGACTCAAAG TTACATGAAA GAAACATTAA AATTTCATGA GGGGCATATG AAGAAACACA AATCAAACCA AGCCAGCAGA CTAAAGATAA ATAATTCAGC GTCCATCAGT TGATTCATGA CAAATAAAAC TTCCACCAGA CAGCAAGGTG GTCCAAAGCA CTGAGAAAAA AGATGGTTTT ATAATACACA TCCAAGAGAA AGAACCTTTT CTCCAGTACT CAGCTCATTT AGCAAATTGT ATTTTGTCAC TGCCTTATTC AACTAATAGA CTGGCAAAAT CAGAATGCTT TACTTCATCA TGCACAAAAT GATACAAAGG ATTAGTATCC CAAAGTGGTA TCCAAGCAGC CAATCAAGAA TGTAGTTTTG CTTACTGAGC TCAAGATTTT CTCAAAAAAA AGAGAAATAT GCAACGTAGT 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 AAGAGAGCTT TGAAACAATT CAGACTCCCA CTAGAAGAAA CAGAACTTGA AAAAAGGATA ATTGTGGATG CTCACACAGA GATTGCCTTA AAGGTATCAT AACTCTTCTC AGTCATTTCT ATGACTGGTG TACAAGAAAG GTTGAATTGC AATGGGTCTC GCGATTAAGT GAAAGCTCTG GGTACTCAGA TTTAAGAAAA GCAATAAATG ACTGAAAGGC CGTACTACTC ATGAAGAAGG CAAAAGAAAA ACACCTCAAC TAGACTACAA CGAGGAGTCA CATTTCCATC ATCTTCCAGC TACAAGGAGC ATCAAAGAGA TTGAGAACAC TTCCAAAAGT CTGGCCATCT GGAATGAAGC CAAA-GACTCC TACCAAAAGA AGGATACCAT AGGGACAAAA TGTGCTCTCA TTCAGTCAGA AAGATTTTGA CACGACACTA CCAGTGGTCC TGAGAAGGAG TAGCATCCCT TATTAGACCT AG CAT CTTAT CAAAAAAAAT GGTTGGCTCC TGTTCTCCCG TCACATTTAT GGATCTCGTG AAACAGACCT CTCCAAGCTA AGAGTGGAAA TTTGTCCCTG TAAGCCCGAA AAACCCACCA TCAAGAGGAA CATTTATGAT TTTTATTGCT AAAAACATGA. AAAGGGGCCA CAAGCAAATA ATATATCTGA AGAAAGAAAG AACCTTTCTT CTGGGGACAA AAACCAGACT CAGAAGGACC GAAGGGAGCC GGAAAAGTTC TTGGATCCTC TCCCAAGAGA AACGCTTGTG ATAGAAGTCA GTCTTGAAAC ATTGACTATG GAGGATGAAA GCAGTGGAGA AACATTTGAC TTACTCAGTC GATCTCCATT CCAGGGTCCT ATTCTGGGGT TAGCCATTCT GTGCCACAAA TGCCCAAAAC TATTCCCTAC TTCTTCAGGG CCTTTCTGAG TTGCTTGGGA AGTCACCAGA AAAGCAATCA CCTGGGCAAA GCCATCAACG ATGATACCAT ATCAGAGCCC GGCTCTGGGA CCCGAGCACC TCCCTTATCA ACCCATTGCA ATTCCAAGAC CCAAGAAAGC AACCTTGGAG TTCAGTCACA ATCTGGCAAA GGAAACTAGC AACAGAGGGA AGTAGCAACA TAACCACTAT AAAAACAGCT TGCAATAGCA GCAAGGTAGG GGAAATAACT ATCAGTTGAA CCGCAGCTTT TTATGGGATG 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 AGTAGCTCCC AAAGTTGTTT CTAAATGAAC ATGGTAACTT TATGAGGAAG ACCAAAACTT TGCAAAGCCT ATTGGACCCC ACAGTACAGG ACTGAAAATA TTTAAAGAGA TTAGTAATGG AACATCCATT AAAATGGCAC AAAGCTGGAA ACACTTTTTC ATTAGAGATT CACATGTTCT TCCAGGAATT ATTTGGGACT TCAGAAATCA ATCAGAGGCA ACTTTTGGAA GGGCTTATTT TTCTGGTCTG AATTTGCTCT TGGAAAGAAA ATTATCGCTT CTCAGGATCA CTATTCATTT TGTACAATCT TTTGGCGGGT TGGTGTACAG TTCAGATTAC AAGAAACAGG TACTGATGGC CCTGGGGCCA GGCCTCTCGT AGGAGCAGAA AGTGCAACAT CTCTGATGTT CCACACTAAC GTTTTTCACC CTGCAGGGCT CCATGCAATC AAGGATTCGA CAGTGGAGAT CTATCCAGGT GGAATGCCTT CAATAAGTGT AGCTTCAGGA GCTCAGAGTG TCCTTTACTC TATATAAGAG CCCTATTCCT CCTAGAAAAA CATATGGCAC GACCTGGAAA ACACTGAACC ATCTTTGATG CCCTGCAATA AATGGCTACA TGGTATCTGC GTGTTCACTG GTTTTTGAGA ATTGGCGAGC CAGACTCCCC CAATATGGAC GCAGTGTCCC AGCCCTTATA CAGAAGTTGA TCTATTCTAG ACTTTGTCAA CCACTAAAGA AAGATGTGCA CTGCTCATGG AGACCAAAAG TCCAGATGGA TAATGGATAC TCAGCATGGG TACGAAAAAA CAGTGGAAAT ATCTACATGC TGGGAATGGC AGTGGGCCCC TCAGTTCAAG CCGTGGAGAA AGATAATATC CCTTATTTCT GCCTAATGAA TGAGTTTGAC CTCAGGCCTG GAGACAAGTG CTGGTACTTC AGATCCCACT ACTACCTGGC CAGCAAkTGAA AGAGGAGTAT GTTACCATCC TGGGATGAGC TTCTGGACAC AAAGCTGGCC 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 AGACTTCATT ATTCCGGATC AATCAATGCC TGGAGCACCA AGGAGCCCTT TTCTTGGATC AAGGTGGATC AAGTTCTCCA TGGCAGACTT TCATCTGGGA CACCCAACTC AAT2AGTTGCA GCTTCATCCT CTCCAAGGGA GTGGACTTCC CT TACCAGCA ACTCTCTTTT CCTGTGGTGA AGTTGGGTGC TACTGAGGGT GGCAGTGTCC TGAAGCCTCC TGCATCCAAT CTTCCAATAT TGTTGGCACC GCCTCTACAT ATCGAGGAAA TAAAACACAA ATTATAGCAT GCATGCCATT ACTTTACCAA GGAGTAATGC AGAAGACAAT TGTATGTGAA TTCAGAATGG ACTCTCTAGA ACCAGATTGC GGCCACTGCA CT CC CTGG CT TGAATTAACT TTAACTTAAC AACTAGGCAA AATGATTATT CTCTCAGTTT TTCCACTGGA TATTTTTAAC TCGCAGCACT GGGAATGGAG TATGTTTGCC CTGGAGACCT GAAAGTCACA GGAGTTC CT C CAAAGTAAAG CCCACCGTTA CCTGAGGATG GCACCTGCCA TGCCTTCTAC ATCATCAGTC TCTTACCTAT AAAGAAGTGA CACGGCATCA ATCATCATGT ACCTTAATGG CCTCCAATTA CTTCGCATGG AGTAAAGCAA ACCTGGTCTC CAGGTGAATA GGAGTAACTA ATCTCCAGCA GTTTTTCAGG CTGACTCGCT GAGGTTCTGG CTGCCGTCAC CTTTGTGCTA CTGCATTTCT TTTCTGCAGC GGAGAAACCT AGACCCAGGG ATAGTCTTGA TCTTCTTTGG TTGCTCGATA AGTTGATGGG TATCAGATGC