WO1999029838A1

WO1999029838A1 - Fibrinogen-converting enzyme hybrids

Info

Publication number: WO1999029838A1
Application number: PCT/US1998/026086
Authority: WO
Inventors: Stewart A. Cederholm-Williams
Original assignee: Bristol-Myers Squibb Company
Priority date: 1997-12-09
Filing date: 1998-12-09
Publication date: 1999-06-17
Also published as: AU753458B2; AU1905899A; NO20002937L; JP2001526027A; NO20002937D0; CA2312476A1; EP1037969A1; EP1037969A4

Abstract

Provided is a fusion protein comprising: a first polypeptide chain comprising a fibrinogen-converting enzyme; and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (1) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, or (2) via a bifunctional linkage moiety linking said groups or functionalities.

Description

FIBRINOGEN-CONVERTING ENZYME HYBRIDS

The present invention is directed to multidomain proteins made up of a first polypeptide that is a fibrinogen-converting enzyme or a snake venom-derived proteinase and a second polypeptide that is a member of a binding pair, where the other member of the binding pair can be used to remove the fusion protein from a fibrin preparation that was formed through the action of the converting enzyme. The invention is further directed to recombinant methods of forming the multidomain protein, to nucleic acids and vectors used in such methods, and to methods of forming fibrin using the fusion protein. The invention further provides a fusion protein between a fibrinogen-converting enzyme and a polypeptide designed to facilitate covalent attachment of one member of a binding pair. Also, the invention provides for an aggregate of (a) the first polypeptide which is covalently linked to a member of a binding pair and (b) the second polypeptide which binds to the first by way of its binding the member of a binding pair.

"Fibrin" sealants are widely used to reduce bleeding in surgery and to seal blood vessels and tissues that have been dissected either in surgery or through wounding. The term "fibrin" can be viewed as a misnomer in this context since historically "fibrin" sealants have been delivered as a material containing the precursor of fibrin, namely fibrinogen. In such sealants, fibrinogen material has been co-delivered at the site to be sealed with a proteinase enzyme that converts the fibrinogen to fibrin. Once a sufficient amount of fibrin is formed from the fibrinogen, the fibrin spontaneously polymerizes into a fibrin polymer which — when sufficient polymer is assembled — forms a fibrin clot. Generally, the conversion enzyme has been bovine-derived thrombin. Recently, however, an effective sealant has been described that delivers fibrin, in a form that is prevented from polymerizating, to the site that is to be sealed. At the site, the polymerization prevention conditions are reversed, and an effective clot forms. See, Edwardson et al., European Patent Application No. EP 592,242. As described in EP 592,242, the fibrin in the sealant can be formed by contacting fibrinogen with a fibrinogen converting enzyme that is bound to a solid support. The solid support allows for the removal of the converting enzyme from the sealant. One of the particular advantages of this fibrin sealant of EP 592,242 is that the sealant can be an autologous sealant that is rapidly prepared from a small amount of a patient's blood only minutes before surgery, and this preparation can be done using standard laboratory equipment. Processes for deriving the fibrinogen material of prior art sealants are much more demanding and more difficult to automate. Specialized tools for preparing fibrin have also recently been described, and these tools allow an autologous sealant to be prepared from a patient in a rapid, highly reproducible, highly reliable, and highly safe manner. See, Holm, "Centrifuge Reagent Delivery System", WO 96/16713, Holm et al., "Method and Device for Separating Fibrin I from Blood Plasma", WO 96/16714 and Holm, "Centrifuge with Annular Filter", WO 96/16715.

The present invention provides additional means to remove the fibrinogen converting enzyme from the fibrin sealant preparation. In one aspect, the invention provides a fusion protein comprising the converting enzyme and another polypeptide that can be used to bind the fusion protein to a solid support after the converting enzyme has been used to form fibrin from fibrinogen. In another aspect, the invention provides a fusion protein comprising the converting enzyme and another polypeptide that can be used to covalently attach binding moieties. In yet another aspect, the converting enzyme and the other polypeptide are aggregated by means of the other polypeptide binding to an member of a binding pair which is covalently linked to the converting enzyme. Summary of the Invention

In a first embodiment, the invention provides a multidomain protein comprising: a first polypeptide chain comprising a fibrinogen-converting enzyme; and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (1) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (2) via a bifunctional linkage moiety linking said groups or functionalities, or (3) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain. In one embodiment, the multidomain protein is a recombinant protein comprising a continuous amino acid sequence that includes the second polypeptide chain and the first polypeptide chain, and the second polypeptide chain comprises a polypeptide with biotin-binding activity. The second polypeptide chain can comprise a multivalent binding entity such as an antibody, Streptavidin or avidin with two or more binding sites. Such a multivalent binding entity can bind to a first polypeptide via a ligand for the binding sites which is covalently linked to the first polypeptide, leaving at least one other binding site available to bind another molecule of ligand.

In a second embodiment, the invention provides a nucleic acid encoding a recombinant fusion protein comprising a continuous amino acid sequence which comprises: a first polypeptide chain comprising a fibrinogen-converting enzyme; and a second polypeptide chain comprising a member of a binding pair, wherein the first and second polypeptides are either fused directly via a peptide bond or fused via a linker polypeptide chain.

