CA2330191A1 - Protease inhibitor peptides - Google Patents

Abstract

Analogues of the Kunitz Protease Inhibitor (KPI) domain of amyloid precursor protein bind to an inhibit activity of serine proteases, including kallikrein, plasmin and coagulation factors such as factors VIIa, IXa, Xa, XIa, and XIIa.
Pharmaceutical compositions containing the KPI analogs, along with methods for using such compositions, are useful for ameliorating and treating clinical conditions associated with increased serine protease activity, such as blood loss related to cardiopulmonary bypass surgery. Nucleic acid sequences encoding these analogs and systems for expression of the peptides of the invention are provided.

Description

WO 99!63090 PCT/US99/12276 PROTEASE INHIBITOR PEPTIDES
Bac~our~ of the Invention The plasma, or serine, proteases of the blood contact system are known to be activated by interaction with negatively charged surfaces. For example, tissue injury during surgery exposes the vascular basement membrane, causing interaction of the blood with collagen, which is negatively charged at physiological pH. This induces a cascade of proteolytic events, leading to production of plasmin, a fibrinolytic protease, and consequent blood loss.
Perioperative blood loss of this type can be particularly severe during cardiopulinonary bypass (CPB} surgery, in which the patient's blood flow is diverted to an artificial heart-lung machine. CPB is an essential component of a number of lifo saving surgical procedures. For example, in the United States, it is estimated that 300,000 patients every year undergo coronary artery bypass grafts involving the use of I S CPB.
Although necessary and generally safe, CPB is associated with a significant rate of morbidity, some of which may be attributed to a "whole body inflammatory response"
caused by activation of plasma protease systems and blood cells through interactions with the artificial surfaces of the heart-lung machine (Butler et al., Ann.
Thorac. Sung.
55:552 (1993); Edmunds et al., J. Card. Sung. 8:404 (1993)). For example, during extracorporeal circulation, exposure of blood to negatively charged surfaces of the artificial bypass circuit, e.g., plastic surfaces in the heart-lung machine, results in direct activation of plasma factor 7dI.
Factor ?QI is a single-chain 80 kDa protein that circulates in plasma as as inactive zymogen. Contact with negatively chargod nonendothelial surfaces, like those of the bypass circuit, causes surface-bound factor 7~ to be autoactivated to the active serine protease factor 3CQa. See Colman, Agents Actions Suppl. 42:125 (1993).
Surfaco-activated factor XIIa then processes prckallila~ein (PK) to active kallikrein, which in tum cleaves more XIIa from 7Qi in a reciprocal activation reaction that results in a rapid amplification of the contact pathway. Factor 3~Ia can also activate the first component of complement C1, leading to production of the anaphylatoxin CSa through the classical complement pathway.
The CPB-induced inflammatory response includes changes in capillary permeability and interstitial fluid accumulation. Cleavage of high molecular weight kininogen (I-BC) by activated ka11i1Qein generates the potent vasodilator bradykinin, which is thought to be responsible for increasing vascular permeability, resulting in edema, especially in the lung. The lung is particularly susceptible to damage associated with CPB, with some patients exhibiting what has been called "pump lung syndrome"
S following bypass, a condition indistinguishable from adult respiratory distress. See Johnson et al., J. Thorac. Cardiovasc. Surg. 107:1193 ( 1994).
Post-CPB pulmonary injury includes tissue damage thought to be mediated by neutrophil sequestration and activation in the microvasculature of the lung.
Butler et al., supra; Johnson, et al., supra. Activated factor XII can itself stimulate neutrophil aggregation. Factor XIIa-generated ka11i1Qein, and complement protein CSa generated by Factor XIIa activation of the complement cascade, both induce neutrophil chemotaxis, aggregation and degranulation. See Edmunds et al., supra.
Activated neutrophils may damage tissue through release of oxygen-derived free radicals, proteolytic enzymes such as elastase, and metabolites of arachidonic acid.
Release of neutrophil products in the lung can cause changes in vascular tone, endothelial injury and loss of vascular integrity.
Intrinsic inhibition of the contact system occurs through inhibition of activated 3CIIa by C1-inhibitor (Cl-INI~. See Colrnan, supra. During CPB, massive activation of plasma proteases and consumption of inhibitors overwhelm this natural inhibitory mechanism. A potential therapeutic strategy for reducing post-bypass pulinonary injury mediated by neutrophil activation would, therefore, be to block the formation and activity of the neutiophil agonists kallikrein, factor XIIa, and CSa by inhibition of proteolytic activation of the contact system.
Protease inhibitor therapy, which partially attenuates the contact system, is currently employed clinically in CPB. Aprotinin, also known as basic pancreaxic protease inhibitor (BPPI), is a small, basic, 58 amino acid polypeptide isolated from bovine Lung. It is a broad-spectrum serine protease inhibitor of the Kunitz type, and was first used during bypass in an attempt to reduce the inflammatory response to CPB. See Butler et a1, supra. Aprotinin treatment results in a significant reduction in blood loss following bypass, but does not appear to significantly reduce neutrophil activation.
Additionally, since aprotinin is of bovine origin, there is concern that repeated administration to patients could lead to the development of an immune response to aprotinin in the patients, precluding its further use.

2 The proteases inhibited by aprotinin during CPB appear to include plasma kallikrein and plasmin. See, e.g., Scott, et al., Blood 69:1431 (1987).
Aprotinin is an inhibitor of plasmin (K; of 0.23nM), and the observed reduction in blood loss may be due to inhibition of fibrinolysis through the blocking of plasmin action.
Although aprotinin inhibits plasma kallikrein (K; of 20nM), it does not inhibit activated factor 3CB, and consequently only partially blocks the contact system during CPB.
Another attractive protease target for use of protease inhibitors, such as those of the present invention, is factor XIIa, situated at the very first step of contact activation.
By inhibiting the proteolytic activity of factor 3CIa, kallikrein production would be prevented, blocking amplification of the contact system, neutrophil activation and bradykinin release. Inhibition of 3QIa would also prevent complement activation and production of CSa. More complete inhibition of the contact system during CPB
could, therefore, be achieved through the use of a better XIIa inhibitor.
Protein inhibitors of factor XIIa are known. For example, active site mutants of a,-antitrypsin that inhibit factor 7~QIa have been shown to inhibit contact activation in human plasma. See Patston et al., J. Biol. Chem. 265:10786 (1990). The large siu and complexity (greater than 400 amino acid residues) of these proteins present a significant challenge for recombinant protein production, since large doses will almost certainly be required during CPB. For example, although it is a potent inhibitor of both kallilaein and plasmin, nearly 1 gram of aprotinin must be infused into a patient to inhibit the massive activation of the kallikrein-kinin and fibrinolytic systems during CPB.
The use of smaller, more potent 7QIa inhibitors such as the com and pumpkin trypsin inhibitors (Wen, et al., Protein Exp. & Purif. 4:215 (1993); Pedersen, et al., J.
Mol. Biol. 236:385 (1994)) could be more cost-effective than the large a,-antitrypsins, but the infusion of high doses of these non-mammalian inhibitors could result in immunologic reactions in patients undergoing repeat bypass operations. The ideal protein 7CIIa inhibitor is, therefore, preferably small, potent, and of human sequence origin.
One candidate for an inhibitor of human origin is found in circulating isoforms of the human amyloid (I-protein precursor (APPn, also known as protease nexin-2.
APPI
contains a Kunitz serine protease inhibitor domain known as KPI (Kunitz Protease Inhibitor). See Ponte et al., Nature, 331:525 (1988); Tanzi et al., Nature 331:528 (1988); Johnstone et al., Biochem. Biophys. Res. Commun. 163:1248 (1989);
Oltersdorf

3 et al., Nature 341:144 (1989). Human KPI shares about 45% amino acid sequence identity with aprotinin. The isolated IS;PI domain has been prepared by recombinant expression in a variety of systems, and has been shown to be an active serine_ protease inhibitor. See, for example, Sinha, et al., J. Biol. Chem. 265:8983 ( 1990).
The measurod in vitro K; of KPI against plasma kallikrein is 45nM, compared to 20nM for aprotinin.
Aprotinin, KPI, and other Kunitz-type serine protease inhibitors have been engineered by site-directed mutagenesis to improve inhibitory activity or specificity.
Thus, substitution of Lysls of aprotinin with arginine resulted in an inhibitor with a K; of 0.32nM toward plasma kallikrein, a 100-fold improvement over natural aprotinin. See PCT application No. 89/10374. See also Norris et al., Biol. Chem. Hoppe Seyler 371:3742 (1990). Alternatively, substitution of position 15 of aprotinin with va.line or substitution of position 13 of KPI with valine resulted in elastase inhibitors with K;s in the 100 pM range, although neither native aprotinin nor native KPI
significantly inhibits eIastase. See Wenzel et al., in: Chemistry of Peptides and Proteins, Yol. 3, (Walter de Gruyter, Berlin, New York, 1986); Sinha et al., supra. Methods for substituting residues 13, 15, 37, and 50 of KPI are shown in general terms in European Patent Application No. 0 393 431, but no specific sequences are disclosed, and no protease inhibition data are given.
Phage display methods have been recently used for preparing and screening derivatives of Kunitz-type protease inhibitors. See PCT Application No.
92115605, which describes specific sequences for 34 derivatives of aprotinin, some of which were reportedly active as elastase and cathepsin inhibitors. The amino acid substitutions in the derivatives were distributed throughout almost all positions of the aprotinin molecule.
Phage display methods have also been used to generate KPI variants that inhibit factor VITa and kallila~ein. See Dennis et al., J. Biol. Chem. 269:22129 and 269:22137 (1994). The residues that could be varied in the phage display selection process were Limited to positions 9-11, 13-17, 32, 36 and 37, and several of those residues were also held constant for each selection experiment. One of those variants was said to have a IC;
of l.2nM for kallila~ein, and had substitutions at positions 9 ('ThrPro), 13 (ArgLys), 15 (MetLeu), and 37 (GlyTyr). None of the inhibitors was tested for the ability to inhibit factor XIIa. PCT application WO 96139515 used phage display methods to vary the residues at positions 11-19 and 34. Certain of those variants were tested for inhibition of kalIikrein; factors Xia, Xa, and VIZa; thrombin; plasmin; and activated protein C. PGT

4 WO 99/63090 PC'T/US99/12276 application WO 96/35788 used phage display methods to vary the residues at positions 9, 11, 13-18, 32, and 37-40. Certain of those variants were tested for inhibition of kaliikrein, plasmin, and factors Xa, Xia, and XIIa.
It is apparent, therefore, that new protease inhibitors that can bind to and inhibit the activity of serine professes are greatly desirable. In particular it is highly desirable to prepare peptides, based on human peptide sequences, that can inhibit selected seriae professes such as kallikrein; chymotrypsins A and B; trypsin; elastase;
subtilisin;
coagulants and procoagulants, particularly those in active form, including coagulation factors such as factors VIIa, IXa, Xa, XIa, and XIIa; plasmin; thrombin;
proteinase-3;
enterokinase; acrosin; cathepsin; urokinase; and tissue plasminogen activator.
It is also highly desirable to prepare novel protease inhibitors that can ameliorate one or more of the undesirable clinical manifestations associated with enhanced serine protease activity, for example by reducing pulmonary damage or blood loss during CPB. In addition, it is highly desirable to prepare such novel protease inhibitors with high expression levels, as well as with high yields.
Summary of the Invention The present invention relates to peptides that can bind to and preferably exhibit inhibition of the activity of serine professes. Those peptides can also provide a means of ameliorating, treating or preventing clinical conditions associated with increased activity of serine proteases. Particularly, the novel peptides of the present invention preferably exhibit a more potent and specific (i.e., greater) inhibitory effect toward serine professes of interest in comparison to known serine protease inhibitors. Examples of such professes include: kallikrein; chymotrypsins A and B; trypsin; elastase;
subtilisin;
coagulants and procoagulants, particularly those in active form, including coagulation factors such as factors V>Za, lXa, Xa, XIa, and XIIa; plasmin; thrombin;
proteinaso-3;
enterokinase; acrosin; cathepsin; urokinase; and tissue plasminogen activator.
In particular, the peptides of the present invention preferably exhibit a greater potency and specificity for inhibiting one or more serine professes of interest (eg., kalIila~ein, plasmin 30' and factors VIZa, IXa, Xa, XIa, and XIIa) than the potency and specificity exhibited by native KPI or other known setirte protease inhibitors.
The novel peptides of the present invention preferably comprise substituting the tyrosine residue at position 48. Such substituted peptides may exhibit an increased level of recombinant expression in comparison to the expression levels of serine professes that