CTTCAAAAGC ATCCAAAAGA CTCAGGGAGT GTCAAGATGC GAAATCAAGA ACCTTCGAAT GCTGCGAGGC CTCTCCCTCC AATCCTAGCA TTGGTGGGGG TGCTCCCAGA GCATGAAAGC TGCCCGTCAG TGGGAAGAAG CAATGTGGAT CATCCGTTTG CTGTGATTTA ACAGATTACT TCGACTTCAC GTGGCTGCAA AAAATCTCTG CCATCAGTGG CTCCTTCACA TCACCCCCAG ACAGGACCTC TCAGCTCCAG GACACTGCCT GCCAGGAGGG TTACTCCTTC ATTCTTCCCT 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 GAAAAGTTAG GCCTCTCAGA GTCACCACTT CCTCTGTTGT AGAAAAACTA TGTGATGAAA CTTTGAAAAA AACTCTCAGT CAATACAATC CAATAACTAC TTAGTCATTA TAGATGGGGT CAAATGTGCA CAATAAAAAA CTTCTTG7tvA CATACATTTA TACAAACTTT CTGAAAATAA GTCCTACTTA GAAAAAACAC CCCCATAAGA CTACACAGAA TGGAGGAAGC GATATTTATG TGTTTATTAT TTGGAGTCAA TACAGTAAAG TGAGGGGCAC TCAAGAATCC TTTTTCTGAC ATAAGTCAGG TTTGTGATGG ATATTTCTGT GTAATTCTAA CACAACAAAA CATAGTTGAA TCCAGTCTGC TTGTGAAGGG CTCTCCTGAT ATCCAAAGAC ATGTTAACAT CCTGATCAAG AAGGCAAATC TCTGTTTCTG ATTCTTATCT CTAAGTCCCC GAGTGTCCAT AGGATGCAAT CCAAGAAAGA GGAAATATGA TAATGCACTC ATGTAACAGG ATATCAAGGA CATATCACCA TTTACTGCTC AGTAAAGGGG TGCAACCCAG TTCAGGTTAA CATGGAACAA ATTTGGACAA CTTCCTTACA CCAAAACTAG TGAAATTATA AGATATAAAG TGTTGAAAGC AAATGATGAT GGAAAATCCA AGTTTACTCT GGAA7ATTATA GGTCAGAAGA CACAATAGGA CTTCCATCTG GCTGGAGGCA GGCAAATGGA GCCTCATACG AGCATGTTTC TCTGCAAAAT CATAGATATA CATTCTTAAA TAAGGCATTC CCATTGGTCT TTTGAAATAA GACATTAGGC TGGTTATCTG CTCCCTCTAC TACCGTGACT AAATTGGACT TCCCCCTTCT CCTGCACCCC AGGATAAGTT AAACAGGAGA TTTAAAATAA AGGATCAGAT GGAGAGAATA ATTATGTTAT CTGAGAATTA TGTATAAATG TAATTCTGAC AATAACATGT TTCTAAAGGA AGATAGGAGA TAATTTCCTG GAAAACTAGA GGTGAAAACA TGCCCTCCAC TTCACTATGA ATAGAGCAGT TCCTAATATG 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 AAAGAAAAAT GGATCCCAAT CTGAGAAAAG GCAAAAGAAT GGCTACTTTT TTCTATGCTG U GAGTATTTTC TAATAATCCT GCTTGACCCT TATCTGACCT CTTTGGAAAC TATAACATAG 8700 CTGTCACAGT ATAGTCACAA TCCACAAATG ATGCAGGTGC AAATGGTTTA TAGCCCTGTG 8760 AAGTTCTTAA AGTTTAGAGG CTAACTTACA GAAATGAATA AGT1TGTTTTG TTTTATAGCC 8820 CGGTAGAGGA GTTAACCCCA AAGG7C"ATAT GGTTTTATTT CCTGTTATGT TTAACTTGAT 8880 AATCTTATTT TGGCATTCTT TTCCCATTGA CTATATACAT CTCTATTTCT CAAATGTTCA 8940 TGGAACTAGC TCTTTTATTT TCCTGCTGGT TTCTTCAGTA ATGAGTTAAA TAAAACATTG 9000 ACACATACA 9009 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 2332 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANTSM: Homo sapicii TISSUE TYPE: Liver cDNA sequence (xi) SEQUENCE DE3CRIPTION: SEQ ID il-. :4: Ala Thr Arg Arg Tyr 1 5 Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr 10 Met Gin Ser Asp Leu Gly Arg Val Pro Lys Ser Phe Thr Lau Phe Val Glu. Phe Arg Pro Pro Trp Met Gly 70 Tyr Asp Thr Val Val -le Ser Leu, His Ala Val Gly 100 Glu Tyr Asp Asp Gin Thr 115 Phe Pro Gly Gly Ser His 130 Gly Pro Met Ala Ser Asp 145 150 His Val Asp Leu, Val Lys 165 Glu Pro Thr 55 Leu. Thr Val Ser Thr 135 Pro Asp Leu Pro Val Asp Ala Arg 25 Phe Asn Thr Ser Val Val 40 Val His Leu Phe Asn Ile Leu Gly Pro Thr Ile Gin 75 Leu Lys Asn Met Ala Ser 90 Ser Tyr Trp Lys Ala Ser 105 Gin Arg Glu Lys Glu Asp 120 125 Tyr Val Trp Gin Val Leu 140 Leu, Cys Leu. Thr Tyr Ser 155 Leu, Asn Ser Gly Leu Ile 170 Phe Pro Pro Tyr Lys Lys Ala Lys Pro Ala Glu Val His Pro Val Giu. Gly Ala 110 Asp Lys Val Lys Giu Asn Tyr Leu Ser 160 Gly Ala Leu 175 Leu. Val Gys Arg Glu Gly Ser Leu Ala Lys Giu Lys Thr Gin Thr Leu. 180 185 190 His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205 His Ser Glu Thr Lys Asn Ser Leu Met Gin Asp Arg Asp Ala Ala Ser 210 215 220 Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Vai Asn Arg 225 230 235 240 Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255 Val Ile Gly Met Gly Thr Thr Pro Giu Val His Ser Ile Phe Leu Giu 260 265 270 Gly His Thr Phe Leu Val Arg Asn. His Arg Gin Ala Ser Leu Glu Ile 275 280 285 Ser Pro Ile Thr Phe Leu Thr Ala Gin Thr Leu Leu Met Asp Lou Gly 290 295 300 Gin Phe Leu Leu Phe Cys His Ile Ser Ser His Gin His Asp Gly Met 305 3i0 315 320 Giu Ala Tyr Val Lys Val Asp Ser Cys Pro Giu Giu Pro Gin Leu Arg 325 330 335 Met Lys Asn Asn Giu Giu Ala Giu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350 Ser Giu Met Asp Val