In a third embodiment, the invention provides a recombinant fusion protein comprising a contiguous polypeptide chain comprising: a first polypeptide comprising a fibrinogen-converting enzyme; and a second polypeptide comprising a two or more of (a) amino acid side chains that can be used to attach a binding partner or (b) O-linked or N- linked polysaccharrides that can be used to attach a binding partner. In one embodiment, the amino acid residues of the second polypeptide are selected to minimize the amount of secondary structure forming adjacent to said amino acids with attachable side chains In a fourth embodiment, the invention provides a method of preparing a fibrin composition, the method comprising: (1) contacting a composition comprising fibrinogen with an enzyme effective to convert fibrinogen to fibrin, forming a fibrin composition, wherein said enzyme comprises a multidomain protein comprising a first polypeptide chain comprising a fibrinogen-converting enzyme, and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (a) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (b) via a bifiinctional linkage moiety linking said groups or functionalities, or (c) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain. Preferably, where the fibrin composition is not a monomeric fibrin composition, the method further comprises: (2) forming a monomeric fibrin composition from the fibrin composition. Preferably, the method further comprises: (3) contacting the monomeric fibrin composition with a solid support having bound thereto a second member of the binding pair effective to bind the first member. In another preferred aspect, the method further comprises after the contacting of step (3): (4) removing the solid support and recovering a resulting monomeric fibrin composition.

In a fifth embodiment, the invention provides a conjugate protein comprising an alpha polypeptide chain comprising a snake venom-derived proteinase effective to convert prothrombin to thrombin, and a second molecule that is a member of a binding pair covalently attached to the alpha polypeptide chain.

In a sixth embodiment, the invention provides a nucleic acid encoding a recombinant fusion protein comprising a continuous amino acid sequence comprising: an alpha polypeptide chain comprising a snake venom-derived proteinase effective to convert prothrombin to thrombin; and a beta polypeptide chain comprising a member of a binding pair, wherein the alpha and beta polypeptides are fused directly via a peptide bond or fused via a linker polypeptide chain.

In a seventh embodiment, the invention provides a method of preparing a thrombin composition, the method comprising: (1) contacting a composition comprising prothrombin with a snake-derived enzyme effective to convert to prothrombin to thrombin, thereby forming a thrombin composition, wherein said enzyme comprises a multidomain protein comprising an alpha polypeptide chain comprising the enzyme, and a beta polypeptide chain comprising a first member of a binding pair, wherein the beta polypeptide chain is linked to the alpha polypeptide chain (a) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (b) via a bifunctional linkage moiety linking said groups or functionalities, or (c) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain. Preferably, the method further comprises: (2) contacting the thrombin composition with a solid support having bound thereto a second member of the binding pair effective to bind the first member. In another preferred aspect, the method further comprises: (3) removing the solid support and thereby recovering a resulting thrombin composition. Definitions

♦ alpha, beta, first, second: Terms such as "first", "second", "alpha" and "beta" are used herein as arbitrary names to help distinguish recitals of similar elements.

♦ bifunctional linkage group: A bifunctional linkage group is a molecule having two sites for attaching to a protein or polypeptide. Preferred bifunctional linkage groups are polypeptides of from one to about 3 amino acids, preferably of from one to about 30 amino acids. Additionally, preferred bifunctional linkage groups can be crosslinking reagents, such as for example reagents having two reactive moieties such as succinimidyl esters, maleimides, iodoactyl groups and nitrophenyl groups ♦ direct bonds: Direct bonds between two protein or polypeptides are bonds that link a nitrogen, carbon, oxygen or sulfur from one protein or polypeptide to a nitrogen, carbon, oxygen or sulfur from the other protein or polypeptide.

♦ fibrinogen-converting enzyme: A fibrinogen-converting enzyme is a substance that catalyzes a conversion of fibrinogen to a derivative that spontaneously polymerizes noncovalently to form fibrin polymer. Generally, the derivative will be fibrin I (desAA- fibrin), fibrin II (desAA-desBB-fibrin) or desBB-fibrin.

♦ high affinity binding: High affinity binding between a first substance and a second substance is binding of sufficient avidity to allow for the first or second substance to be used as an affinity ligand for the isolation of the other substance. Typically, high affinity binding is reflected in a association constant of about 10^ M"l or more, preferably 10°^"

M"l or more, more 10^ M^~l or more.

♦ monomeric fibrin: Monomeric fibrin is fibrin that has been prevented from polymerization so that, for a solution form of a fibrin composition, when examined by such techniques as ultracentrifugation or gel filtration substantially all of the fibrin chain molecules in the composition behave as the non-polymerized hexamers (o-2β2Y2)- The phrase "substantially all" in this context means at least about 80% of the fibrin chain molecules, preferably at least about 90%, more preferably at least about 95%. For solid forms, the monomeric form in some instances will be indicated by the fact that the solid was formed from a solution form of monomeric fibrin monomer by lyophilization or another dehydration method. If necessary, more involved analytical techniques can be applied to assertain if the fibrin hexamers are not aggregated with polymer-forming non- covalent bonds of type and strength involved in the formation of fibrin clots. Solid forms can include suspended solids within a liquid in which the fibrin is not soluble, such as a suspension in acetone. ♦ peptide or polypeptide: The term "polypeptide" as used herein includes shorter polypeptides such as those often referred to a "peptides," for instance polypeptides of less than about 100 amino acid residues.