5 do not have that substitution. The effect of this substitution may be manifested not only on the substituted ICPI peptides of the present invention, but on wild-type ICPI as well.
Also, the peptides of the present invention that comprise the N-terminal sequence Glu-Val-Val-Arg (residues -4 to -I ) may also preferably exhibit increased yields via a substitution of that N-terminal sequence to Asp-Val-Val-Arg.
In achieving the inhibition of serine protease activity, the invention provides protease inhibitors that can ameliorate one or more of the undesirable clinical manifestations associated with enhanced serine protease activity, for example, by reducing pulmonary damage or blood loss during CPB.
The present invention relates to protease inhibitors comprising the following sequences: X'-Val-Cys-Ser-Glu-Gln-Ala-Glu-Xz-Gly-X3-Cys-Arg-Aia-X°-XS-3C6-X~-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Xa-Tyr-Gly-Gly-Cys-X9-X~°-X~ ~-X~~-Asn-Asn-Phe-Asp-Thr-GIu-Glu-X~3-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile, wherein X~ is selected from Glu-Va1-Val-Arg-Glu-, Asp-Val-Val-Arg-GIu-, Asp, and Glu; XZ is selected from Thr, Val, Ile and Ser; X' is selected from Pro and Ala; X'~ is selected from Arg, Ala, Leu, Gly, and Met; Xs is selected from Ile, His, Leu, Lys, Ala, and Phe; X6 is selected from Ser, IIe, Pro, Phe, Tyr, Tip, Asn, Leu, His, Lys, and Glu; X' is selected from Arg, His, and Ala; X$ is selected from Phe, Val, Leu, and Gly; X9 is selected from Gly, Ala, Lys, Pro, Arg, Leu, Met, and Tyr; X'° is selected from Ala, Arg, and Gly; Xl1 is selected from Lys, Ala, and Asn; X~z is selected from Ser, Ala, and Arg, X13 is selected from His, Gln, Ala, and Asp.
A further aspect of the present invention provides protease inhibitors wherein X' is Asp-Val-Val-Arg-Glu-, XZ is Thr, Val, or Ser, X3 is Pro, X° is Ala or Met, Xs is Ile, X6 is Ser or Tyr, X7 is His, X$ is Phe, X9 is Gly, X'° is Gly, X~~ is Asn, and X~~ is Arg.
Another aspect of the present invention provides protease inhibitors wherein Xt is Asp-Val-Val-Arg-Glu-, Xi is Pro, X'' is Ala, Xs is Ile, X6 is Phe, X7 is Arg, X$
is Phe, Xs is Giy, X~° is Gly, X~i is Asn, and X1Z is Arg. Yet another aspect of the present invention provides protease inhibitors wherein XZ is Thr or Val. Another aspect of the present invention provides protease inhibitors wherein Xi is Thr. A further aspect of the present invention provides protease inhibitors wherein XZ is Val. Another aspect of this invention provides protease inhibitors wherein X2 is Thr or Val, and X°
is Ala. A further aspect of the present invention provides protease inhibitors wherein XZ is Thr or Val, and X4 is Met. Yet another aspect of the present invention provides protease inhibitors wherein XZ is Thr, J~C4 is Ala, ?~ is Tyr, and X~3 is His. A further aspect of the present

6 invention provides protease inhibitors wherein Xz is Thr, X'' is Ala, X6 is Tyr, and X13 is Gln. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X' is Ala, X6 is Tyr, and X'3 is Ala. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X° is Ala, X~ is Tvr, and X13 is Asp.
Another aspect of the present invention provides protease inhibitors wherein Xi is Thr, X4 is Met, 3C6 is Ser, and X13 is selected from His, Ala, or Gln. Another aspect of the present invention provides protease inhibitors wherein XZ is Val, X; is Ala, X6 is Tyr, and X13 is selected from His, Ala, or Gln. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X4 is Ala, X6 is Tyr, and X'3 is selectal from His, Ala, or Gln. Another aspect of the present invention provides protease inhibitors wherein X'3 is selected from His or Ala. Another aspect of the present invention provides protease inhibitors wherein X'3 is selected from His or Ala. Anoiher aspect of the present invention provides protease inhibitors wherein X13 is His. A further aspect of the present invention provides protease inhibitors wherein X13 is Ala.
A further aspect of the present invention provides an isolated DNA molecule comprising a DNA sequence encoding a protease inhibitor of the invention.
Another aspect of the present invention provides an isolated DNA molecule operably linked to a regulatory sequence that controls expression of the coding sequence of the protease inhibitor in a host cell. Another aspect of the present invention provides an isolated DNA molecule operably linked to a regulatory sequence that controls expression of the coding sequence of the protease inhibitor in a host cell fiuther comprising a DNA
sequence encoding a secretory signal peptide. That secretory signal peptide may preferably comprise the signal sequence of yeast x-mating factor. Another aspect of the present invention provides a host cell transformed with a DNA molecule.
Another aspect of the present invention provides a host cell transformed with any of the DNA
molecules defined above. Such a host cell may preferably comprise E. coli or a yeast cell. When said host cell is a yeast cell, the yeast cell may preferably be Saccharonry~aes cerevisiae. When said host cell is a yeast cell, the yeast cell may preferably be Piehia pastoris.
Another aspect of this invention provides a method for producing a protease inhibitor, comprising the steps of culturing a host cell as defined above and isolating and purifying said protease inhibitor.

7 A further aspect of this invention provides a pharma;,eutical composition, comprising a protease inhibitor together with a pharmaceutically acceptable sterile vehicle.
An additional aspect of the present invention provides a method of treatment of a clinical condition associated with increased activity of one or more serine proteases, comprising administering to a patient suffering from said clinical condition an effective amount of a pharmaceutical composition comprising a protease inhibitor of the present invention together with a pharmaceutically acceptable sterile vehicle. That method of treatment may preferably be used to treat the clinical condition of blood loss during surgery.
Yet another aspect of the present invention provides a method for inhibiting the activity of serine proteases of interest in a mammal comprising administering a therapeutically effective dose of a pharmaceutical composition comprising a protease inhibitor of the present invention together with a pharmaceutically acceptable sterile vehicle, wherein said serine proteases are selected from the group consisting of kallikrein; chymotrypsins A and B; trypsin; elastase; subtilisin; coagulants aad procoagulants, particularly those in active form, including coagulation factors such as factors VIIa, iXa, Xa, XIa, and XIIa; plasmin; thrombin; proteinase-3;
enterokinase;
acrosin; cathepsin; urokinase; and tissue piasminogen activator.
Another aspect of the present invention provides protease inhibitors comprising the sequence: X'-Val-Cys-Ser-Glu-Gln-Ala-Glu-Xz-Gly-Pm-Cys-Arg-Ala-Ala-Ile-Tyr-His-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-Gfy-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-X;-Cys-Met-Ala-Val-Cys-Gly-Set Ales-Ile, wherein X~ is selected from Glu-Val-Val-Arg-Glu-, Asp-Val-Val-Arg-Glu-, Asp, or Glu; XZ is selected from Thr and Val; X3 is selected from His, Gln, Ala, or Asp.
A further aspect of the present invention relates to protease inhibitors wherein X' is Glu-Val-Val-Arg-Glu. Yet another aspect of the present invention provides for protease inhibitors wherein XZ is Thr. An additional aspect of the present invention provides protease inhibitors wherein Xi is Val. Yet another aspect of the present invention provides protease inhibitors wherein X3 is His. Another aspect of the present invention provides protease inhibitors wherein X3 is Gln. Another aspect of the present invention provides protease inhibitors wherein X' is AIa. Another aspect of the present invention provides protease inhibitors wherein X3 is Asp. Another aspect of the present invention provides protease inhibitors wherein X~ is Asp-Val-Val-Arg-Glu.
Another

8 aspect of the present invention provides proteaseXz is Another inhibitors wherein Thr.

aspect of the present invention provides proteaseX~ is Another inhibitors wherein Val.

aspect of the present invention provides proteaseX3 is Another inhibitors wherein His.

aspect of the present invention provides proteaseX3 is Another inhibitors wherein Gln.

aspect of the present invention provides X3 is Another protease inhibitors wherein Ala.

aspect of the present invention provides proteaseX3 is Another inhibitors wherein Asp.

aspect of the present invention provides protease A
inhibitors wherein X~ is Glu. further aspect of the present invention provides proteaseXz is Another inhibitors wherein Thr.

aspect of the present invention provides proteaseXz is Another inhibitors wherein Val.

aspect of the present invention provides X3 is Another protease inhibitors wherein His.

aspect of the present invention provides protease inhibitors wherein X3 is Gln.
Another aspect of the present invention providesX3 protease inhibitors wherein is Ala. Another aspect of the present invention providesX3 protease inhibitors wherein is Asp. Another aspect of the present invention providesXt protease inhibitors wherein is Another aspect of the present invention providesX=
Asp. protease inhibitors wherein is Thr. Another aspect of the present invention providesXi protease i_~hibitors wherein is Val. Another aspect of the present invention providesXl protease inhibitors wherein is His. Another aspect of the present invention providesX3 protease inhibitors wherein is Gln. Another aspect of the present invention providesX3 protease inhibitors wherein is Another aspect of the present invention providesX3 Ala. protease inhibitors wherein is Asp.
Another aspect of the present invention provides protease inhibitors wherein Xt is Glu-Val-Val-Arg-Glu-, Xi is Thr, Val, or Ser, X3 is Pro, X~ is Ala or Met, XS is Ile, X6 is Ser or Tyr, X' is His, X$ is Phe, X9 is Gly, Xi° is Gly, Xt ~ is Asn, and Xt= is Arg.
Another aspect of the present invention provides protease inhibitors wherein Xi is Thr or Val. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr. Another aspect of the present invention provides protease inhibitors wherein X= is Val. Another aspect of the present invention provides protease inhibitors wherein X2 is Thr or Vat, and X° is Ala Another aspect of the present invention provides protease inhibitors wherein XZ is Thr or Val, and X° is Met. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X4 is AIa, X6 is Tyr, and Xt3 is His. Another aspect of the present invention provides protease inhibitors wherein X= is Thr, X° is Ala, X6 is Tyr, and X~; is Gln. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X; is Ala, X6 is Tyr, and Xt3 is Ala.