Vai Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365 Ile Gin Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380 Tyr Ile 385 Ala Pro Gin Arg Asp Glu Leu Gly 450 Phe Lys 465 Thr Asp His Leu Trp Thr Leu Thr 530 Ser Gly 545 Ala Asp Ie Thr 435 Pro Asn Val Lys Val 515 Arg Leu Ala Giu Giu 390 Asp Arg Ser 405 Gly Arg Lys 420 Phe Lys Thr Leu Leu Tyr Gin Ala Ser 470 Arg Pro Leu 485 Asp Phe Pro 500 Thr Val Glu Tyr Tyr Ser Ile Gly Pro 550 Giu Asp Trp Asp Tyr Ala Pro Leu Val Leu 395 400 Tyr Tyr Arg Gly 455 Arg Tyr Ile Asp Ser 535 Leu Lys Lys Giu 440 Glu Pro Ser Leu Gly 520 Phe Leu Ser L, rs 425 Ala Val Tyr Arg Pro 505 Pro Val Ile Gin 410 Val. Ile Giy Asn Arg 490 Gly Thr Asn Cys Lys 570 Tyr Leu Arg Phe Gin His Asp Thr 460 Ile Tyr 47"'5 Leu Pro Giu Ile Lys Ser Met Giu 540 Tyr Lys 555 2:-sn Met Giu 445 Leu Pro Lys Phe Asp 525 Arg Giu Asn Ala 430 Ser Leu His Gly Lys 510 Pro Asp Ser Gly 415 Tyr Giy Ile Gly Val 495 Tyr Arg Leu Val Pro Thr Ile Ile Ile 480 Ljys Lys Cys Ala Asp Gin Arg Giy Asn Gin Ile Met Ser Asp 565 Arg Asn Val IleLeu 1-ne 575 Ser Arg Gin Leu 625 Ser Thr Phe Ile Leu 705 Asp Ile Gin Val Phe Aia 610 Gin Ile Phe Ser Leu 690 Leu Ser Giu Lys Phe Leu 595 Ser Leu Gly Lys Gly 675 Gly Lys Tyr Pro Gin 755 Asp Giu 580 Pro Asn Asn Ile Ser Val Ala Gin 645 His Lys 660 Glu Thr Cys His Val Ser Giu Asp 725 Arg Ser 740 Phe Asn Asn Arg Pro Ala Met Hi!, 615 Cys Leu 630 Thr Asp Met Vai Val Phe Asn Ser 695 Ser Cys 710 Ile Ser Phe Ser Ala Thr Ser Gly 600 Ser His Phe Tyr Met 680 Asp Asp Ala Gin Thr 760 Trp 585 Val Ile Giu Leu Giu 665 Ser Phe Lys Tyr Asn 745 Ile Tyr Lev. Gin Leu Asn Giy Val Ala 635 Ser Val 650 Asp Thr Met Glu Arg -Asn Asn Thr 71i5 Leu Leu 730 Ser Arg Pro Glu Thr Glu Tyr 620 Tyr Phe Leu Asn Arg 700 Giy Ser His Asn Giu Asp 605 Val Trp Phe Thr Pro 685 G iy Asp Lys Pro Asp 765 Asn Ile 590 Pro Giu Phe Asp Tyr Ile Ser Gly 655 Leu Phe 670 Gly Leu Met Thr Tyr Tyr Asn Asn 735 Ser Thr 750 Ile Giu Gin Phe Ser Leu 640 Tyr Pro Trp Aia Glu 720 Aia Arg Lys Thr Asp Pro Trp Phe Ala His 770 775 Val Ser Ser Ser Asp Leu Leu 785 790 His Gly Leu Ser Leu Ser Asp 805 Ser Asp Asp Pro Ser Pro Gly 820 Glu Met Thr His Phe Arg Pro 835 Phe Thr Pro Giu Ser Gly Leu 850 855 Thr Thr Ala Ala Thr Glu Leu 865 870 Thr Ser Asn Asn Leu Ile Ser 885 Gly Thr Asp Asn Thr Ser Ser 900 Tyr Asp Ser Gin Leu Asp Thr 915 Leu Thr Glu Ser Gly Gly Pro 930 935 Ser Lys Leu Leu Giu Ser Gly 945 950 Arg Thr Pro Met Pro Lys Ile Gin Asn 780 Met Leu Leu Arg Gin Ser Pro Thr Pro 795 800 Leu Gin Giu Ala Lys Tyr Giu Thr Phe 810 815 Ala Ile Asp Ser Asn Asn Ser Leu Ser 825 830 Gin Leti His~ His Ser Gly Asp Met Val 840 845 Gin Leu Arg Leu Asn Giu Lys Leu Gly 860 Lys Lys Leu Asp Phe Lys Vai Ser Ser 875 880 Thr Ile Pro Ser Asp Asn Leu Ala Ala 890 895 Leu Gly Pro Pro Ser Met Pro Vai His 905 910 Thr Leu Phe Giy Lys Lys Ser Ser Pro 920 925 Leu Ser Leu Ser Giu Giu Asn Asn Asp 940 Leu Met Asn Ser Gin Giu Ser Ser Trp 955 960 Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975 Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990 Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn ASn Ser Ala 995 1000 1005 Thr Asn Arg Lys Thr His Ile Asp Giy Pro Ser Leu Leu Ile Gi-u Asn 1010 1015 1020 Ser Pro Ser Val Trp Gln Asn Ile Leu Giu Ser Asp Thr Giu Phe Lys 1025 1030 1035 1040 Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala 1045 1050 1055 Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys 1060 1065 1070 Asn Met Giu Met Val Gin Gin Lys Lys Giu Gly Pro Ile Pro Pro Asp 1075 1080 1085 Ala Gin Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Giu 1090 1095 1100 Ser Ala Arg Trp Ile Gin Arg Thr His Gly Lys Asn Ser Leu Asn Ser 1105 1110 1115 1120 Gly Gin Gly Pro Ser Pro Lys Gin Leu Val Ser Leu Giy Pro Glu Lys 1125 1130 1135 Ser Vai Giu Gly Gin Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val 1140 1145 1150 Gly Lys Gly Giu Phe Thr Lys Asp Val Gly Leu Lys Giu Met Val Phe 1155 1160 1165 Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Giu 1170 1175 1180 Asn Asn Thr His Asn Gin Glu Lys Lys Ile Gin