♦ recombinant protein: A recombinant protein is a protein in a form or a protein expressed in a cell in which it would not be found but for the cell or organism that expresses the recombinant protein, or an ancestor of that cell or organism, having been transformed by the introduction of extrinsic nucleic acid material.

^♦ snake venom-derived proteinase: A snake venom-derived proteinase is a proteinase found in snake venom, a proteinase prepared from snake venom, a recombinant proteinase prepared from a cDNA for a snake venom proteinase or a portion of that cDNA which recombinant proteinase retains the proteolytic activity of the snake venom proteinase of the cDNA, a synthetically prepared snake venom proteinase or a portion of any of the foregoing that retains the proteolytic activity of the parent molecule. Detailed Description Enzyme-to-Binding Polypeptide Fusion Protein

The fibrinogen-converting enzyme is preferably batroxobin ("Btx"), i.e., a proteinase from the snake venom of snakes of the genus Bothrops. Other proteinases of appropriate specificity can also be used. Snake venom proteinases are particularly suitable, including without limitation the venom enzymes from Agkistrodon acutus, Agkistrodon ancrod, Agkistrodon bilineatus, Agkistrodon contortrix contortrix,

Agkistrodon halys pallas, Agkistrodon (Calloselasma) rhodostoma, Bothrops asper, Bothrops atrox, Bothrops insularis, Bothrops jararaca, Bothrops Moojeni, Lachesis muta muta, Crotalus adamanteus, Crotalus atrox, Crotalus durissus terrificus, Trimeresurus flavorviridis, Trimeresurus gramineus, Trimeresurus mucrosquamatus and Bitis gabonica. In one embodiment, enzymes from Bothrops are used.

The sequence for a fibrinogen-converting enzyme from Agkistrodon rhodostoma is described in Bach et al., WO90/06362. Two sequences from Agkristrodon c. contortrix are described in Valenzuela et al., EP O 323 722. The sequence of batroxobin (from Bothrops atrox moojeni), is described in Japanese Patent Application (Kokai) 2-124092. Sequences for batroxobin, an enzyme from Trimeresurus flavorviridis, and an enzyme from Crotalus horridus are reported in Pirkle and Theodor, "Structure of Thrombin-like snake venom Proteinases", in Medical use of Snake Venom Proteins. Sequences from Agkristrodon c. contortix, Russell's viper, Bothrops atrox moojeni and Trimeresurus flavoviridis are described in McMullen et al., Biochemistry 28:674-679, 1989.

Sequences for enzymes from Trimeresurus mucrosquamatus are available from the National Center for Biotechnology Information (NCBI, Bethesda, MD) under accession numbers 602596, 602598, 602600, 602602 and 602604. Sequence for an enzyme from Protobothrops mucrosquamatus is available from the NCBI under accession number 951152. Sequence for an enzyme from Agkistrodon ancrod (Malayan pit viper, also known as Calloselasma rhodostoma) is available from the SWISS-PROT protein database (accessible through the NCBI) under accession number P47797. Sequence for an enzyme from Bothrops atrox is available from the NCBI under accession number 211031 or Genbank under accession number J02684. Sequence for an enzyme from Bothrops jararaca is available from the PIR protein database (accessible through the NCBI) under accession number A54361. Sequences for enzymes from Russell's viper (Vipera Russellϊ) are available from the SWISS-PROT protein database (accessible through the NCBI) under accession numbers P 18964 and P 18965. Sequences for an enzyme from Trimeresurus flavoviridis is available from the SWISS-PROT protein database (accessible through the NCBI) under accession number P05620. Sequence for an enzyme from Crotalus atrox is available from the PIR protein database (accessible through the NCBI) under accession number A45655. Sequence for an enzyme from Agkistrodon bilineatus is available from the NCBI under accession number 211031. Sequences for enzymes from Agkistrodon contortrix are available from the NCBI under accession numbers 603215 and 603217 and from GenBank (accessible through the NCBI) under accession numbers 106680, 106681, 106724 and 106751.

When the fibrinogen-converting enzyme is thrombin, generally the fusion protein of the invention will be formed by chemical methods. Recombinant methods must account for the proteolytic processing reactions required to generate thrombin from prothrombin, as was done by Falkner et al., International Patent Application WO91/11519 ("Recombinantly Produced Blood Factors").

Snake-derived proteinases that convert prothrombin to thrombin are, for example, of use in large-scaled processes for producing thrombin, in methods for producing autologous thrombin. These methods are improved by the use of enzyme preparations that can be removed through the use of a binding-partner relationship. The snake-derived prothrombin-converting enzyme is preferably from the venom oϊEcchis carinatus. Other snake venom proteinases that are suitable, include without limitation the venom enzymes from Australian tiger snake and Akistrodon hallys pallys. For example, the sequence for a prothrombin activator (ecarin) from Kenyan Echis carinatus venom is described in Kawabata, Biochemistry 34: 1771-1778, 1995 (GenBank Accession No. D32212). The polypeptide that is a member of a binding pair is preferably avidin or Streptavidin, which polypeptides each bind with high affinity to biotin. An amino acid sequence for avidin is described in Dayhoff, Atlas of Protein Sequence, Vol. 5, National Biomedical Research Foundation, Washington, DC, 1972 (see also, DeLange and Huang, J Biol. Chem. 246: 698-709, 1971), and an amino acid sequence for Streptavidin is described in Argarana et ., Nucl. AcidRes. 14: 1871-1882, 1986. Nucleic acid sequences are available, for example, as follows: (1) chicken mRNA for avidin, Gene Bank Ace. No. X5343, Gore et al., Nucl. AcidRes. 15: 3595-3606, 1987; (2) chicken, strain White Leghorn mRNA for avidin, Gene Bank Ace. No. L27818 (3) streptavidin from Strep, avidinii, Gene Bank Ace. No. X03591, Argarana et al., Nucl. AcidRes.