9 Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X4 is Ala, 3.'° is Tyr, and X~~ is Asp. Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X~ is Met, ?C6 is Ser, and X~3 is selected from His, Ales, or Gln. Another aspect of the present invention provides a protease inhibitors wherein Xz is teal, X4 is Ala, X6 is Tyr, and X'3 is selected from His, Ales, or Gln.
Another aspect of the present invention provides protease inhibitors wherein XZ is Thr, X° is Ales, X6 is Tyr, and X~3 is selected from His, Ala, or Gln.
Another aspect of the present invention provides protease inhibitors wherein X'3 is selected from His or Ales.
Another aspect of the present invention provides protease inhibitors wherein X'3 is selected fi-om His or Ala. Another aspect of the present invention provides protease inhibitors wherein X~3 is His. Another aspect of the present invention provides protease inhibitors wherein X'3 is Ala.
Another aspect of the present invention provides an isolated DNA molecule comprising a DNA sequence encoding a protease inhibitor. Another aspect of the present invention provides an isolated DNA molecule operably linked to a regulatory sequence that controls expression of the coding sequence in a host cell.
Another aspect of the present invention provides an isolated DNA molecule further comprising a DNA
sequence encoding a secretory signal peptide. Another aspect of the present invention provides an isolated DNA molecule wherein said secretory signal peptide comprises the signat sequence of yeast x-mating factor. Another aspect of the present invention provides a host cell transformed with any of the DNA molecules defined above.
Such a host cell may preferably comprise E. coli or a yeast cell. When said host cell is a yeast cell, the yeast cell may preferably be Saccharomyces cereviriae. When said host cell is a yeast cell, the yeast cell may preferably be Pichia pastoris.
Another aspect of the present invention provides for a method for producing a protease inhibitor, comprising the steps of culturing a host cell as defined above and isolating and purifying said protease inhibitor.
A further aspect of this invention provides a pharmaceutical composition, comprising a protease inhibitor together with a pharmaceutically acceptable sterile vehicle.
An additional aspect of the present invention provides a method of treatment of a clinical condition associated with increased activity of one or more serine proteases, comprising administering to a patient suffering from said clinical condition an effective amount of a pharmaceutical composition comprising a protease inhibitor of the present invention together with a pharmaceutically acceptable sterile vehicle. That method of treatment may preferably be used to treat the clinical condition of blood loss during surgery.
S Another aspect of the present invention provides a method for inhibiting the activity of serine proteases of interest in a mammal comprising administering a therapeutically effective dose of a pharmaceutical composition comprising a protease inhibitor of the present invention together with a pharmaceutically acceptable sterile vehicle, wherein said seiine proteases are selected from the group consisting of ka11i1Qein; chymotrypsins A and B; trypsin; elastase; subtilisin; coagulants and procoaguiants, particularly those in active form, including coagulation factors such as factors VBa, IXa, Xa, XIa, and XIIa; plasmin; thrombin; prnteinase-3;
enterokinase;
acrosin; cathepsin; urokinase; and tissue plasminogen activator.
Yet another aspect of the present invention provides a method for increasing the.
expression levels of recombinant protease inhibitors comprising the step of culturing a host cell transformed with an isolated DNA molecule comprising a DNA sequence encoding a protease inhibitor. Such a host cell is E. coli or a yeast cell.
When such a host cell is a yeast cell, the yeast cell may preferably be Saccharomyces cerevisiae.
When such a host cell is a yeast ccll, the yeast cell may preferably be Pichia pastoris.
Another aspect of the present invention provides a method for increasing the yield of recombinant protease inhibitors comprising the step of culturing a host cell transformed with an isolated DNA molecule comprising a DNA sequence encoding a protease inhibitor according to claim 1, wherein X~ is Asp-Val-Val-Arg-Glu-, and isolating and purifying said protease inhibitor. When said host cell is a yeast cell, the yeast cell may preferably be Saccharomyces cerevisiae. When said host cell is a yeast cell, the yeast cull may preferably be Pichia pastoris.
Another aspect of the present invention provides protease inhibitors comprising the sequence: X'-Val-Cys-Ser-Glu-Gln-Ala-Glu-Xi-Gly-Pro-Cys-Arg-Ala-X3-DaX'~-Xs-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phc-Tyr-Gly-Gly-Cys-Gly-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-7C6-Cys-Met-Ala-Val-Cys-Gly Ser-Ala-Ile, wherein: X~ is selected from Glu-Val-Val-Arg-Glu-, Asp-Val-Val-Arg-Glu-, Asp, or Glu; X~ is selected from Thr or Val; X3 is selected from Arg and Met;
X4 is selected from Ser and Tyr; Xs is selected from Arg, His, or Ala; and X6 is selected from His, Gln, Ala or Asp.

A further aspect of the present invention provides protease inhibitors comprising the sequence: X~-Val-Cys-Ser-Glu-Gln-Ala-Glu-Thr-Gly-Pro-Cys-Arg-Ala-Leu-Phe-Lys-Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Giy-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-Leu-Gly-Asp-Arg-Asn-Asn-Phe-Asp-Thr-Glu-GIu-XZ-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile, wherein: X' is selected from Glu-Val-Val-Arg-Glu-, Asp-Val-Val-Arg-Glu-, Asp, and Glu; XZ is selected from His, Gln, Ala, and Asp.
Yet a further aspect of the present invention provides protease inhibitors wherein X~ is Asp-Val-Val-Arg-Glu. Another aspect of the present invention provides protease inhibitors wherein Xi is His. Another aspect of the present invention provides protease inhibitors wherein XZ is Gln. Another aspect of the present invention provides protease inhibitors wherein XZ is Ala. Another aspect of the prrsent invention provides protease inhibitors wherein Xi is Asp. Another aspect of the present invention provides protease inhibitors wherein X~ is Glu-Val-Val-Arg-Glu. Yet another aspect of the present invention provides protease inhibitors wherein X2 is His. A further aspect of the present invention provides protease inhibitors wherein XZ
is Gln. Another aspect of the present invention provides protease inhibitors wherein X~
is Ala. Another aspect of the present invention provides protease inhibitors wherein X~
is Asp.
Yet another aspect of the present invention provides protease inhibitors wherein X' is Asp. A further aspect of the present invention provides protease inhibitors wherein Xz is His. Another aspect of the present invention provides protease inhibitors whettin XZ is Gln. Another aspect of the present invention provides protease inhibitors wherein Xz is Ala. t~nother aspect of the present invention provides protease inhibitors wherein X= is Asp. Yet another aspect of the present invention provides protease inhibitors wherein X' is Glu. Another aspect of the present invention provides protease inhibitors wherein XZ is His. A further aspect of the present invention provides protease inhibitors wherein X~ is Gln. Another aspect of the present invention provides protease inhibitors wherein X~ is Ala. Another aspect of the present invention provides protease inhibitors wherein XZ is Asp. Another aspect of the present invention provides protease inhibitors comprising the sequence: Asp-Val-Val-Arg-Glu-Val-Cys-Ser-Glu-Gln-Ala-Glu-Thr-Gly-Pro-Cys-Arg-Ala-Leu-Phe-Lys-Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-G~s-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-Leu-Gly-Asp-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-Tyr-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent io those skilled in the art from this detailed description.
Brief Description of the Drawing Figure 1 shows the strategy for the construction of plasmid pTW lO:KPI.
Figure 2 shows the sequence of the synthetic gene for KPI (1-r57) fused to the bacterial phoA secretory signal sequence.
Figure 3 shows the strategy for construction of plasmid pKPI-61.
Figure 4 shows the 192 by XbaI-Hindl>I synthetic gene fragment encoding KPI
(1-X57) and four amino acids from yeast «-mating factor.
Figure 5 shows the synthetic 201 by XbaI-HindIII fragment encoding KPI
(-4-i57) in PKPI-61.
Figure 6 shows the strategy for the construction ofplasnud pTW113.
Figure 7 shows plasmid pTW113, encoding the 445 by synthetic gene for yeast «-factor-KPI(-4-~57) fusion.
Figure 8 shows the amino acid sequence for KPI (-4-~57).
Figure 9 shows the strategy for constructing plasmid pTW6165.
Figure 10 shows piasmid, pTW6165, encoding the 445 by synthetic gene for yeast «-factor-KPI(-4-~57; M15A, S17V~ fusion.
Figure I 1 shows the sequences of the annealed oligonucleotide pairs used to construct plasmids pTW6165, pTW6166, pTW6175, pBG028, pTW6183, pTW6184, pTW6185, pTW6I73, and pTW6174.
Figure 12 shows the sequence of plasmid pTW6166 encoding the fusion of yeast «-factor and KPI(-4-~57; M15A, S171~.
Figure 13 shows~the sequence of plasmid pTW6175 encoding the fusion of yeast «-factor and KPI(-4-~57; MISL, S17F).
Figure 14 shows the sequence of plasmid pBG028 encoding the fusion of yeast «-factor and KPI(-4157; M15L, S17I~.

WO 99/63090 PCT/CfS99/12276 Figure 15 shows the sequence of plasmid pTW6183 encoding the fusion of yeast x-factor and IkPI(-4-~ 57; I 16H, S 17F).
Figure 16 shows the sequence of plasmid pTW6184 encoding the fusion of yeast x-factor and KPI(-4-~57; I1GH, S17Y).
Figure 17 shows the sequence of plasmid pTW6185 encoding the fusion of yeast x-factor and KPI(-4-~57; I16H, S 17W).
Figure 18 shows the sequence of plasmid pTW6173 encoding the fusion of yeast x-factor and KPI(-4-~57; M15A, I16H).
Figure i 9 shows the sequence of plasmid pTW6174 encoding the fusion of yeast x-factor and KPI(-4-~57; M15L, I16H).
Figure 20 shows the sequence of plasmid pBG022 encoding the fusion of yeast x-factor and KPI (-4-~57; M15A, S17Y, R18H, Y48H).
Figure 21 shows the sequence of plasmid pBG033 encoding the fusion of yeast x-factor and KPI (-4-~57; T9V, hilSA, R18H, Y48H).
Figure 22 shows the sequence of plasmid pBG048 encoding the fusion of yeast x-factor and KPI {-4-~57; Y48H).
Figure 23 shows the sequence of plasmid pBG049 encoding the fusion of yeast x-factor and KPI (-4--~57; MISA, S17Y, R18H).
Figure 24 shows the sequence of piasmid pBG050 encoding the fusion of yeast x-factor and KPI (-4-~57; T9V, M15A, S17Y, R18H).
Figure 25 shows the sequence of the coding region for phoA signal: KPI-BG022: gIII protein contained within the phage display vector pDW 1-L6-16.
Figure 26 shows the sequence of the coding region for yeast x-factor and KPI-P48 library contained within the P4.8 library.
Figure 27 shows the amino acid sequence of KPI (-4--X57; M15A, S17W).
Figure 28 shows the amino acid sequence of KPI (-4-X57; M15A, S17Y).
Figure 29 shows the amino acid sequence of KPI (-4--X57; M15L, S17F).
Figure 30 shows the amino acid sequence of KPI (-4--~57; M15L, S17Y).
Figure 31 shows the amino acid sequence of KPI (-4--~57; I16H, S17F).
Figure 32 shows the amino acid sequence of KPI (-4-X57; I16H, S17Y).
Figure 33 shows the amino acid sequence ofKPI (-4-X57; I16H, S17W).
Figure 34 shows the amino acid sequence of KPI (-4-~57; M15A, SI7F).
Figure 35 shows the amino acid sequence of KPI (-4-a57; M15A, I16H).

Figure 36 shows the amino acid sequence ofICPI (-4--~57; MISL, I16H).
Figure 37 shows the amino acid sequence of ICPI (-4-i57; M15A, S17Y, RIBH, Y48H).
Figure 38 shows the amino acid sequence of ICPI (-4-X57; T9V, M15A, RIBH, Y48H).
Figure 39 shows the amino acid sequence of KPI (-4-X57; Y48H).
Figure 40 shows the amino acid sequence of KPI (-4--57; MISA, S17Y, R18H).
Figure 41 shows the amino acid sequence ofICPI (-4-X57; T9V, M15A, S17Y, R18H).
Figure 42 shows the amino acid sequence of KPI-P48 library (-4-X57; M15A, S 17Y, Rt 8H, Y28X) encoded by the P48 library.
Figure 43 shows the construction of plasmid pSP26:Amp:Fl.
Figure 44 shows the construction of plasmid pgllI.
Figure 45 shows the construction of plasmid pPhoA:KPI:gIlI.
Figure 46 shows the construction of plasmid pLGI.
Figure 47 shows the construction of plasmid pAL51.
Figure 48 shows the construction of plasmid pAL53.
Figure 49 shows the construction of plasmid pSP26:Amp:FI :PhoA:KPI:gIII.
Figure 50 shows the construction ofplasmid pDWI #14.
Figure 51 shows the construction of plasmid pBG022.
Figure 52 shows the construction of plasmid pBG048.
Figure 53 shows the construction of plasmid pBG049.
Figure 54 shows the construction of plasmid pBG050.
Figure 55 shows the construction of the P48 library.
Figure 56 shows the coding region for the fusion ofphoA-KPI (155)-geneIll.
Figure 57 shows the construction of plasmid pDW 1 14-2.
Figure 58 shows the construction of KPI Library ICrl9.
Figure 59 shows the expression unit encoded by the members of KPI Library 16-19.
Figure 60 shows the phoA-ICPI(155)-geneItl region encoded by the most frequently occurring randomized KPI region.
Figure 61 shows the construction ofpDD185 KPI (-4-X57; M15A, S17F).