Giu Glu Ile Giu Lys 1185 1190 1195 1200 Lys Giu Thr Leu Ile Gin Giu Asn Val Vai Leu Pro Gin Ile His Thr 1205 1210 1215 Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr 1220 1225 1230 Arg Gin Asn Val Glu Gly Ser Tyr Giu Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245 Gin Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His 1250 1255 1260 Thr Ala His Phe Ser Lys Lys Gly Glu Giu Giu Asn Leu Giu Gly Leu 1265 1270 1275 1280 Gly Asn Gin Thr Lys Gin Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg 1285 1290 1295 Ile Ser Pro Asn Thr Ser Gin Gin Asn Phe Val Thr Gin Arg Ser Lys 1300 1305 1310 Arg Ala Leu Lys Gin Phe Arg Leu Pro Leu Glu Giu Thr Glu Leu Glu 1315 1320 1325 Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gin Trp Ser Lys Asn Met 1330 1335 1340 ~s IILC C Lys His Leu Thr Pro Ser Thr Leu Thr Gin Ile Asp Tyr Asn Glu Lys 1345 1350 1355 1360 Glu Lys Gly Ala Ile Thr Gin Ser Pro Leu Ser Asp Cys Leu Thr Arg 1365 1370 1375 Ser His Ser Ile Pro Gin Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys 1380 1385 1390 Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu 1395 1400 1405 Phe Gin Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1410 1415 1420 Asp Ser Gly Val Gin Glu Ser Ser His Phe Leu Gin Gly Ala Lys Lys 1425 1430 1435 1440 Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gin 1445 1450 1455 Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr 1460 1465 1470 Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gin Lys Asp 1490 1495 1500 Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu 1505 1510 1515 1520 Val Glu Gly Ser Leu Leu Gin Gly Thr Glu Gly Ala Ile Lys Trp Asn 1525 1530 1535 I L I -I II I Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu 1540 1545 1550 Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp 1555 1560 1565 Asn His Tyr Gly Thr Gin Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu 1570 1575 1580 Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser 1585 1590 1595 1600 Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly 1605 1610 1615 Gin Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gin Gly Arg Thr 1620 1625 1630 Glu Arg Leu Cys Ser Gin Asn Pro Pro Val Leu Lys Arg His Gln Arg 1635 1640 1645 Glu Ile Thr Arg Thr Thr Leu Gin Ser Asp Gin Glu Glu Ile Asp Tyr 1650 1655 1660 Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr 1665 1670 1675 1680 Asp Glu Asp Glu Asn Gin Ser Pro Arg Ser Phe Gin Lys Lys Thr Arg 1685 1690 1695 His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser 1700 1705 1710 Ser Ser Pro His Val Leu Arg Asn Arg Ala Gin Ser Gly Ser Val Pro 1715 1720 1725 Ls ~ILL I I- Gin Phe Lys Lys Val Val Phe Gin Glu Phe Thr Asp Gly Ser Phe Thr 1730 1735 1740 Gin Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly 1745 1750 1755 1760 Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg 1765 1770 1775 Asn Gin Aia Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr 1780 1785 1790 Glu Glu Asp Gin Arg Gin Giy Ala Giu Pro Arg Lys Asn Phe Val Lys 1795 1800 1805 Pro Asn Giu Thr Lys Thr Tyr Phe Trp Lys Val Gin His His Met Ala 1810 1815 1820 Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp 1825 1830 1835 1840 -4 C:' Val Asp Leu Giu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu 1845 1850 1855 Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gin Val Thr 1860 1865 1870 Val Gin Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Giu Thr Lys Ser 1875 1880 1885 Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1890 1895 1900 Ile Gin Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala 1905 1910 1915 1920 I I_ Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gin 1925 1930 1935 Asp Gin Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn 1940 1945 1950 Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965 Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu 1970 1975 1980 Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys 1985 1990 1995 2000 Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val 2005 2010 2015 Tyr