14: 1871-1882, 1986; (4) synthetic gene for streptavidin from Strep, avidinii, Gene Bank Ace. No. A00743, Edwards, WO89/03422; and (5) synthetic gene for streptavidin, Gene Bank Ace. No. X65082, Thompson et al., Gene 136: 243-246, 1993.

Avidin and Streptavidin are preferably used in a tetrameric form, although monomers can be used. In reciting herein that an avidin or Streptavidin protein retains biotin-binding activity, it is of course envisioned that this may involve the protein forming multimeric associations with like proteins. Other useful members of a binding pair can include an antibody specific for a polypeptide or other molecule, any polypeptide to which an antibody is available or can be prepared, thioredoxin, which binds phenylarsine oxide (expression vectors include, for example, the thioredoxin fusion protein vector pTrxFus available from Invitrogen, Carlsbad, CA, or Invitrogen B.V., Netherlands), poly-His sequences that bind to divalent cations such as nickel II (expression vectors include, for example, the pThioHis vectors A, B and C available from Invitrogen), glutathione-S- transferase vectors that bind to glutathione (vector for example available from Pharmacia Biotech, Piscataway, NJ). Methods of producing such antibodies are available to those of ordinary skill in light of the ample description herein of polypeptide expression systems and of known antibody production methods. For antibody preparation methods, see, for example, Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons, New York, 1992. The binding-pairs used in the invention preferably display high affinity binding even at relatively low pH, such as a pH of about 5. In some embodiments of the invention, the member of a binding pair that is attached to an enzyme is not limited to polypeptide members of binding pairs. In this case, biotin is the most preferred such member of a binding pair.

In many embodiments of the invention, the polypeptide chains making up the fusion proteins will be manufactured by recombinant means, as described further below. These recombinant techniques allow for the polypeptide chains to be modified by amino acid substitutions and sequence deletions such as deletions of internal or terminal sequences. Further, the N-terminal leader sequence can be modified as appropriate to promote export of the protein from the host cell. Such modified recombinant products can be readily synthesized on a small pilot scale and tested, for instance for enzymatic activity or binding activity. These pilot tests can generally be conducted without strenuous purification procedures since the organism used to produce the recombinant substance can be selected to lack the relevant activity, allowing for crude lysates or unpurified culture medium to be tested for the activity.

Mutational and deletional approaches can be applied to all of the nucleic acid sequences encoding relevant polypeptide chains. Conservative mutations are preferred. Such conservative mutations include mutations that switch one amino acid for another within one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp. A preferred listing of conservative variations is the following:

Original Residue Variation

Ala Gly, Ser

Arg Lys - l ι

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala, Pro

His Asn, Gin

He Leu, Val

Leu He, Val

Lys Arg, Gin, Glu

Met Leu, Tyr, He

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp, Phe

Val He, Leu

The types of variations selected may be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer- Verlag, 1978, on the analyses of structure- forming potentials developed by Chou and Fasman, Biochemistry 13, 211, 1974 and Adv. Enzymol, 47, 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81, 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157, 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15, 321-353, 1986. All of the references of this paragraph are incorporated herein in their entirety by reference.

In a preferred embodiment of the invention, the association between the fibrinogen-converting enzyme or snake-derived proteinase and the polypeptide that is a member of a binding pair is effected by recombinantly expressing the two components of the fusion proteins with (a) the two polypeptide encoding nucleic acids are directly fused such that in the synthesized protein the C-terminal amino acid of one polypeptide is directly linked by a peptide bond to the N-terminal amino acid of the other or (b) the two polypeptide encoding nucleic acids are fused via a linker nucleic acid encoding an amino acid or polypeptide, such that in the synthesized protein the C-terminal amino acid of one polypeptide is linked by a peptide bond to the N-terminal of the linker amino acid or polypeptide, which linker amino acid or polypeptide is linked by a peptide bond at its C- terminal to the N-terminal of the other polypeptide of the fusion protein.

In other embodiments, the fusion between the fibrinogen-converting enzyme or snake-derived proteinase and the polypeptide that is a member of a binding pair is effected by other types of bonds including disulfide bonds between cysteine residues of the respective polypeptides, amide bonds between amine and carboxylate functionalities of the two polypeptides, and bonds formed by bifunctional crosslinking reagents. Such bifunctional reagents include compounds with activated acyl esters such as N- hydroxysuccinimide esters, mercuric ion, other mercury compounds, compounds containing maleimide functionalities, compounds containing iodoacetyl functionalities, compounds containing fluoro-nitro-aryl functionalities, compounds containing alkylimidate functionalities, compounds containing arylsulfonyl chloride functionalities, compounds containing isocyanate functionalities, aldehyde or dialdehyde compounds and compounds containing diazoaryl functionalities. Crosslinking methods using such reagents are reviewed in Means and Feeney, Chemical Modification of Proteins, Holden- Day, San Francisco, 1971, which document is incorporated herein in its entirety by reference.