Figure 62 shows the sequence of yeast x-factor fused to ICPI (-4-~57; M15A, S 17F).
Figure 63 shows the inhibition constants (K;s) determined for purified KPI
variants against the selected serine proteases kallikrein, factor Xa, and factor XIIa.
S Figure 64 shows the inhibition constants (K;s) determined for KPI variants against kallikrein, plasmin, and factors Xa, XIa, and XIIa.
Figure 65 shows the post-surgical blood loss in pigs in the presence (KPI) and absence (NS) of KPI 185-1 (M15A, S17F).
Figure 66 shows the post-surgical hemoglobin loss in pigs in the presence (KPI) and absence (NS) of KPI 185-1 (M15A, S17F).
Figure 67 shows the oxygen tension in the presence and absence of KPI, before CPB, immediately after CPB, and at 60 and 180 minutes after the end of CPB.
Figure 68 summarizes the results shown in Figures 65-67.
Figure 69 shows the inhibitor constants (Kis) determined for KPI variants against kallikrein in nM and expression levels (mg/ml) of those variants.
Figure 70 shows a comparison of the survival time of rat xenografts in the presence and absence of KPI-BG022.
Figure 71 shows a comparison of damage in a rat model of TNBS
(trinitrobenzene sulfonic acid) induced colitis in the presence and absence of KPI-BG022.
Figure 72 shows a comparison of the HPLC traces, after lyophiiization, of KPI
having the N-tem~inus sequence Glu-Val-Val-Arg (E-KPI) and KPI having the N-tecminus sequence Asp-Val-Val-Arg (D-KPI).
Detailed Description The present invention provides peptides that can bind to and preferably inhibit the activity of serine proteases. These inhibitory peptides can also provide a means of ameliorating, treating or preventing clinical conditions associated with increased activity of serine proteases. The novel peptides of the present invention preferably exhibit a more potent and specific (i.e., greater) inhibitory effect toward serine proteases of interest than known serine protease inhibitors. Examples of such proteases include:
kallikrein; chymotrypsins A and B; trypsin; elastase; subtilisin; coagulants and procoagulants, particularly those in active form, including coagulation factors such as factors VIIa, IXa, Xa, XIa, and. XIIa; plasmin; thrombin; proteinase-3;
enterokinase;
acrosin; cathepsin; urokinase; and tissue plasminogen acrivator. In particular, the peptides of the present invention preferably exhibit a greater potency and specificity for inhibiting one or more serine proteases of interest (e.g., ka11i1Qein, plasmin and factors VIIa, IXa, Xa, XIa, and XIIa) than the potency and specificity exhibited by native KPI or other known serine protease inhibitors. In addition, the peptides of the present invention preferably comprise a substitution at position 48. Such position 48 substituted peptides may exhibit an increased level of expression in comparison to the expression levels of serine proteases that do not have that substitution. The effect of this substitution may be manifested not only on the substituted KPI peptides of the present invention, but on wild-type KPI as well. Also, the peptides of the present invention that comprise the N-temunal sequence Glu-Val-Val-Arg (residues -4 to -1 ) may also preferably exhibit increased yields via a substitution to Asp-Val-Val-Arg.
Peptides of the present invention may be used to reduce the tissue damage caused by activation of the proteases of the contact pathway of the blood doting surgical procedures such as cardiopulmonary bypass (CPB). Inhibition of contact pathway proteases reduces the 'whole body inflammatory response" that can accompany contact pathway activation, and that can lead to tissue damage, and possibly death.
The peptides of the present invention may also be used in conjunction with surgical procedures to ZO roduce activated seiine protease-associated perioperative and postoperative blood loss.
For instance, perioperative blood loss of this type may be particularly severe during CPB
surgery. Pharmaceutical compositions comprising the peptides of the present invention may be used in conjunction with surgery such as CPB; administration of such compositions may occur preoperatively, perioperatively or postoperatively.
Examples of other clinical conditions associated with increased serine protease activity for which the peptides of the present invention may be used include: CPB-induced inflammatory response; post-CPB pulmonary injury; pancreatitis; allergy-induced protease release;
deep vein thrombosis; thrombocytoptnia; rheumatoid arthritis; adult respiratory distress syndrome; chronic inflammatory bowel disease; psoriasis; hyperfibrinolytic hemorrhage;
organ preservation; wound healing; and myocardial infarction. Other examples of preferable uses of the peptides of the present invention are described in U.S.
Patent No.
5,187,153.
The invention is based upon the novel substitution of amino acid residues in the peptide corresponding to the naturally occurring KPI protease inhibitor domain of human arnyloid p-amyloid precursor protein (APPn. These substitutions produce peptides that can bind to serine proteases and preferably exhibit an inhibition of the activity of serine proteases. The peptides also preferably exhibit a more potent aad specific serine protease inhibition than known serine protease inhibitors. In accordance with the invention, peptides are provided that may exhibit a more potent and specific inhibition of one or more serine proteases of interest, e.g., kallila~ein, plasmin and factors Xa, XIa, XIIa, and XIIa.
The present invention also includes pharmaceutical compositions comprising an effective amount of at least one of the peptides of the invention, in combination with a pharmaceutically acceptable sterile vehicle, as described in REMINGTONS
PHARMACEUTICAL SCIENCES: DRUG RECEPTORS AND RECEPTOR
THEORY, (18th ed.), Mack Publishing Co., Easton, PA (1990).
A. Selection of sequences of KPI variants The sequence of KPI is shown in Table 1. Table 2 shows a comparison of this sequence with that of aprotinin, with which it shares about 45% sequence identity. The numbering convention for KPI shown in Table 1 and used hereinafter designates the first glutamic acid residue of KPI as residue 1. This corresponds to residue number 3 using the standard numbering convention for aprotinin.
The crystal structure for KPI complexed with trypsin has been determined. See Perona et al., J. Mol. Biol. 230:919 (1993). The three-dimensional structure reveals two binding loops within KPI that contact the protease. The first loop extends from residue Thr9 to Ile~6, and the second loop extends from residue Phe32 to Gly3~. The two protease binding loops are joined through the disulfide bridge extending from Cys~Z t0 Cys36.
KPI contains two other disulfide bridges, between Cys3 and Cys33, and between Cys~ to Cys°9. ' This structure was used as a guide to inform our strategy for making the amino acid residue substitutions that will be most likely to affect the protease inhibitory properties of KPI. Our examination of the structure indicated that certain amino acid residues, including residues 9, 11, 13-18, 32, and 37-40 appear to be of particular significance in determining the protease binding properties of the KPI
peptides of the present invention. It was also found that certain position 48 substitutions positively affected the expression levels of the peptide by the transformed host. In a preferred embodiment of the invention one or more of those KPI peptide residues are substituted, such substitutions preferably occurring among residues 9, 11, I3-18, 32, 37-40, and 48.
In particular, those substituted peptides, including peptides comprising substitutions at position 9, substitutions of at least two of the four residues at positions IS-18 and substitutions at position 48 may exhibit more potent and specific serine protease inhibition toward selected serine proteases of interest than exhibited by the natural KPI
peptide domain.
Specifically, replacement of arginine at position 18 of the native KPI peptide with histidine (R18H) in combination with one or more additional substitutions at residues 9, 15 and 17 were found to exhibit more potent and specific setine protease inhibition toward selected serine proteases of interest than the native KPI
peptide. In particular, the specific substitutions T9V, M15A, S17Y and M15A ,S17Y in the context of the R18H substitution exhibited such potent serine protease inhibition. See Figures 63, 64 and 69D.
In addition, the peptides of the present invention preferably comprise a substitution at position 48. Such position 48 substituted peptides may exhibit an increased level of expression in comparison to the expression levels of serine proteases that do not have that substitution. The effect of this substitution may be manifested not only on the substituted KPI peptides of the present invention, but on wild-type KPI as well. Also, the peptides of the present invention that comprise the N-terminal soquenee Glu-Val-Val-Arg (residues -4 to -1) may also preferably exhibit increased yields via a substitution to Asp-Val-Val-Arg.
Specifically, substitutions at position 48 may exhibit an increased level of expression of KPI peptides in comparison to the expression levels of such peptides not having such a substitution. These substituted peptides exhibiting an increased level of expression also may preferably comprise one or more additional substitutions at residues 9, 11, 13-18, 32 and 37-40; in particular, such peptides may preferably comprise a substitution at positions 9 or 37 and/or substitution of at least two of the four residua at positions 15-18. Those additionally substituted peptides may exhibit more potent and specific setine protease inhibition toward selected serine proteases of interest than exhibited by the natural KPI peptide domain as well as increased expression levels.
One specific embodiment of the invention is based upon a finding that as expression vector prepared to express the KPI variant M15A, S17Y, R18H
underwent a spontaneous mutation at position 48 which changed the native tyrosine to histidine (Y48H) and that this mutation conferred beneficial properties. To assess the effect of this mutation, the KPI variant M1 SA, S 17Y, Rl 8H (pBG049) was constructed using methods known to those skilled in the art and its expression levels compared with the KPI variant M15A, S17Y, R18H, Y48H (pBG022). As detailed infra, the expression level of KPI variant M15A, S17Y, R18H was increased over five-fold by replacing the native tyrosine at position 48 with histidine. See Figures 69A aad B.
Moreover, it has been determined that this Y48H substitution confers improvements in expression levels upon KPI variants as well as upon native sequence KPI.
As an additional example of the position 48 substitution effect on expression of the recombinant peptides of the present invention, and as delineated in detail infra, the expression level of wild-type KPI (pTW 113) was increased on the average approximately five to six-fold by replacing the native tyrosine at position 48 with histidine (pBG048;Y48H), glutamine (pBG072; Y48Q) or alanine (pBG073; Y48A).
See Figures 69B and F.
In an additional preferred embodiment of the invention, it was found that replacement of arginine at position 18 of the native KPI peptide with histidine (R18H), in combination with one or more additional substitutions at residues 9, 15 aad 17, exhibited more potent and specific serine protease inhibition toward selected serine proteases of interest than the native KPI peptide. In particular, the specific substitutions T9V, M15A, S17Y and M15A; S17Y exhibited particularly potent serine protease inhibition in the context of the R18H substitution. See Figures 63 and 64.
Additionally, the R18H substitution conferred an increased level of expression in comparison to tire expression levels of the corresponding peptides lacking the position 48 substitution. To assess the effect of the position 48 substitution on these R18H substituted peptides, Library P48 was constructed for expression of KPI (M15A, S17Y, R18H) in which the amino acids exhibiting at position 48 are randomized. See Figure 55. The amino acid sequences of the KPI-P48 Library contained within the P48 Library are shown in Figure 26. Those substituted peptides included substituting the native tyrosine at position 48 with histidine (pBG022; SOD4, SOB6,Y48H), glutamine (SOB6, SOL1, SOM1; Y48Q), alanine (SOPS, SOC4; Y48A) and aspartic acid (SON1; Y48D). See Figures 69B, E
and F.
In yet another preferred embodiment, the KPI peptides of the present invention may also comprise a substitution at its N-terminus. Specifically, such a substitution was found to alleviate the problems associated with the purification and subsequent isolation of the expressed peptides of the present invention having a giutamic acid residue at its N-terminus. This specific substitution changes the additional N-terminal amino acids from the ICPI protein sequence (Glu-Val-Val-Arg, designated residues -4 to -1) immediately proceeding the KPI domain in APPI to Asp-Val-Val-Atg. Specifically, this substitution is thought to prevent cyclization of the N-terminus glutamic acid during purification of . the expressed peptides of the present invention. In a preferred embodiment of the invention, and as described supra, one or more additional KPI peptide residues are substituted, such substitutions preferably occurring among residues 9, 11, 13-18, 32, 37 40, and 48. in particular, those substituted peptides, including peptides comprising substitutions at position 9, substitutions of at least two of the four residues at positions 15-18 and substitutions at position 48 preferably exhibit the desired potency and specificity as well as an increased level of expression in comparison to the expression levels of other serine pretenses W thout those specific substitutions.
In particular, the peptides of the present invention preferably exhibit a greater potency and specificity for inhibiting one or more serine pretenses of interest (eg., kallila~ein, plasmin and factors VIIa, IXa, Xa, XIa, and XIIa) than the potency and specificity exhibited by native ICPI or other known serine protease inhibitors as well as an increased level of expression in comparison to the~expression levels of other setine pmteases without those specific substitutions. That greater potency and specificity may be manifested by the peptides of the present invention by exhibiting binding constants for serine pretenses of interest that are less than the binding constants exhibited by native KPI, or other known serine protease inhibitors, for such proteases.
By way of example, and as set forth in greater detail below, the serine protease inhibitory properties of peptides of the present invention were measured for the serine proteases of interest kallikrein, piasmin and factors Xa, XIa, and XIIa.
Methodologies for measuring the inhibitory properties of the KPI variants of the present invention are known to those skilled in the art, e.g., by determining the inhibition constants of the variants toward serine pretenses of interest, as described in Example 4, infra. Such studies measure the ability of the novel peptides of the present invention to bind to one or more serine pretenses of interest and to preferably exhibit a greater potency and specificity for inhibiting one or more serirte protease of interest than known setine protease inhibitors such as native KPI.
The ability of the peptides of the present invention to bind one or more serine pretenses of interest, particularly the ability of the peptides to exhibit such greater potency and specificity toward serine proteases of interest, manifest the clinical and therapeutic applications of such peptides. The clinical and therapeutic efficacy of the peptides of the present invention can be assayed by in vitro and in vivo methodologies lrnown to those skilled in the art, e.g., as described in Examples 5-8, infra.
Tabte 1: SEQUENCE OF KPI:

VREVCSEQAETGPCRAMISRWYFDVTEGKCAP

FFYGGCGGNRNNFDTEEYCMAVCGSAI
Table 2: COMPARISON OF KPI AND APROTININ SEQUENCES:

KPI: VREVCSEQAETGPCRAMISRWYFDVTEGKCAPF~,NRNNFDTEEYCMAVCGSAI
BP'TI: RPDFCLEPPYTGPCKARIIRYFYNAKA GL CQTFV~'Q~CRNNFfCSAEDCI~TCGGA

B. Methods of producing KPI variants The peptides of the present invention can be created by synthetic techniques or recombinant techniques which employ genomic or cDNA cloning methods.
1. Production by chemical synthesis Peptides of the present invention can be routinely synthesized using solid phase or solution phase peptide synthesis. Methods of preparing relatively short peptides such as KPI by chemical synthesis are well known in the art. ICPI variants could, for example be produced by solid-phase peptide synthesis techniques using commercially available equipment and reagents such as those available from Milligen (Bedford, MA) or Applied Biosystems-Perkin Elmer (Foster City, CA). Alternatively, segments of KPI
variants could be prepared by solid-phase synthesis and linked together using segment condensation methods such as those described by Dawson et al., Science 266:776 (1994). During chemical synthesis of the KPI variants, substitution of any amino acid can be achieved simply by replacement of the residue that is to be substituted with a different amino acid monomer.
2. Production by recombinant DNA technology (a) Preparation ojgenes encoding KPI variants In a preferred embodiment of the invention, KPI variants are produced by recombinant DNA technology. See PCT application WO 96/35788, hereby incorporated in its entirety. This requires the preparation of genes encoding each ICPI
variant that is to be made. Suitable genes can be constructed by oligonucleotide synthesis using commercially available equipment, such as that provided by Milligen and Applied Biosystems, supra. The genes can be prepared by synthesizing the entire coding and non-coding strands, followed by annealing the two strands. Alternatively, the genes can be prepared by ligation of smaller synthetic oligonucleotides by methods well known in the art. Genes encoding ICPI variants are produced by varying the nucleotides introduced at any step of the synthesis to change the amino acid sequence encoded by the gene.
Preferably, however, KPI variants are made by site-directed mutagenesis of a gene encoding KPI. Methods of site-directed mutagenesis are well known in the art.
See, for example, Ausubel et al., (eds.) CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (Wiley Interscience, 1987); PROTEIN ENGINEERING (Oxender & Fox eds., A. Liss, Inc. 1987). These methods require the availability of a gene encoding KPI
or a variant thereof, which can then be mutagenized by known methods to produce the desired ICPI vaaants. In addition, linker-scanning and polymerase chain reaction ("PCR'~ mediated techniques can be used for purposes of mutagenesis. See PCR
TECHNOLOGY (Erlich cd., Stockton Press 1989); CURRENT PROTOCOLS IN
MOLECLTL,AR BIOLOGY, vols. 1 & 2, loc. cit.
A gene encoding KPI can be obtained by cloning the naturally occurring gene, as described for example in U.S. Patents Nos. 5,223,482 and 5,187,153, which are hereby incorporated by reference in their entireties. In particular, see columns 6-9 of U.S.
Patent No. 5,187,153. See aLro PCT Application No. 93/09233. In a nreferned embodiment of the invention a synthetic gene encoding KPI is produced by chemical synthesis, as described above. The gene may encode the 57-amino acid KPI
domain shown in Table 1, or it may also encode additional N-terminal amino acids from the APPI protein sequence, such as the four amino acid sequence (Glu-Val-Val-Arg or Asp-Val-Val-Arg, designated residues -4 to -I) immediately preceding the ICPI
domain in APPI.
Production of the gene by synthesis allows the codon usage of the KPI gene to be altered to introduce convenient restriction endonuciease recognition sites, without altering the sequence of the encoded peptide. In a preferred embodiment of the invention, the synthetic ICPI gene contains restriction endonuclease recognition sites that facilitate excision of DNA cassettes from the KPI gene. These cassettes can be replaced with small synthetic oligonucleotides encoding the desired changes in the KPI
peptide sequence. See Ausubel, supra.
This method also allows the production of genes encoding KPI as a fusion peptide with one or more additional peptide or protein sequences. The DNA
encoding these additional sequences is arranged in-frame with the sequence encoding KPI
such that, upon translation of the gene, a fusion protein of KPI and the additional peptide or protein sequence is produced. Methods of making such fusion proteins are well known in the art. Examples of additional peptide sequences that can be encoded in the genes are secretory signal peptide sequences, such as bacterial leader sequences, for example ompA and phoA, that direct sxretion of proteins to the bacterial periplasmic space. Ia a preferred embodiment of the invention, the additional peptide sequence is a yeast secretory signal sequence, such as a-mating factor, that directs secretion of the peptide when produced in yeast.
Additional genetic regulatory sequences can also be introduced into the synthetic gene that are operably linked to the coding soquence of the gene, thereby allowing synthesis of the protein encoded by the gene when the gene is introduced into a host cell.
Examples of regulatory genetic sequences that can be introduced are: promoter attd eahancer sequences and transcriptional and translational control sequences.
Other regulatory sequences are well known in the art. See Ausubel et al., supra, and Sambrook et al., supra.
Sequences encoding other fusion proteins and genetic elements are well known to those of skill in the art. In a preferred embodiment of the invention, the ICPI sequence is prepared by ligating together synthetic oligonucleotides to produce a gene encoding an in-frame fusion protein of yeast a-mating factor with either KPI (1-i57) or KPI
(-4-i ST).

The gene constructs prepared as described above are conveniently manipulated in host cells using methods of manipulating recombinant DNA techniques that are well known in the art. See, for example Sambrook et al., MOLECULAR CLONING: A
LABORATORY MANUAL, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989), and Ausubel, supra. In a preferred embodiment of the invention the host cell used for manipulating the KPI constructs is E. toll.
For example, the construct can be ligated into a cloning vector and propagated in E. toll by methods that are well known in the art. Suitable cloning vectors are described in Sambrook, supra, or are commercially available from suppliers such as Promega (Madison, WI), Stratagene (San Diego, CA) and Life Technologies (Gaithersburg, MD).
Once a gene construct encoding KPI has been obtained, genes encoding KPI
variants are obtained by manipulating the coding sequence of the construct by standard methods of site-directed mutagenesis, such as excision and replacement of small DNA
cassettes, as described supra. See Ausubel, supra, and Sinha et al., supra.
See also U.S.
I S Patent 5,373,090, which is herein incorporated by reference in its entirety. See particularly, columns 4-12 of U.S. Patent 5,272,090. These genes are then used to produce the KPI variant peptides as described below.
Alternatively, KPI variants can be produced using phage display methods. See, for example, Dennis et al., supra, which is hereby incorporated by reference in its entirety. See also U.S. Patent Nos. 5,223,409 and 5,403,484, which are hereby also incorporated by reference in their entireties. In these methods, libraries of genes encoding variants of KPI are fused in-frame to genes encoding surface proteins of Slamentous phage, and the resulting peptides are expressed (displayed) on the surface of the phage. The phage are then screened for the ability to bind, under appropriate conditions, to serine proteases of interest immobilized on a solid support.
Large libraries of phage can be used, allowing simultaneous screening of the binding properties of a large number of KPI variants. Phage that have desirable binding properties are isolated and the sequences of the genes encoding the corresponding KPI variants is determined.
These genes are then used to pmduce the KPI variant peptides as described below.
(b) Expression ofKPl variant peptides Once genes encoding KPI variants have bcen prepared, they are inserted into an expression vector and used to produce the recombinant peptide. Suitable expression vectors and corresponding methods of expressing recombinant proteins and peptides are well known in the art. Methods of expressing KPI peptides are described in U.S. Patent 5,187,153, columns 9-11, U.S. Patent 5,223,482, columns 9-11, PCT application 93/09233, pp. 49-67, and PCT application 96/35788, pp. 31-33. See also Ausubel et al., supra, and Sambrook et al., supra. The gene can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the KPI
variant, which can then be purified and tested for its ability to bind to and inhibit serine proteascs of interest. Within the context of the present invention, substitutions at position 48 may exhibit an increased level of expression of KPI peptides, both wild-type and substituted, in comparison to the expression levels of such peptides not having such a substitution. Such peptides having a substitution at position 48 also may preferably comprise one or more additional substitutions at residues 9, 11, 13-18, 32 and 37-40; in particular, such peptides may preferably comprise a substitution at positions 9. or 37 and/or substitution of at least two of the four residues at positions 15-18.
Those additionally substituted peptides may exhibit more potent and specific serine protease inhibition toward selected serine proteases of interest than exhibited by the natural KPI
peptide domain as well as increased expression levels.
In particular, replacement of arginine at position 18 of the native KPI
peptide with histidine (R18H) in combination with one or more additional substitutions at residues 9, 15 and 17 was found to exhibit more potent and specific serine protease inhibition toward selected serine proteases of interest than the native KPI
peptide. In particular, the specific substitutions T9V, M15A, S17Y and M15A, S17Y in the context of the R18H substitution exhibited such potent serine protease inhibition.
Placing this position 48 substitution in such substituted peptides resulted in increased expression levels of these peptides in comparison to the expression levels of the peptides without the position 48 substitution.
Examples of expression systems lrnown to the skilled practitioner in the art include bacteria such as E. colt, yeast such as Saccharamyces cerevisiae and Pichia pastoris, baculovirus, and mammalian expression systems such as in Cos or CHO
cells.
In a preferred embodiment, KPI variants are expressed in S cerevisiae. In another preferred embodiment the KPI variants are cloned into expression vectors to produce a chimeric gene encoding a fusion protein of the KPI variant with yeast a-mating factor.
The mating factor acts as a signal sequence to direct secretion of the fusion pmtein from the yeast cell, and is then cleaved from the fusion protein by a membrane-bound protease WO 99/63090 PIrT/US99/12276 during the secretion process. The expression vector is transformed into S.
cereviriae, the transformed yeast cells are cultured by standard methods, and the KPI variant is purified from the yeast growth medium.
Recombinant bacterial cells expressing the peptides of the present invention, for example, E. colt, are grown in any of a number of suitable media, for example LB, and the expression of the recombinant antigen induced by adding IPTG to the media or switching incubation to a higher temperature. After culturing the bacteria for a ftuther period of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media. The bacterial calls are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed. If the recombinant peptide is expressed in inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to assist in the removal of any contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g., SM) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as (i-mercaptoethanol or DTT (dithiothreitol).
At this stage it may be advantageous to incubate the peptides of the present invention for several hours under conditions suitable for the peptides to undergo a refolding process into a conformation which more closely resembles that of native KPI.
Such conditions generally include low protein concentrations less than 500 uglml, low levels of reducing agent, concentrations of urea less than 2M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulphide bonds within the protein molecule. The refolding process can be monitored, for example, by SDS-PAGE or with antibodies, which are specific for the native molecule (which can be obtained from animals vaccinated with the native molecule isolated from parasites). Following refolding, the peptide can then be purif ed ftuther and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.
Purification of KPI variants can be achieved by standard methods of protein purification, e.g., using various chromatographic methods including high performance liquid chromatography and adsorption chromatography. The purity and the quality of the peptides can be confirmed by ami.-~o acid analyses, molecular weight determination, sequence determination and mass spectrometry. See, for example, PROTEIN
PURIFICATION METHODS: A PRACTICAL APPROACH, Hams et a1, eds. (IRL
Press, Oxford, 1989). In a preferred embodiment, the yeast cells are removed from the growth medium by filtration or centrifugation, and the ICPI variant is purified by amity chromatography on a column of trypsin-agamse, followed by reversed-phase HPLC.
In yet another embodiment, the ICPI peptides of the present invention may also comprise a substitution at its N-terminus. Placing the amino acid sequence Asp-Val Val-Arg (designated residues -4 to -I ) immediately before the ICPI domain was found to alleviate the problems associated with the purification and subsequent isolation of the expressed peptides of the present invention having a glutamic acid residue at its N-terminus. In a preferred embodiment, this substitution changes the additional N-terminaI amino acids from the KPI protein sequence (Glu-Val-Val-Arg, designated residues -It to -I) immediately proceeding the ICPI domain to Asp-Val-Val-Arg.
Specifically, this substitution is thought to prevent cyclization of the N-terminus glutamic acid in the unsubstituted variant during purification of the expressed peptides of the present invention and is thought to be applicable to the substituted KPI
variants of the present invention, as well as wild-type KPI. By way of example, Figure 72 provides a comparison of the HPLC traces, aRer lyophilization, of KPI having the N-terminal sequence Glu-Val-Val-Arg (E-KPI) and ICPI having the N-terminus sequence Asp-Val-Val-Arg (D-ICPI). Those ICPI samples were injected onto a YMC-Phenyl HPLC column (Cat. No.:PH12S030504WTA, 4x50 mm, 3p particle size, 50 angstrom pore size). Mobile Phase A was 40 nM ammonium phosphate (pH 6.5),