Ser Asn Lys Cys Gin Thr Pro Leu Gly Met Ala Ser Gly His Ile 2020 2025 2030 Arg Asp Phe Gin Ile Thr Ala Ser Gly Gin Tyr G3y Gin Trp Ala Pro 2035 2040 2045 Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr 2050 2055 2060 Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile 2065 2070 2075 2080 Ile His Gly Ile Lys Thr Gin Gly Ala Arg Gin Lys Phe Ser Ser Leu 2085 2090 2095 Tyr Ile Ser Gin Phe Ile Ile Met Tyr Ser Lev Asp Gly Lys Lys Trp 2100 2105 2110 -dl, I Lsl I I' L Gin Thr Tyr Arq Gly Asn Ser Thr Gly Thr Leu Met Vai Phe Phe Gly 2115 2120 2125 Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2130 2135 2140 Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser 2145 2150 2155 2160 Thr Leu Arg Met Giu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met 2165 2170 2175 Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gin Ile Thr Ala 2180 2185 2190 Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205 Arg Leu His Leu Gin Gly Arg Ser Asn Ala Trp Arg Pro Gin Val Asn 2210 2215 2220 Asn Pro Lys Glu Trp Leu Gin Val Asp Phe Gin Lys Thr Met Lys Val 2225 2230 2235 2240 Thr Gly Val Thr Thr Gin Gly Val Lys Ser Leu Leu Thr Ser Met Ty~r 2245 2250 2255 Val Lys Giu Phe Leu Ile Ser Ser Ser Gin Asp Gly His Gin Trp Thr 2260 2265 2270 Leu Phe Phe Gin Asn Gly Lys Val Lys Val Phe Gin Gly Asn Gin Asp 2275 2280 2285 Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg 2290 2295 2300 ~lrsRms~aRlaraarrry~rmCC~s~$ll~ere* ~I~ Tyr Leu Arg Ile His Pro Gin Ser Trp Val His Gin Ile Ala Leu Arg 2305 2310 2315 2320 Met Glu Val Leu Gly Cys Glu Ala Gin Asp Leu Tyr 2325 2330 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1260 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Porcine (xi) SEQUENCE DESCRIPTION: SEQ ID GAATTCTTCA CTCAGATTCT CCTGTTCACA GTAGAAATTC AGTATTGTTA GCACTCTTTT AGTTACCTGT ATCCTAAACC TAAGTCCTGC TCCCTTATAC TTACTCATCC TACAAATTGG 120 TCAGAGTATG TGTTTGGCAT TATGTTATGT GATTTGAATG CATTATCAGA TACTACTAGT 180 CTCATTTACA AATTAGAAAA CTGGAGCTCA GAGAGTTCCT TGGACTTGCT TAAAGCAACA 240 CAGCTGGTAA ATTGTATAGC TAGGATTCGA ACCGAGGCAA TCGTACTCTA GAACCCATGC 300 CACTATGTTG CATAGCATAA TAGCCCGCCT ATATAAACTT GGCTGAATTA AGTCACGATC 360 C -II ~t LC -LI~I TATCATCACC CTCCATTTTT AGGTACACTA GATATAATGG AGAGAACCTC CTTCCTTATC GGATGACAGC ACACTTATGA, GCTTTGCCCA GTCCAGAAGA AACTAAGTGT GCTCATCCTC CAAGAGAAAG GTGCCGAAAG TGTCTAGTTC A.AAGAGTCCG ATCCCAATAT GAGCCATGGT TACCGACTAG TA: CACAGAT TCCAGGTCTC CTTACTGAAC AGATATTCCA GAATTCAAGA TGACGTGGAG CCCCl1CTGGT ArCTGATCTT AAACACGGCC AGTACTTACT ATCAGACCTT TGTGACTAAG ATATTCATTT TGGGCTGCAG TGTTTTGTTT CTTGCTTGGG TGGGTCCTAG GTGTATAGTT GGCTTCTTGC CCCCCTAGTG CTTGACCCGC GATGGGTC(6A CAAGAAGCCA CCATCCGCAG CCTGAGCCAG CTAAAGACT AGTCTCAACT AAATCACAGC TCCATGGTGT TTGTTTTTGT ACCTGGGCTG GGTGCCACAA GTGACAGGGA TGAGTGGAAA CGAGCCAAAA AGTCTGGAGA TGCTCI2'GGG GGAATGNGGC CGGCACGTCT AGAAAGAGTT CGCCAACAAT ATTGTATGTC CCTT. CTTGT ACATTTAACC TTCATTT[TTC TGAGTAACCA CTCAGACTTG CACTGGTGAT GAATGTCATT GCAATTCCAA GAGAACCCAA ACAGAATCCT TGATGAT.LTAT CAGACCAGAG GAAGAAACT TCCATCAGAC AATTATATTT GGTCACAAAC CAAC1GACCTC TGGGAATAGA GAGTTTTATT CGGAACAGAG TATTATGACA GAACCCAGAA ACCATCACAA GCACTGGAAG GCTCCACATG; TTACCTGGAG CTGCATCACA GATTCTAAA ACGTTGTCAG 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 INFORM4ATIONI FOR SEQ ID NO: 6: ()SEQUENCE CHARACTERISTICS: LENGTH: 868 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) M4OLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: N-termainal (vi) ORIGINAL SOURCE: ORGANISM: Porcine (xi) SEQUENCE DESCRIPTION: SEQ ID NO,,6: Gly Leu Trp Val Leu Gly Cys His Met S'er Asp Leu Arg Asn Arg Gly 1 5 10 Met Thr Ala Leu Leu Lys Val Tyr Ser Cys, Asp Arg Asp Thr Gly Asp c 25 300 Tyr Tyr Asp Asn Thr Tyr Giu Asp Leu Pro Gly Phe Leu Leu 'Per Gly 40 Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gin Asn Ser Arg Pro Pro 55r Ser Ala Ser Gin Lys Gin Phe Gin Thr Ile Thr Ser Pro Glu Asp Asp 70 75 Val Giu Leu Asp Pro Gin Ser Gin Giu Arg Thr Gin Ala Leu Glu Giu 90 Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gin Asn Pro 100 