Among the modifications that can be added to a protein by recombinant methods are sequences that are glycosylated when the protein is expressed in an appropriate cell. N-linked glycosylations typically occur at the asparagine of Asn-Xaa-Ser/Thr tripeptide subsequences of glycoproteins. Converting Enzyme-Attachable Polypeptide Fusion Protein

In one embodiment of the invention, the fibrinogen converting enzyme is fused not with the member of a binding pair, but with a polypeptide designed to facilitate linkage with a member of a binding pair. Such a facilatitive polypeptide can, for instance, comprise a polylysyl polypeptide, or another repetitive polypeptide that is rich in an amino acid whose side chain is useful in linking the member of a binding pair. Preferably, the facilitative polypeptide comprises between about 10 and about 50 amino acid residues, more preferably between about 20 and about 30 amino acid residues. Preferably, the linkable amino acid comprises lysine, arginine, histidine, aspartic acid, glutamic acid or cysteine, more preferably lysine or cysteine, and yet more preferably lysine. For methods on how to link biotin to the facilatitive polypeptide can be found, for example, in Savage et al., Avidin. Biotin Chemistry: A Handbook, Pierce Chemical Co., 1992. A preferred method of linking a member of a binding pair to a carbohydrate structure is oxidation with periodate followed by reductive alkylation. Recombinant Nucleic Acids. Cells and Methods Where nucleic acid sequence is known, or sufficient amino acid sequence is known to predict useful primers, nucleic acid amplification methods, such as polymerase chain reaction (PCR) methods, can be used to amplify useful polypeptide-encoding nucleic acids from the RNA of a tissue that expresses such a polypeptide. Such PCR methods are well described in PCR Protocols, Cold Spring Harbor Press, 1991. In some cases, PCR methods directly applied will only isolate internal sequences. Fortunately, methods have been developed to amplify and isolate sequences extended from such internal sequences so as to encompass all useful sequence. One such method is referred to as PCR-RACE, and protocols for this method are available, for example, from Gibco BRL (Gaithersburg, MD). Where amplification methods can be used to isolate two or more overlapping nucleic acids that together encode all of the needed nucleic acid, these can be pieced together using natural restriction sites or by designing restriction sites by use of appropriate PCR primers. Where restriction sites are designed into the PCR primers, it can be necessary to change the codons used to encode particular amino acid residues or to make mutational changes (preferably conservative) to design the restriction site. In some cases, it may be necessary to subclone a particular nucleic acid fragment and use well- established site directed mutagenesis techniques to engineer the needed restriction sites. See, Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1995, pp.8.1.1-8.1.6.

To construct non-naturally occurring enzyme- or binding polypeptide-encoding nucleic acids, the native sequences can be used as a starting point and modified to suit particular needs. For instance, the sequences can be mutated to incorporate useful restriction sites. See Maniatis et al. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989). Such restriction sites can be used to create "cassettes", or regions of nucleic acid sequence that are facilely substituted using restriction enzymes and ligation reactions. The cassettes can be used to substitute synthetic sequences encoding mutated enzyme or binding polypeptide amino acid sequences. Alternatively, the enzyme or binding polypeptide-encoding sequence can be substantially or fully synthetic. See, for example, Goeddel et al., Proc. Natl. Acad. Sci. USA, 76, 106-110, 1979. For recombinant expression purposes, codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic enzyme or binding polypeptide-encoding nucleic acid. For example, a nucleic acid sequence incorporating prokaryotic codon preferences can be designed from a mammalian-derived sequence using a software program such as Oligo-4, available from National Biosciences, Inc. (Plymouth, MN).

The nucleic acid sequence embodiments of the invention are preferably deoxyribonucleic acid sequences, preferably double-stranded deoxyribonucleic acid sequences. However, they can also be ribonucleic acid sequences.

Numerous methods are known to delete sequence from or mutate nucleic acid sequences that encode a protein and to confirm the function of the proteins encoded by these deleted or mutated sequences. Accordingly, the invention also relates to a mutated or deleted version of a nucleic acid sequence that encodes a protein that retains (a) the ability to bind specifically another molecule or (b) the intended enzymatic activity. These analogs can have N-terminal, C-terminal or internal deletions, so long as appropriate function is retained.

A suitable expression vector is capable of fostering expression of the included polypeptide in a host cell, which can be eukaryotic (including fungal), or prokaryotic. Useful expression vectors include pRc/CMV (Invitrogen, San Diego, CA), pRc/RSV (Invitrogen), pcDNA3 (Invitrogen), Zap Express Vector (Stratagene Cloning Systems, LaJolla, CA); pBk/CMV or pBk-RSV vectors (Stratagene), Bluescript II SK +/- Phagemid Vectors (Stratagene), LacSwitch (Stratagene), pMAM and pMAM neo (Clontech, Palo Alto, CA), pKSVIO (Pharmacia, Piscataway, NJ), pCRscript (Stratagene) and pCR2.1 (Invitrogen), among others. Useful yeast expression systems include, for example, pYEUra3 (Clontech). Useful baculovirus vectors, for expression in insect cells, include several viral vectors from Invitrogen (San Diego, CA) such as pVL1393, pVL1392, pBluBac2, pBluBacHis A, B or C, and pbacPACό from Clontech. Some of these vectors will utilize inducible promoters such as the lac promoter. In one aspect of the invention, inducible promoters are desirable, such as promoters responsive to zinc or other metal ions, to metabolites or metabolite mimics such as isopropylthio- -galactoside, or to hormones such as estrogen or ecdyson (for instance, found in expression systems available from Invitrogen, San Diego, CA). Inducible systems help to minimize the adverse effects that can flow from the expressed protein having toxic effects on the expression cells.