10% acetonitrile, and 90% water. Mobile Phase B was 40 nM ammonium phosphate (pH 6.5), 60% acetonitrile, and 40% water. The KPI-185 elution point was at approximately 21% acetonitrile. The HPLC of E-KPI exhibits an additional peak after 10 minutes, which is the product of the cyclization of the N-temunus glutamic acid of E-ICPI. The HPLC of D-KPI exhibits no such peak and thus no such cyclization pmduct.
C. Measurement of protease inhibitory properties of KPI variants Once ICPI variants have been purified, they are tested for their ability to bind to and inhibit serine proteases of interest in vitro. The peptides of the present invention preferably exhibit a more potent and specific inhibition of serine proteases of interest than known serine protease inhibitors, such as the natural ICPI peptide dcmain. Such binding and inhibition can be assayed for by determining the inhibition constants for the peptides of the present invention toward serine proteases of interest and comparing those constants with constants determined for known serine protease inhibitors, e.g., the native ICPI domain, toward those proteases. Methods for determining inhibition constants of protease inhibitors are well known in the art. See Fersht, ENZYME STRUCT(JRE
AND MECHANISM, 2nd ed., W.H. Freeman and Co., New York, ( i 985).
In a preferred embodiment the inhibition experiments are carried out using a chromogcnic synthetic protease substrate, as described, for example, in Render et al., J.
Amer. Chem. Soc. 88:5890 (1966). Measurements taken by this method can be used to calculate inhibition constants (K; values) of the peptides of the present invention toward serine proteases of interest. See Bieth in BAYER-SYMPOSIUM V "PROTEINASE
INHIBITORS", Fritz et al., eds., pp. 463-69, Springer-Verlag, Berlin, Heidelberg, New York, (1974). ICPI variants that exhibit potent and specific inhibition of one or more . serine proteases of interest may subsequently be tested in vivo. In vitro testing, however, is not a prerequisite for in vivo studies of the peptides of the present invention.
D. Testing of KPI variants in vivo The peptides of the present invention may be tested, alone or in combination, for their therapeutic efficacy by various in vivo methodologies known to those skilled in the art, e.g., the ability of ICPI variants to reduce postoperative bleeding can be tested in standard animal models. For example, cardiopulmonary bypass surgery can be carried out on animals such as pigs in the presence of ICPI variants, or in control animals where the ICPI variant is not used. The use of pigs as a model for studying the clinical effects associated with CPB has previously been described. See Redmond et al., Ann.
ThoraG
Surg. 56:474 (1993).
The ICPI variant is supplied to the animals in a pharmaceutical sterile vehicle by methods known in the art, for example by continuous intravenous infusion. Chat tubes can be used to collect shed blood for a defined period of time. The shed blood, together with the residual intrathoracic blood found after sacrifice of the animal can be usod to calculate hemoglobin (Hgb) loss. The postoperative blood and Hgb loss is then compared between the test and control animals to detemnirte the effect of the ICPI
vanants.

E. Therapeutic use of KPI variants ICPI variants of the present invention found to exhibit therapeutic efficacy (eg., reduction of blood loss following surgery in animal models) may preferably be used anti administered, alone or in combination or as a fusion protein, in a manner analogous to that currently used for aprotinin or other known serine protease inhibitors.
See Butler et aL, supra. Peptides of the present invention generally may be administered in the manner that natural peptides are administered. A therapeutically effective dose of the peptides of the present invention preferably affects the activity of the serine proteases of interest such that the clinical condition may be treated, ameliorated or prevented Therapeutically effective dosages of the peptides of the present invention can be determined by those skilled in the art, e.g., through in vivo or in vitro models. Generally, the peptides of the present invention may be administered in total amounts of approximately 0.01 to approximately 500, specifically 0.1 to 100 mg/kg body weight, if desired in the form of one or more administrations, to achieve therapeutic effect. It may, however, be necessary to deviate from such administration amounts, in particular depending on the nature and body weight of the individual to be treated, the nature of the medical condition to be treated, the type of preparation and the administration of the peptide, and the time interval over which such administration occurs. Thus, it may in some cases be sufficient to use less than the above amount of the peptides of the present invention, while in other cases the above amount is preferably exceeded. The optimal dose required in each case and the type of administration of the peptides of the present invention can be determined by one skilled in the art in view of the circumstances surrounding such administration. Such peptides can be administered by intravenous injections, in situ injections, local applications, inhalation, oral administration using coated polymers, dermal patches or other appropriate means. Compositions comprising peptides of the present invention are advantageously administered in the form of injectable compositions. Such peptides may be preferably administered to patients via continuous intiavtnous infusion, but can also be administered by single or multiple injections. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described in REMIT1GTONS PF-IARMACEUTICAL SCIENCES, pp. 1405-12 and 1461-87 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association ( 1975). Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobials, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the composition are adjusted according to routine skills in the art. See GOODMAN AND GILMANS 'ITS
PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th ed.). The peptides of the present invention may be present in such pharmaceutical preparations in a concentration of approximately 0.1 to 99.5% by weight, specifically 0.5 to 95% by weight, relative to the total mixture. Such pharmaceutical preparations may also comprise other pharmaceutically active substances in addition to the peptides of the present invention.
Other methods of delivering the peptides to patients will be readily apparent to the skilled artisan.
Examples of mammalian serine proteases that may exhibit inhibition by the peptides of the present invention include: kallikrein; chymotrypsins A and B;
trypsin;
elastase; subtilisin; coagulants and procoagulants, particularly those in active form, including coagulation factors such as thrombin and factors V>Za, IXa, Xa, XIa, and XBa;
plasmin; proteinase-3; enterokinase; acrosin; cathepsin; urokinase; and tissue plasminogen activator. Examples of conditions associated with increased serine protease activity include: CPB-induced inflammatory response; post-CPB pulmonary injury;
pancreatitis; allergy-induced protease release; deep vein thrombosis;
thrombocytopenia;
rheumatoid arthritis; adult respiratory distress syndrome; chronic inflammatory bowel disease; psoriasis; hyperfibtinolytic hemorrhage; organ preservation; wound healing; and myocardial infarction. Other examples of the use of the peptides of the present invention are described in U.S. Patent No. 5,1$7,153.
The inhibitors of the present invention may also be used for inhibition of serine protease activity in vitro, for example during the preparation of cellular extracts to prevent degradation of cellular proteins. For this purpose the inhibitors of the preseat invention may preferably be used in a manner analogous to the way that aprotinin, or other known serine protease inhibitors, are used. The use of aprotinin as a protease inhibitor for preparation of cellular extracts is well known in the art, and aprotinin is sold commercially for this purpose.

The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are nut intended to be limiting of the present invention.
EXAMPLES
Example I. Expression of wild-type KPI (-4-~5'n A. Construction of pTWIO:KPl Plasmid pTWIO:KPI is a bacterial expression vector encoding the 57 amino acid form of ICPI fused to the bacterial phoA signal sequence. The strategy for the construction of pTW l O:ICPI is shown in Figure 1.
Plasmid pcDNAII (Invitrogen, San Diego, CA) was digested with PvuII and the larger of the two resulting PvuII fragments (3013 bp) was isolated. Bacterial expression plasmid pSP26 was digested with MIuI and RrrII, and the 409 by MIuI-RsrII
fragment containing the pTrp promoter element and transcription termination signals was isolated by electrophoresis in a 3% NuSieve Agarose geI (FMC Corp., Rockland, ME).
Plasmid pSP26, containing a heparin-binding EGF-tike growth factor (I~-EGF) insert between the NdeI and HindllI sites, is described as pNA28 in Thompson et aL, J. Biol.
Chem.
269:2541 (1994). Plasmid pSP26 was deposited in host E. coli W3110, pSP26 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Mantissas, Virginia 20110-2209, USA under the conditions specified by the Budapest Treaty on the International Recognition of the Deposit of Microorganisms (Budapest Treaty).
Host E.
coli W3110, pSP26 was deposited on 3 May 1995 and given Accession No. 69$00.
Availability of the deposited plasmid is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
The ends of the MIuI-RsrII fragment were blunted using DNA polymerise Klenow fragment by standard techniques. The blunted fiagrrtent of pSP26 was then ligated into the large PvuII fragment of plasmid pCDNAII, and the ligation mixture was used to transform E. coli strain MC1061. Ampicillin-resistant colonies were selected and used to isolate plasmid pTW 10 by standard techniques.
A synthetic gene was constructed encoding the bacterial phoA secretory signal sequence fused to the amino terminus of KPI(1-X57). The synthetic gene contains cohesive ends for NdeI and HindBI, and also incorporates restriction endonuclease recognition sites for AgeI, RrrII, AatII and BamHI, as shown in Figure 2. The synthetic WO 99!63090 PCT/US99/12276 phoA-KPI gene was constructed from 6 oligonucleotides of the following sequences (shown 5'-3'):
S 6167:
TATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTAC
CCCTGTGACAAAAGCCGAGGTGTGCTCTGAA
6169:
CTCGGCTTTTGTCACAGGGGTAAACAGTAACGGTAAGAGTGCCAGTG
CAATAGTGCTTTGTTTCATA
6165:
CAAGCTGAGACCGGTCCGTGCCGTGCAATGATCTCCCGCTGGTACTTT
GACGTCACTGAAGGT__AAGTGCGCTCCATTCTTT
6166:
GCACTTACCTTCAGTGACGTCAAAGTACCAGCGGGAGATCATTGCAC
GGCACGGACCGGTCTCAGCTTGTTCAGAGCACAC
6168:
TACGGCGGTTGCGGCGGCAACCGTAACAACTTTGACACTGAAGAGTA
CTGCATGGCAGTGTGCGGATCCGCTATTTAAGCT
6164:
AGCTTAAATAGCGGATCCGCACACTGCCATGCAGTACTCTTCAGTGTC
AAAGTTGTTACGGTTGCCGCCGCAACCGCCGTAAAAGAATGGAGC
The oligonucleotides were phosphorylated and annealed in pairs: 6167 + 6169, 6165 + 6166, 6168 + 6164. In 20 ~.1 T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA), 1 g of each oiigonucleotide pair was incubated with 10 U T4 Polynucleotide Kinase (New England Biolabs) for 1 h at 37°C, then heated to 95°C for 1 minute, and slow-cooled to room temperature to allow annealing. All three annealed oligo pairs were then mixed for ligation to one another in a total volume of DNA Ligase Buffer, and incubated with 400 U T4 DNA Ligase (New England Biolabs) overnight at I S°C. The ligation mixture was extracted with an equal volume of phenol:CHCl3 (1:1), ethanol-precipitated, resuspended in SO Restriction Endonuclease Buffer #4 (New England Biolabs) and digested with NdeI and Hindllt. The annealod, ligated and digested oligos were then subjected to electrophoresis in a 3%
NuSieve Agarose gel, and the 240 by NdeI-HindlTI fragment was excised. This gel-ptuified synthetic gene was ligated into plasn~.id pTWlO, which had previously been digested with NdeI and HindllI, and the ligation mixture was used to transform E. coli strain MC1061. Ampicillin-resistant colonies were selected and used to prepare plasmid pTWIO:KPI. This plasmid contains the phoA-KPI(1-X57)-fiuion pmtein inserted between the pTrp promoter element and the transcription termination signals.
B. Constraction of pKPI 61 The strategy for constructing pKPI-61 is shown in Figure 3. Plasmid pTWIO:KPI
was digested with AgeI and HindIll; the resulting 152 by AgeI-HindllI fragment containing a portion of the KPI synthetic gene was isolated by preparative gel electrophoresis. An oligonucleotide pair (I29 + 130) encoding the 9 amino-terminal residues of KPI(1-~57) and 4 amino acids of yeast a-mating factor was phosphorylated and annealed as described above.
129: CTAGATAAAAGAGAGGTGTGCTCTGAACAAGCTGAGA
130: CCGGTCTCAGCTTGTTCAGAGCACACCTCTCTITI'AT
The annealed oligonucieotides were then ligated to the AgeI-HindJII fragarent of the KPI (1-X57) synthetic gene. The resulting 192 by XbaI-Hi~tdIII synthetic gene (shown in Figure 4) was purified by preparative gei electrophoresis, and ligated into plasmid pUC 19 which had previously been digested with Xbal and HindlTI. The ligation products were used to transform E. coli strain MC1061. Ampicillin-resistant colonies were picked and used to prepare plasmid pKPI-57 by standard methods. To crate a synthetic gene encoding KPI(-4-~ 57), pKPI-57 was digested with ~'baI and Agel and the smaller fragment replaced with annealed oligos 234 + 235, which encode 4 amino acid residues of yeast a-mating factor fused a 4 amino acid residue amino-terminal extension of KPI(1-~57).
234:
CTAGATAAAAGAGAGGTTGTTAGAGAGGTGTGCTCTGAACAAGCTGAGA
235: CCGGTCTCAGCTTGTTCAGAGCAGACCTCTCTAACAACCTCTCI'ITI'AT
The 4 extra amino acids are encoded in the amyloid [3-protein precursor/protease nexin-2 (APPn which contains the KPI domain. The synthetic 201 by XbaI-HindIB
fragment encoding KPI(-4-X57) in pKPI-61 is shown in Figure 5.
C. Assembly of pTWI !3 The strategy for the construction of pTW113 is shown in Figure 6. Plasmid pSP35 was constructed from yeast expression plasmid pYES2 (Invitrogen, San Diego, CA) as follows. A 267 by PvuII XbaI fragment was generated by PCR from yeast a mating factor DNA using oiigos 6274 and 6273:
6274: GGGGGCAGCTGTATAAACGATTAAAA
6273: GGGGGTCTAGAGATACCCCTTCTTCTTTAG
This PCR fragment, encoding an 82 amino acid portion of yeast a-mating factor, including the secrctory signal peptide and pro-region, was inserted into pYES2 that had been previously digested with PvuII and XbaI. The resulting plasmid is denoted pSP34.
Two oligonucleotide pairs, 6294 + 6292 were then ligated to 6290 + 6291, and the resulting 135 by fragment was purified by gel electrophoresis.
6294: CTAGATAAAAGAGAGGCTGAGGCTCACGCTGAAGGTACTTTCACTTC
6290:
TGACGTCTCTTCTTACTTGGAAGGTCAAGCTGCTAAGGAATTCATCG
CTTGGTTGGTCAAAGGTAGAGGTTAAGCTTA
6291:

CTAGTAAGCTTAACCTCTACCTTTGACCAACCAAGCGATGAATTCCT
TAGCA
6292:
GCTTGACCTTCCAAGTAAGAAGAGACGTCAGAAGTGAAAGTACCTT
CAGCGTGAGCCTCAGCCTCTCTTTTAT
The resulting synthetic fragment was ligated into the XbaI site of pSP34, resulting in plasmid pSP35. pSP35 was digested with XbaI and HindBI to remove the insert, and ligated with the 201 by .YbaI-HindBI fragment of pKPI-61, encoding KPI
(-4-X57). The resulting plasmid pTW 113, encodes the 445 by synthetic gene for the a-factor-KPI(-4-X57) fusion. See Figure 7.
D. Transformation of yeast with pTWll3 Saccharomyces cerevisiae strain ABLi 15 was transformed with plasmid pTW113 by electroporation by the method of Becker et al., Methods Enrymol. 194:182 (1991).
An overnight culture of yeast strain ABL115 was used to inoculate 200 ml YPD
medium. The inoculated culture was grown with vigorous shaking at 30°C
to an ODD
of 1.3-1.5, at which time the cells were harvested by centrifugation at 5000 rpm for 5 minutes. The cell pellet was resuspended in 200 ml ice-cold water, respun, and resuspended in 100 ml ice-cold water, then pelleted again. The washed cell pellet was resuspended in 10 ml ice-cold 1M sorbitol, recentrifuged, then resuspended in a final volume of 0.2 ml ice-cold IM sorbitol. A 40 ml aliquot of cells was placed into the chamber of a cold 0.2 cm electroporation cuvette (Invitrogen), along with 100 ng plasmid DNA for pTW 113. The cuvette was placed into an Invitrogen Electroporator II
and pulsed at 1500 V, 25F, 100 s2. Electroporated cells were diluted with 0.5 mI 1M
sorbitol, and 0.25 ml was spread on an SD agar plate containing 1M sorbitol.
After 3 days' growth at 30°C, individual colonies were streaked on SD + CAA
agar plates.
E. Induction ofpTWll3/ABLIIS, purification ofKPl(-4-~57) Yeast cultures were grown in a rich broth and the galactose promoter of the KPI
expression vector induced with the addition of galactose as described by Sherman, Methods Enzymol. 194:3 (1991). A single well-isolated colony of pTW113/ABL115 was used to inoculate a 10 ml overnight culture in Yeast Batch Medium. The next day, IL Yeast Batch Medium which had been made 0.2% glucose was inoculated to an ODD
of 0.1 with the overnight culture. Following 24 hours at 30°C with vigorous shaking, the 1L culture was induced by the addition of 20 ml Yeast Galactose Feed Medium.
Following induction, the culture was fed every 12 hours with the addition of 20 ml Yeast Galactose Feed Medium. At 48 hours after induction, the yeast broth was harvested by centrifugation, then adjusted to pH 7.0 with 2M Tris, pH 10. The broth was subjected to trypsin-Sepharose affinity ~ chromatography, and bound KPI(-4-~57) was eluted with 20mM Tris pH 2.5. See Schilling et al., Gene 98:225 (I991). Final purification of ICPI(-4-~57) was accomplished by HPLC chromatography on a semi-prep Vydac C4 column in a gradient of 20% to 35% acetonitrile. The sample was dried and resuspended in PBS
at 1-2 mg/ml. The amino acid sequence of KPI(-4-X57) is shown in Figure 8.
Example 2. Recombinant Expression of site-directed KPI(-4--X57) variants Expression vectors for the production of specific variants of KPI(-4-~57) were all constructed using the pTW 113 backbone as a starting point. For aach KPI
variant, an expression construct was created by replacing the 40 by RsrII AatlI fi-agment of the synthetic ICPI gene contained in pTW 113 with a pair of anneaked oligonucleotides which encode specific codons mutated from the wild-type KPI(-4-X57) sequence. In the following examples, the convention used for designating the amino substituents in the ICPI variants indicates first the single letter code for the amino acid found in wild-type ICPI, followed by the position of the residue, followed by the code for the replacement amino acid. Thus, for example, M15R indicates that the methionine residue at position 15 is replaced by an arginine.
A. Corutruction of pTW6165 The strategy for constructing pTW6165 is shown in Figure 9. Plasmid pTW113 was digested with RsrlI and AatII, and the larger of the two resulting fragments was isolated. An okigonucleotide pair (812 + 813) was phosphorylated, annealed and gel-purified as described above.
812: GTCCGTGCCGTGCAGCTATCTGGCGCTGGTACTTTGACGT
813: CAAAGTACCAGCGCCAGATAGCTGCACGGCACG

The annealed oligonucleotides were ligated into the RrrII and AatII-digested pTW113, and the ligation product was used to transform E. coli strain MC1061.
Transformed colonies were selected by ampicillin resistance. The resulting plasmid, pTW616S, encodes the 44S by synthetic gene for the a-factor-KPI(-4--57; M1SA, S17VV) fusion.
S See Figure 10.
B. Corutruction of pTW6166, pT'W6175, pBG028, pTW6183, pTW6184, pTW6185, pT'W6173, pTW6174.
Construction of the following KPI(-4-i57) variants was accomplished exactly as outlined for pTW6165. The oligonucleotides utilized for each construct are denoted below, and the sequences of annealed oligonucleotide pairs are shown in Figure

11.
Figures 12-19 show the synthetic genes for the a-factor fusions with each KPI(-4-iS7) variant.
1S pTW6166: KPI(-4-~S7; MISA, S17~ See Figure 12.
814: GTCCGTGCCGTGCAGCTATCTACCGCTGGTACTTTGACGT
815: CAAAGTACCAGCGGTAGATAGCTGCACGGCACG
pTW6175: KPI(-4-iS7; M1SL, S17F) See Figure 13.
2S 867: GTCCGTGCCGTGCATTGATCTTCCGCTGGTACTTTGACGT
868: CAAAGTACCAGCGGAAGATCAATGCACGGCACG
pBG028: KPI(-4-~57; M15L, S17I~ See Figure 14.
1493: GTCCGTGCCGTGCTTTGATCTACCGCTGGTACTTTGACGT

1494: CAAAGTACCAGCGGTAGATCAAAGCACGGCACG
pTW6183: KPI(-4-X57; I 16H, S 17F j See Figure 15.
925: GTCCGTGCCGTGCAATGCACTTCCGCTGGTACTTTGACGT
926: CAAAGTACCAGCGGAAGTGCATTGCACGGCACG
pTW6184: KPI(-4-~57; I16H, S 17I~ See Figure 16.
927: GTCCGTGCCGTGCAATGCACTACCGCTGGTACTTTGACGT
928: CAAAGTACCAGCGGTAGTGCATTGCACGGCACG
pTW6185: KPI(-4-~57; I16H, S17W) Sec Figure 17.
929: GTCCGTGCCGTGCAATGCACTGGCGCTGGTACTTTGACGT
930: CAAAGTACCAGCGCCAGTGCATTGCACGGCACG
pTW6173: KPI(-4-~57; M15A, I16H) See Figure 18.
863: GTCCGTGCCGTGCAGCTCACTCCCGCTGGTACTTTGACGT
864: CAAAGTACCAGCGGGAGTGAGCTGCACGGCACG
pTW6174: KPI(-4--~57; M15L, I16H) See Figure 19.
865: GTCCGTGCCGTGCATTGCACTCCCGCTGGTACTTTGACGT