105 110 Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gin Giu Ala Arg Asn Glu 115 120 125 Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Asn Thr Ala Pro Ser 130 135 140 Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Giu Arg Val 145 150 155 160 Leu Thr Pro Glu Pro Glu Lys Giu Leu Lys Lys Leu Asp Ser Lys Met 165 170 175 Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp 180 185 190 Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His 195 200 205 Pro Gin Val Asn Phe Arg Ser Gin Leu Gly Ala Ile Val Leu Gly Lys 210 215 220 0 Asn Ser Ser His Phe Ile Gly Ala Gly Val Pro Leu Gly Ser Thr Glu 225 230 235 240 Phe Asp His Glu Ser Ser Leu Gly Giu Asn Val Ser Pro Val Glu Ser 245 250 255 Asp Gly Ile Phe Giu Lys Giu Arg Ala His Gly Pro Ala Ser Leu Thr 260 265 270 Lys Asp Asp Vai Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn 275 280 285 Lys Aj~a Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp 290 295 300 Ala Ala Leu Leu Thr Glu Asn Arg Ala 305 Asn Pro Leti Gly Gly 385 Ser Arg Pro Phe His 465 Thr Leu Leu Gin 370 Lys Ala Glu Gln His 450 Ala Thr Gly Thr 355 Gly Glu Asp Glu Val 435 Gln Pro Ala Lys 340 Ser Arg Met Val Met 420 Tyr Ser Val Ser 325 Asn Ser Ile Met Gln 405 Giu Thr Thr Pro 310 Gly Leu Pro Leu Gly Ser Arg Val 375 Leu Pro 390 Gly Asn Arg Arg Ala Thr Giu Pro 455 Gin Asp 470 Asn Ser Gly 360 Ala Asn Asp Giu Gly 440 Ser Ser His Ser 345 Lys Val Ser Thr Leu 425 Thr Val Arg Ser Val 330 Glu Ser Giu Giu His 410 Val Lys Giu S er Ala 315 Ser Arg Val GiU Leu 395 Ser Gin Asn Gly Leu 475 Thr Phe Met Asp Asn Gly Lys Giu 380 Thr Gin Giu Phe Phe 460 Asn Trp Pro Gly 365 Glu Phe Gly Lys Leu 445 Asp Asp Ile Ser 350 Gin Leu Leu Liys Val 430 Arg Gly Ser Lys 335 Pro Ser Ser Thr Lys 415 Asp Asn Giy Ala Lys 320 Gly GlU Ser Lys Asn 400 Ser Leu Ile Ser Giu 480 Arg Ala Giu Thr His Ile Ala His Phe Ser Ala Ile Arg Giu Giu Ala 495 485 490 Pro Leu Gin Ala Pro Gly Asn Phe Thr Gly Pro Gly Pro Arg Ser Ala 510 500 Val Gin Arg 545 Len Ie Len Lys Pro 625 Gin Asn Pro Gin 530 Trp Ser Ser Giu Ala 610 Gin Ile Arg Arg 515 Ile S er Len Ala Lys 595 Gin Lys Phe Pro Arg Lys Glun Pro Len 580 Ala Phe Thr Leu Gly 660 Val Pro Ser Phe 565 Gly Val Leu Ser Gin 645 Arg Lys Gin Ser 550 Len Lys Len Pro Asn 630 Lys Thr Gin Arg 535 Pro Thr Ser Ser Lys 615 Val Thr Pro Ser 520 Gly Ile Len Ala Ser 600 Val Ser Arg Ser Lys 680 505 Len Val Leu Gin Ala 585 Ala Arg Cys Giy Lys 665 Lys Val Gin Met 570 Giy Gly Vai Ala Pro 650 Leu Gin Leu Gly 555 Ala Pro Len His His 635 Vai Len Ile Asn 540 Ala Gly Len Ser Arg 620 Gly Asn Gly Arg 525 Ala Lys Gly Ala Glu 605 Gin Asp Len Pro Leu Thr Arg Gin Ser 590 Ala Asp Ile Asn Pro 670 Pro Ser Asn Gly 575 Gly Ser Leu Gly Lys 655 Met Len Thr Asn 560 Lys Lys Gly Len Gin 640 Val Pro Lys Gin Trp 675 Gin Ser Len Gin Ser Pro Lys Ser Thr 685 Ala Len Arg Thr Lys 690 Asp Ile Leu Ser Leu Pro Leu Asp Arg 695 His Glu Ser Asn His 700 S er 705 Ala Pro Pro Lys Arg 785 Gin Arg Glu Asn Asp 865 Ile Trp Val Glu Gly 770 Ser Leu Ala Phe Lys 850 Ala Thr Leu Giu 755 Glu Phe Trp Gin Ala 835 His Ala Lys Arg 740 Asp Asp Gin Asp Asn 820 Asp Lys Asn 710 Gin Gly 725 Arg His Lys Met Phe Asp Lys Arg 790 Tyr Gly 805 Gly Giu Gly Ser Giu Giy Gin Asp Ile 775 Thr Met Val Phe Gly Pro Arg Tyr 760 Tyr Arg Ser Pro Thr 840 Gin G ly Asp 745 Asp Gly His Giu Arg 825 Asn Ala Arg 730 le Asp Glu Tyr S er 810 Phe Pro Phe 715 Leu Ser le Asp Phe 795 Pro Lys Ser Tkr Gin Cys Ala Leu Pro Phe Ser 765 Giu Asn 780 le Ala Arg Ala Lys Val Tyr Arq 845 Arg Pro Thr 750 Thr Gin Ala Leu Val 830 Gly Glu Lys 735 Pro Giu Asp Val Arg 815 Phe Giu Ala 720 Pro Gin Thr Pro Glu 800 Asn Argj Leu Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Giu Val Giu 855 860 Asn Ile Met Claims (21) 1. A purified hybrid factor VIII molecule comprising 1porcine and human amino acid sequences, wherein tile molecule has procoagulant activity in an in vitro coagulation assay and wherein the molecule is selected from the group consisting of a molecule consistinlg of a human factor VIII in which the porcine A2 domain is substituted for the homologous hlumnl factor VIII A2 domain; a molecule consisting of a porcine factor VIII ill whlich the human A2 domain is suibstituted for the hiomologous porcine factor VIII A2 domain; a molecule consisting of a human light chain factor VIII subunit and a porcine heavy chain factor VIII subunit; and a molecule consisting of a porcine light chain factor VIII subunit and a human heavy chain factor VIII subunit. 