In one embodiment of the invention, the polypeptides are preferably expressed in a mammalian cell line, preferably a transformed cell line with an established cell culture history. In this embodiment, suitable cell lines include COS-1, COS-7, LM(tk"), HeLa, HEK293, CHO, Rat-1 and NIH3T3.

In another embodiment, the polypeptides are expressed in a cell line that is more inexpensively maintained and grown than are mammalian cell lines, such as a bacterial cell line or a fungal cell line such as a yeast cell line. In this aspect of the invention, E. coli bacterial cells are particularly preferred.

In all aspects of recombinant methodology referred to herein, ample guidance can be found in a number of widely recognized authorities including: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989; and Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons, New York, 1992. Methods of Producing Recombinant Fusion Proteins

One simplified method of isolating polypeptides synthesized by an organism under the direction of one of the nucleic acids of the invention is to recombinantly express a version of the fusion protein having a fusion component that is facilely affinity purified. This fusion component can be simply the polypeptide chain that is a member of a binding pair. Or, this fusion component can be another fused polypeptide. A useful component for purification is, for instance, glutathione S-transferase, which is encoded on commercial expression vectors (e.g., vector pGEX4T3, available from Pharmacia, Piscataway, NJ). Another useful purification component is, for instance, thioredoxin. This glutathione S-transferase-containing fusion protein can then be purified on a glutathione affinity column (for instance, that available from Pharmacia, Piscataway, New Jersey). If extra fusion partners are used, the extra fusion partner can be removed by partial proteolytic digestion approaches that preferentially attack unstructured regions such as the linkers between the extra fusion partner and the desired fusion protein. The linkers can be designed to lack structure, for instance using the rules for secondary structure forming potential developed, for instance, by Chou and Fasman, Biochemistry 13, 211, 1974 and Chou and Fasman, Adv. in Enzymol. 47, 45-147, 1978. The linker can also be designed to incorporate protease target amino acids, such as, arginine and lysine residues, the amino acids that define the sites cleaved by trypsin. To create the linkers, standard synthetic approaches for making oligonucleotides can be employed together with standard subcloning methodologies. Other fusion partners besides GST can be used. Procedures that utilize eukaryotic cells, particularly mammalian cells, are preferred since these cells will post-translationally modify the protein to create molecules highly similar to or identical to native proteins. Additional purification techniques can be applied, including without limitation, preparative electrophoresis, FPLC (Pharmacia, Uppsala, Sweden), HPLC (e.g., using gel filtration, reverse-phase or mildly hydrophobic columns), gel filtration, differential precipitation (for instance, "salting out" precipitations), ion-exchange chromatography and affinity chromatography. Preferably, the protein is substantially pure, meaning a purity of at least 20%> w/w with respect to other proteins, more preferably at least about 50%>, yet more preferably at least about 70%>, still more preferably at least about 90%>. For the purposes of this application, the fusion protein is "isolated" if it has been separated from other proteins or other macromolecules of the cell or tissue from which it is derived or prepared. Methods of Preparing Fibrin Compositions

In using the fusion protein of the invention, blood is for example drawn from a patient and mixed with an anticoagulant, such as trisodium citrate to a final concentration of about 0.5% w/v. Blood and liver cell cultures, recombinant cultures or milk from transgenically modified mammals are examples of suitable sources of fibrinogen. Plasma is isolated by centrifugation, which removes the cellular components of the blood. The fusion protein is added to the fibrinogen containing solution, for example, at a concentration approximately corresponding, on a molar basis, to a concentration of baxtroxobin of about 0.1 μg/ml to about 100 μg/ml, preferably to a concentration of about 0.5 μg/ml to about 50 μg/ml. A precipitate of fibrin polymer forms from the reaction of fibrinogen with enzyme incorporated into the fusion protein. Where that fibrinogen-converting enzyme is batroxobin, the polymer is generally made up of fibrin I. The fibrin polymer is isolated by centrifugation or filtration, and then dissolved in a low pH buffer, such as 0.2 M sodium acetate, pH 4.0, preferably in the presence of calcium ions, for example at a concentration of about 20 mM. Biotin covalently bound to a solid support such as agarose is added in sufficient quantity to bind in excess of about 99% of the fusion protein. The biotinylated support can be prepared, for example, by reacting one of the biotinylating agents available from Boehringer Manheim (Indianapolis, IN) or Clontech (Palo Alto, CA) with a solid support having primary amino groups. The biotinylating reagents typically have a biotinyl substituent, one to two aminocaproyl spacer groups, and a reactive N-hydroxysuccinimide group. The solid support can be, for example, the amino-derivatized agarose resins available from Sigma (St. Louis, MO) or an amino-derivatized chromatography matrix available from Pharmacia (Uppsala, Sweden). The solubilized fibrin is removed from the support-bound fusion protein by centrifugation or filtration. The solubilized fibrin is now ready for use in a sealant, for instance as described in Edwardson et al., European Patent Application No. 592,242. Preparation of Solid Supports The solid support to which the second member of the binding pair is bound is, for example made up of beaded or non-beaded particles of carbohydrate-based material such as agarose, cross-linked agarose or cross-linked dextran, or a non-porous material such as polystyrene. Methods for covalently coupling molecules to solid supports are well known in the art, and include for example creating reactive sites on the solid supports with cyanogen bromide or reacting the solid supports with bifunctional reagents such as diglycidyl ethers. See, for example, "Attachment to Solid Supports" in Means and Feeney, Chemical Modification of Proteins, Holden-Day, San Francisco, 1971, pp. 40-43 ox Affinity Chromatography: A Practical Approach, Dean et al., eds., IRL Press, Oxford, 1991, the disclosures of which two references are incorporated herein in there entirety by reference. For coupling with silica-based materials, alkyloxysilane moieties, for example, can provide the silica-reactive moiety of a bifunctional coupling reagent. For example, γ-glycidoxypropyltrimethoxysilane can be reacted with the silica-based material, which is then directly reacted with the protein (via the glycidic ether moiety), or a second step is employed such as reacting the glycidic ether with an amine and subsequently attaching by reductive alkylation a glycoprotein that is mildly oxidized (for instance with periodate) to contain aldehyde moieties. A preferred coupling chemistry reacts a carbohydrate-based solid support with a hydrazide group, and then coupling by reductive alkylation a glycoprotein that is mildly oxidized (for instance with periodate) to contain aldehyde moieties. See, Axelsson et al., Thromb. Haemost. 36: 517, 1976, which document is incorporated herein in its entirety by reference.

The following examples further illustrate the present invention, but of course, should not be construed as in any way limiting its scope. Example IA - Mammalian Vector Encoding Batroxobin

A EcoRT-Xbal fragment encoding batroxobin is excised from a pUC18 clone (R&D Systems, Inc., Minneapolis, MN) and cloned into the multiple cloning site of pCI- neo (Promega, U.K., Southampton, UK). The expression sequence is made up of the -24 to 228 sequence of batroxobin which includes the leader sequence. The batroxobin enzyme is expressed in CHO cells from the resulting vector. Example IB - Bacterial Vector Encoding Batroxobin-thioreductase fusion Protein

A Bsal-Xbal fragment encoding batroxobin was excised from a pCI/neo clone (R&D Systems, Inc., Minneapolis, MN) and cloned into the multiple cloning site of pTrxFus (Invitrogen). The resulting fusion protein expressed in E. coli was made up of thioredoxin fused at its 5' end via linker with an enterokinase cleavage site to the 1 to 228 amino acid sequence of batroxobin. The fusion protein was purified phenylarsine oxide column (Invitrogen, B.V., Netherlands). Example 2 - Chemical Formation of a Batroxobin- Avidin Fusion Protein Covalent complexes may be formed between batroxobin and avidin using

N-succinimidy-3-(2-pyridyldithio)proprionate (SPDP, Pierce Chemical Co., Rockford, IL) as follows: 3mg of batroxobin was reacted with 1.9 mg SPDP dissolved in 0.75 ml ethanol for 60 minutes at room temperature. 6 mg avidin was reacted identically in a separate container. The protein product of each reaction was separately desalted on Sephadex G25 in 50 mM sodium phosphate, 20 mM NaCl, pH 7.0 buffer. The derivatised proteins were then activated by reduction, which exposed thiol groups derived from the SPDP, mixed together, and the mixed proteins were again desalted by gel filtration on Sephadex G25. Batroxobin-avidin conjugates were isolated (away from non- conjugated protein) by gel filtration on Sephadex G100.

Sequence Listing SEQ ID NO: 1 is of the Gallus gallus cDNA for avidin, and SEQ ID NO: 2 is the corresponding protein. Amino acids 1-24 are believed to be the leader sequence, and amino acids 25-152 the mature protein. SEQ ID NO:3 is an cDNA for streptavidin, and SEQ ID NO:4 is the corresponding protein. Amino acids 1-24 are believed to be the leader sequence, and amino acids 25-183 the mature protein. SEQ ID NO:5 is of the Bothrops atrox cDNA for batroxobin, and SEQ ID NO: 6 is the corresponding protein. Amino acids 1-18 are believed to be the leader sequence, and amino acids 25-255 the mature protein.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references. While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations in the preferred devices and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims that follow.

Claims

What is claimed:

1. A multidomain protein comprising: a first polypeptide chain comprising a fibrinogen-converting enzyme; and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (1) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, (2) via a bifunctional linkage moiety linking said groups or functionalities, or (3) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain.

2. The multidomain protein of claim 1 which is a fusion protein, comprising a recombinant protein comprising a continuous amino acid sequence that includes the second polypeptide chain and the first polypeptide chain.

3. The multidomain protein of claim 1, wherein the second polypeptide chain comprises a polypeptide with biotin-binding activity.

4. The multidomain protein of claim 3, wherein the second polypeptide chain comprises at least a portion of a Streptavidin protein or an avidin protein sufficient to retain biotin-binding activity.