2. The molecule of claim 1, wherein the molecule has a specific activity greater than 20,000 U/A 2 Oprotein in aqueous solution when human plasma is used as the standard in a one-stage coagulation assay. 3. The molecule of claim 1, wherein the hybrid humnan/porcine factor VIII is combined with a pharmaceutically acceptable carrier, 4. The molecule of claim 3, wherein the carrier is selected from the group consisting of stabilising agents and delivery vehicles. The molecule of claim 4, wherein the stabilising agents are selected from the group consisting of proteins and polysaccharides. 6. The molecule of claim 3, further comprising clotting factors selected from the group consisting of von Willebrand factor, vitamin K dependent clotting factors, and coagulant tissue factor, The molecule of claim 4, wherein the delivery vehicles are liposomes. 8. The molecule of claim 1, wherein the molecule consists of a human factor VIII in which the porcine AZ domain is subs tituted for the homologous human factor VIII A2 domain. 9. The molecule of claim 1, wherein the molecule consists of a porcine factor VIII which the huuman AZ domain is substituted for the homologous porcine factor VIII A2 domain. The molecule of claim 1, wherein tile molecule consists of a human light chain factor VIII subunit and a porcine heavy chain factor VIII subunit. 11. The molecule of claim 1 wherein the molecule consists of a porcine light chain factor VIII subunit and a human heavy chain factor VIII subunit. 12. A method of preparing purified hybrid/human/porcine factor VIII comprising combining primary amino acid sequence derived from porcine factor VIII with primary amino acid sequence derived from human factor VIII to form a hybrid factor VIII molecule having procoagulant activity in an in vitro coagulation assay, wherein the molecule is selected from the group consisting of a molecule consisting of a human light chain factor VII subunit and a porcine heavy chain factor VIII subunit; and a molecule consisting of a porcine light chain factor VIII subunit and a human heavy chain factor VIII subunit. 13. The method of claim 12 herein the hybrid human/porcine factor VIII molecule is formed by isolating and purifying heavy and light chain subunits of human factor VIII and porcine factor VIII, then mixing the human and porcine subunits to form the hybrid hunnan/porcine factor VIII, 14. The method of claim 13 herein the human and porcine factor VIII subunits are isolated from human and porcine plasma. 15. The method of claim 13 herein the hybrid human/porcine factor VIII molecule is formed by mixing human light chain factor VIII subunits and porcine heavy chain factor VIII subunits, 16. The method of claim 13, wherein the hybrid human/porcine factor VIII molecule is formed by mixing porcine light chain factor VIII subunits and human heavy chain factor VIII subunits. 17. A method of preparing purified hybrid human/porcine factor VIII comprising expressing recombinant DNA encoding domains in the light chain and heavy chain subunits of porcine and human factor VIII, further comprising substituting A2 domains of porcine and human factor VIII, to form the purified hybrid human/porcime factor VIII having primary amino acid sequence derived from porcine factor VIII and primary amino acid sequence derived from human factor VIII and having procoagulant activity in an in vitro coagulation assay. I 18. The method of claim 17, wherein the hybrid human/porcine factor VIII molecule is formed by substituting the porcine A2 domain in human factor VIII, 19. The method of claim 17, wherein the hybrid human/porcine factor VIII molecule is formed by substituting the human A2 domain in porcine factor VIII. An isolated nucleic acid molecule encoding the hybrid human/porcine factor VIII molecule as claimed in any one of claims claim 1 to 11. 21. The nucleic acid molecule of claim 20, wherein the molecule lacks the B domain. 22. An isolated nucleic acid molecule encoding porcine factor VIII A2 domain, 23. A method of treating human factorVoldeficiency in a subject having factor vwideficiency comprising administering to the subject an effective amount of a hybrid human/porcine factor VIII molecule as claimed in any one of claims 1 to 11. 24. The method of claim 23 wherein the subject has antibodies to factor VIII that inhibit coagulant activity. 25. The method as claimed in claim 24, wherein the clotting activity of the hybrid human/porcine factor VIII molecule is not inhibited by the antibodies to factor VIII, Dated this 30th day of June 1997 EMORY UNIVERSITY Patent Attorneys for the Applicant: F.B. RICE CO. AU42799/93A 1992-04-07 1993-04-07 Hybrid human/porcine factor VIII Ceased AU682147B2 (en) Applications Claiming Priority (3) Application Number Priority Date Filing Date Title US07/864,004 US5364771A (en) 1992-04-07 1992-04-07 Hybrid human/porcine factor VIII US864004 1992-04-07 PCT/US1993/003275 WO1993020093A1 (en) 1992-04-07 1993-04-07 Hybrid human/porcine factor viii Publications (2) Publication Number Publication Date AU4279993A AU4279993A (en) 1993-11-08 AU682147B2 true AU682147B2 (en) 1997-09-25 Family ID=25342308 Family Applications (1) Application Number Title Priority Date Filing Date AU42799/93A Ceased AU682147B2 (en) 1992-04-07 1993-04-07 Hybrid human/porcine factor VIII Country Status (12) Country Link US (2) US5364771A (en) EP (2) EP1359222B1 (en) JP (2) JP3440097B2 (en) AT (2) ATE284963T1 (en) AU (1) AU682147B2 (en) CA (1) CA2133203C (en) DE (2) DE69333724T2 (en) DK (2) DK1359222T3 (en) ES (2) ES2261866T3 (en) HK (1) HK1058946A1 (en) PT (1) PT1359222E (en) WO (1) WO1993020093A1 (en) Families Citing this family (61) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title US6180371B1 (en) 1996-06-26 2001-01-30 Emory University Modified factor VIII US5859204A (en) * 1992-04-07 1999-01-12 Emory University Modified factor VIII US5663060A (en) * 1992-04-07 1997-09-02 Emory University Hybrid human/animal factor VIII US5563045A (en) * 1992-11-13 1996-10-08 Genetics Institute, Inc. 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PP 12481-12486 * Also Published As Publication number Publication date HK1058946A1 (en) 2004-06-11 ATE322544T1 (en) 2006-04-15 EP1359222A2 (en) 2003-11-05 CA2133203C (en) 2002-07-02 ES2235154T3 (en) 2005-07-01 US5583209A (en) 1996-12-10 EP1359222B1 (en) 2006-04-05 US5364771A (en) 1994-11-15 EP0638088B1 (en) 2004-12-15 DE69334003T2 (en) 2007-01-25 WO1993020093A1 (en) 1993-10-14 DE69333724T2 (en) 2005-11-10 DK0638088T3 (en) 2005-04-25 EP0638088A1 (en) 1995-02-15 JP2003174876A (en) 2003-06-24 DK1359222T3 (en) 2006-06-19 JP3440097B2 (en) 2003-08-25 DE69334003D1 (en) 2006-05-18 JP3495365B2 (en) 2004-02-09 EP0638088A4 (en) 1996-06-26 JPH07508404A (en) 1995-09-21 DE69333724D1 (en) 2005-01-20 PT1359222E (en) 2006-07-31 EP1359222A3 (en) 2003-11-19 ES2261866T3 (en) 2006-11-16 AU4279993A (en) 1993-11-08 CA2133203A1 (en) 1993-10-14 ATE284963T1 (en) 2005-01-15 Similar Documents Publication Publication Date Title AU682147B2 (en) 1997-09-25 Hybrid human/porcine factor VIII AU693837B2 (en) 1998-07-09 Hybrid human/animal factor VIII US6376463B1 (en) 2002-04-23 Modified factor VIII AU717686B2 (en) 2000-03-30 Modified factor VIII CA2400295C (en) 2012-01-10 Modified factor viii AU747644B2 (en) 2002-05-16 Modified factor VIII AU2001238416A1 (en) 2001-12-06 Modified factor VIII US20040249134A1 (en) 2004-12-09 Factor viii c2 domain variants US20040005670A1 (en) 2004-01-08 Compositions and methods for the treatment of hemophilia A
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