5. The multidomain protein of claim 4, wherein Streptavidin protein or avidin protein comprises at least two biotin binding sites, and the Streptavidin protein or avidin protein is associated with the first polypeptide chain by binding to a biotin molecule covalently linked to the first polypeptide chain.

6. The multidomain protein of claim 1, wherein the first polypeptide chain comprises a snake venom enzyme.

7. The multidomain protein of claim 6, wherein the first polypeptide chain comprises a venom enzyme from Agkistrodon acutus, Agkistrodon contortrix contortrix, Agkistrodon halys pallas, Agkistrodon (Calloselasma) rhodostoma, Bothrops asper, Bothrops atrox, Bothrops insularis, Bothrops jararaca, Bothrops Moojeni, Lachesis muta muta, Crotalus adamanteus, Crotalus durissus terrificus, Trimeresurus flavorviridis, Trimeresurus gramineus or Bitis gabonica.

8. A nucleic acid encoding a recombinant fusion protein comprising a continuous amino acid sequence comprising a first polypeptide chain comprising a fibrinogen-converting enzyme, and a second polypeptide chain comprising a member of a binding pair, wherein the first and second polypeptides are fused directly via a peptide bond or fused via a linker polypeptide chain.

9. A vector comprising the nucleic acid of claim 8 and sufficient regulatory sequences for expressing the fusion protein in a cell.

10. A cell comprising the vector of claim 9.

11. A method of producing a fusion protein comprising a fibrinogen- converting enzyme and a first member of a binding pair comprising the steps of: growing the cell of claim 10 in a culture medium; and recovering from the cell or the culture medium the fusion protein.

12. A recombinant fusion protein comprising a contiguous polypeptide chain comprising: a first polypeptide comprising a fibrinogen-converting enzyme; and a second polypeptide comprising a two or more of (a) amino acid side chains that can be used to attach a binding partner or (b) O-linked or N-linked polysaccharrides that can be used to attach a binding partner.

13. A method of preparing a fibrin composition, the method comprising: (1) contacting a composition comprising fibrinogen with an enzyme effective to convert fibrinogen to fibrin, thereby forming a fibrin composition, wherein said enzyme comprises a multidomain protein comprising a first polypeptide chain comprising a fibrinogen-converting enzyme, and a second polypeptide chain comprising a first member of a binding pair, wherein the second polypeptide chain is linked to the first polypeptide chain (a) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, or (b) via a bifunctional linkage moiety linking said groups or functionalities, or (c) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the first polypeptide chain..

14. The method of preparing a fibrin composition of claim 13, further comprising:

(2) forming a monomeric fibrin composition from the fibrin composition.

15. The method of preparing a fibrin composition of claim 14, further comprising: (3) contacting the monomeric fibrin composition with a solid support having bound thereto a second member of the binding pair effective to bind the first member.

16. The method of claim 15, comprising after the contacting of step (3):

(4) removing the solid support and thereby recovering a resulting monomeric fibrin composition.

17. A conjugate protein comprising an alpha polypeptide chain comprising a snake venom-derived proteinase effective to convert prothrombin to thrombin, and a beta polypeptide chain that is a member of a binding pair covalently attached to the alpha polypeptide chain.

18. The conjugate protein of claim 17, wherein the alpha polypeptide chain is linked to the beta polypeptide chain (1) directly by bonds utilizing the N-terminal amino groups, the C-terminal carboxy groups, or side-chain functionalities, or (2) via a functional linkage moiety linking said groups or functionalities.

19. The conjugate protein of claim 17, comprising a recombinant protein comprising a continuous amino acid sequence that includes the alpha polypeptide and the beta polypeptide.

20. A nucleic acid encoding a recombinant fusion protein comprising a continuous amino acid sequence comprising an alpha polypeptide chain comprising a snake venom-derived proteinase effective to convert prothrombin to thrombin, and a beta polypeptide chain comprising a member of a binding pair, wherein the alpha and beta polypeptides are fused directly via a peptide bond or fused via a linker polypeptide chain.

21. A vector comprising the nucleic acid of claim 20 and sufficient regulatory sequences for expressing the fusion protein in a cell.

22. A cell comprising the vector of claim 19.

23. A method of producing a fusion protein comprising a fibrinogen- converting enzyme and a first member of a binding pair comprising the steps of: growing the cell of claim 22 in a culture medium; and recovering from the cell or the culture medium the fusion protein.

24. A method of preparing a thrombin composition, the method comprising: (1) contacting a composition comprising prothrombin with a snake-derived enzyme effective to convert to prothrombin to thrombin, thereby forming a thrombin composition, wherein said enzyme comprises a multidomain protein comprising an alpha polypeptide chain comprising the enzyme, and a beta polypeptide chain comprising a first member of a binding pair, wherein the beta polypeptide chain is linked to the alpha polypeptide chain (a) directly by bonds utilizing the N-terminal amino groups, the C- terminal carboxy groups, or side-chain functionalities, (b) via a bifunctional linkage moiety linking said groups or functionalities, or (c) by the first member binding to the second member of the binding pair, wherein the second member of the binding pair is covalently attached to the alpha polypeptide chain.

25. The method of preparing a thrombin composition of claim 24, further comprising:

(2) contacting the thrombin composition with a solid support having bound thereto a second member of the binding pair effective to bind the first member.

26. The method of claim 25, comprising after the contacting of step (2): (3) removing the solid support and thereby recovering a resulting thrombin composition.