WO 99/63090 PCTlUS99/12276 866: CAAAGTACCAGCGGGAGTGCAATGCACGGCACG
C. Transformation ojyeast with expression vectors S Yeast strain ABL115 was transformed by electroporation exactly according to the protocol described for transformation by pTW 113.
D. Induction of transformed yeast strains, puriftcation of KPI(-4-iS7) variants.
Cultures of yeast strains were grown and induced, and recombinant secreted ICPI
(-4-X57) variants were purified according to the procedure described for KPI {-4-X57).
The amino acid sequences of KPI(-4--~57) variants are shown in Figures 27-36.
Example 3. Identification of ICPI (-4-~57; M15A, S17F) DD185 by phage display.
A. Construction of vector pSPZ6:Amp:F1 The construction of pSP26:Amp:F1 is outlined in Figure 43. Vector pSP26:Amp:F1 contributes the basic plasmid backbone for the construction of the phage display vector for the phoA:KPI fusion, pDWI #14. pSP26Amp:F1 contains a low-copy number origin of replication, the ampicillin-resistance gene (Amp) and the Fl origin for production of single-stranded phagemid DNA.
The ampicillin-resistance gene (Amp) was generated through poiymerase chain reaction (PCR) amplification from the plasmid genome of PUC19 using oligonucleotides 176 and 177.
176: GCCATCGATGGTTTCTTAAGCGTCAGGTGGCACTTTTC
177: GCGCCAATTCTTGGTCTACGGGGTCTGACGCTCAGTGGAACGAA
The PCR amplification ofAmp was done according to standard techniques, using Taq polymerase (Perkin-Elmer Cetus, Norwallc, CT). Amplification from plasmid pUCl9 with these oligonucleotides yielded a fragment of 1159 bp, containing P,~INB and CIaI restriction sites. The PCR product was digested with PJIIvB and CIaI and purified by agarose gel electrophoresis in 3% NuSieve Agarose (FMC Corp.). Bacterial expression vector pSP26 (supra) was digested with PfINl1 and CIaI and the larger vector fragment was purified. The PJIMI-CIaI PCR fi~agment was ligated into the previously digested pSP26 containing the Amp gene. The ligation product was used to transform E.
call strain MC1061 and colonies were selected by ampicillin resistance. The resulting plasmid is denoted pSP26Amp.
The F1 origin of replication firom the mammalian expression vector pcDNAII
(Invitrogen) was isolated in a 692 by Earl figment. Piasmid pcDNAII was digested with Earl and the resulting 692 by figment purified by agarose gel electrophoresis.
Earl-NotI adapters were added to the 692 by Earl fragment by ligation of two annealed oligonucleotide pairs, 179 + 180 and 181 + 182. The oligo pairs were annealed as described above.
179: GGCCGCTCTTCC
180: AAAGGAAGAGC
181: CTAGAATTGC
182: GGCCGCAATTC
The oligonucleotide-ligated fragment was then ligated into the single NotI
site of PSP26:Amp to yield the vector pSP26:Amp:Fl.
B. Constraetion of vector pglll The construction of pgIl1 is outlined in Figure 44. The portion of the phage gene)TI protein gene contained by the PDW1 #14 phagemid vector was originally obtained as a PCR amplification product from vector m13mp8. A portion of m13mp8 geneIll encoding the carboxyl-terminal 158 amino acid rcsiducs of the geneIT1 prnduct was isolated by PCR amplification of m13mp8 nucleotide residues 2307-2781 using PCR oligos 6162 and 6160.
6162: GCCGGATCCGCTATTTCCGGTGGTGGCTCTGGTTCC
6160: GCCAAGCTTATTAAGACTCCTTATTACGCAG
The PCR oligos contain BamHI and HindIII restriction recognition sites such that PCR from ml3mp8 plasmid DNA with the oligo pair yielded a 490 by BamHI-HindIII figment encoding the appropriate portion of geneIlT. The PCR product was ligated between the BamHI and Hina~I sites within the poiylinker of PUC19 to yield plasmid pgIII.
C Cor~straciion ojpPhoA:KPLglll Construction of pPhoA:KPI:gIII is outlined in Figure 45. A portion of the phoA
signal sequence and KPI firsion encoded by the phage display vector PDWI #14 originates with pPhoA:KPI:gIIl. The 237 by NdeI-HindIII fiagment of pTWIO:KPI
encoding the entire phoA:KPI (I-X57) fission was isolated by preparative agarose gel electrophoresis, and inserted between the NdeI and HindIII sites of pUCI9 to yield plasmid pPhoA:KPI. The 490 by BamHI-HindIII fi-agment of pgllI encoding the C-terminal portion of the geneIII product was then isolated and ligated between the BamHI
and Hina'ITI sites of pPhoA:KPI to yield vector pPhoa:KPI:gITI. The pPhoA:KPI:gIIi vector encodes a 236 amino acid residue fission of the phoA signal peptide, KPI (1-~57) and the carboxyl-terminal portion of the geneIII product.
D. Constraction oJpLGI
Construction of pLGI is illustrated in Figure 46. The exact geneBI sequences contained in vector PDWI #14 originate with phage display vector pLGI. A
modified genellI segment was generated by PCR amplification of the genellI region from pgIl1 using PCR oligonucleotides 6308 and 6305.
6308: AGCTCCGATCTAGGATCCGGTGGTGGCTCTGGTTCCGGT
6305: Gc:AGCGGCCGTTAAGCTTATTAAGACTCCT
PCR amplification fi-om pgIlI with these oligonucleotides yielded a 481 by BamHI-HinaHI fiagment encoding a geneIII product shortened by 3 amino acid residues at the amino-terminal portion of the segment of the genellI fiagment encoded by pgIll.
A 161 by NdeI-BamHI fragment was generated by PCR amplification firm bacterial expression plasmid pTHW05 using oligonucleotides 6306 and 6307.
6306: GATCCTTGTGTCCATATGAAACAAAGC
6307: CACGTCGGTCGAGGATCCCTAACCACGGCCTTTAACCAG

The 161 by NdeI-BamHI fragment and the 481 by BamHI-HindIll fragment were gel-purified, and then iigated in a three-way ligation into pTW 10 which had previously been digested with NdeI and HindIII. The resulting plasmid pLGl encodes. a phoA
signal peptide-insert-geneIII fusion for phage display purposes.
E. Construction ojpALS!
Construction of pALS 1 is illustrated in Figure 47.
Vector pAL51 contains the geneIII sequences of pLGI which are to be incorporated in vector pDW 1 # 14.
A 1693 by fragment of plasmid pBR322 was isolated, extending from the BamHI site at nucleotide 375 to the PvuII site at position 2064. Plasmid pLGI
was digested with Asp718I and BamHI, removing an 87 by fiagment. The overhanging Asp718I end was blunted by treatment with Klenow fragment, and the PvuII-BamHI
fragment isolated from pBR322 was ligated into this vector, resulting in the insertion of a 1693 by "stuffer" region between the Asp718I and BamHI sites. The 78 by NdeI-Asp718I region of the resulting plasmid was removed and replaced with the annealed oligo pair 6512 + 6513.
6512:
TATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTITA
CCCCGGTGACCAAAGCCCACGCTGAAG
6513:
GTACCTTCAGCGTGGGCTI"TGGTCACCGGGGTAAACAGTAACGGTAA
GAGTGCCAGTGCAATAGTGCTITGTTTCA
The newly created 74 by NdeI Asp718I fragment encodes the phoA signal peptide, and contains a BstEII cloning site. The resulting plasmid is denoted pALS I.
F. Construction ojpAL53 Construction of pAL53 is outlined in Figure 48. Plasmid pAL53 contributes most of the vector sequence of pDWI #14, including the basic vector backbone with Amp gene, F1 origin, low copy number origin of replication, genellI segment, phoA
promoter and phoA signal sequence.

Plasmid pAL51 was digested with NdeI and Hindlil and the resulting 2248 by NdeI-HindIII fragment encoding the phoA signal peptide, stuffer region and genellI
region was isolated by preparative agarose gel electrophoresis. The NdeI-HindllI
fragment was ligated into plasmid pSP26:Amp:F1 between the NdeI and HindIII
sites, resulting in plasmid pALS2.
The phoA promoter region and signal peptide was generated by amplification of a portion of the E. coli genome by PCR, using oligonucleotide primers 405 and 406.
405: CCGGACGCGTGGAGATTATCGTCACTG
406: GCTTTGGTCACCGGGGTAAACAGTAACGG
The resulting PCR product is a 332 by MIuI-BstEII fragment, which contains the phoA promoter region and signal peptide sequence. This fragment was used to replace the 148 by MIuI-BstEII segment of pAL52, resulring in vector pALS3.
G. Construction ofpSP26:Amp:Fl:PhoA:KPLglll Construction of pSP26:Amp:Fl:PhoA:ICPI:gIII is illustrated in Figure 49. This particular vector is the source of the KPI coding sequence found in vector pDWI #14.
Plasmid pPhoa:ICPI:gIII was digested with NdeI and HindIll, and the resulting 714 by NdeI-HindIll fragment was purified, and then inserted into vector pSP26:Amp:F1 between the NdeI and HindIII sites. The resulting plasmid is denoted pSP26:Amp:F 1:PhoA:KPI:gIII.
H. Construction of pDWI #l4 Construction ofpDWl #14 is illustrated in Figure 50. The sequences encoding KPI were amplified from plasmid pSP26Amp:Fl:PhoA:KPI:gIl1 by PCR, using oligonucleotide primers 424 and 425.
424: CTGTTTACCCCGGTGACCAAAGCCGAGGTGTGCTCTGAACAA
425: AATAGCGGATCCGCACACTGCCATGCAGTACTCTTC
The resuiting 172 by BstEII-BamHI fragment encodes most of KPI (155). This fragment was used to replace the stuff'er region in pAL53 between the BstEII
and BamHI
sites. The resulting piasmid, pDWI #14, is the parent ICPI phage display vector for preparation of randomized itPI phage libraries. The coding region for the phoA-KPI
(155)-geneIII fusion is shown in Figure 55.
I. Corutruction ojpDWl l4-2 Construction of pDWI 14-2 is illustrated in Figure 57. The first step in the construction of the KPI phage libraries in pDW 1 #14 was the replacement of the AgeI-BamHI fragment within the KPI coding sequence with. a stuffer fragment. This greatly aids in preparation of randomized KPI libraries, which are substantially fi~ee of contamination of phagemid genomes encoding wild-type KPI sequence.
Plasmid pDWI #14 was digested with AgeI and BamHI, and the 135 by AgeI-BamHI fragment encoding KPI was discarded. A stuffer fiagment was created by PCR
amplification of a portion of the pBR322 Tet gene, extending from the BamHI
site at nucleotide 375 to nucleotide 1284, using oligo primers 266 and 252.
266: GCTTTAAACCGGTAGGTGGCCCGGCTCCATGCACC
252: CGAATTCACCGGTGTCATCCTCGGCACCGTCACCCT
The resulting 894 by AgeI-BamHI stuffer fi~agment was then inserted into the Age1/BamHI-digested pDWI #14 to yield the phagcmid vector pDWI 14-2. This vector was the starting point for construction of the randomized KPI libraries.
J. Construction of KPI Library l619 Construction of KPI Library 16-19 is outlined in Figure 58. Library 16-19 was constructed to display KPI-genelTI fi~sions in which amino acid positions Ala~4, Met~s, Ile~b and Sere are randomized. For preparation of the library, plasmid pDWl 14-2 was digested with AgeI and BamHI to remove the stuffcr region, and the resulting vector was purified by preparative agarose gel elxtrophoresis. Plasmid pDWI #14 was usod as template in a PCR amplification of the KPI region extending from the AgeI site to the BamHI site. The oligonucleotide primers used were 544 and 551.
544: GGGCTGAGACCGGTCCGTGCCGT(NNS)4CGCTGGTACTTTGACGTC
551: GGAATAGCGGATCCGCACACTGCCATGCAG

Oligonucleotide primer 544 contains four randomized colons of the sequence NNS, where N represents equal mixtures of A/G/C/T and S an equal mixture of G
or C.
Each NNS colon thus encodes all 20 amino acids plus a single possible stop colon, in 32 different DNA sequences. PCR amplification fi-om the wild-type KPI gene resulted in the production of a mixture of 135 by AgeI-BamHI fragments all containing diffet~ent sequences in the randomized region. The PCR product was purified by preparative agarose gel electrophoresis and ligated into the AgeIlBamHI digested pDW 1 14-2 vector.
The ligation mixture was used to transform E. coli TopIOF~ cells (Invitrogtn) by electroporation according to the manufacturer's directions. The resulting Library 16-19 contained approximately 400,000 independent clones. The potential size of the library, based upon the degeneracy of the priming PCR oligo #544 was 1,048,576 members.
The expression unit encoded by the members of Library 16-19 is shown in Figure 59.
K. Selection ofLibrary 16-19 with human plasma kallikrein KPI phage were prepared and amplified by infecting transformed cells with M13K07 helper phage as described by Matthews et a1, Science 260:1113 (1993).
Human plasma kallikrein (Enzyme Research Laboratories, South Bend, INJ, was coupled to Sepharose 6B resin. Prior to phage binding, the immobilized kallilQein resin was washed three times with 0.5 ml assay buffer (AB = 100mM Tris-HCI, pH 7.5, O.SM
NaCI, SmM each of KCI, CaCIZ, MgCIZ, 0.1% gelatin, and 0.05% Triton X-100).
Approximately 5x109 phage particles of the amplified Library 16-19 in PBS, pH
7.5, containing 300mM NaCI and 0.1% gelatin, were bound to 50 pl ka11i1Qein resin containing 15 pmoles of active human plasma kallila~ein in a total volume of 250 pl.
Phage were allowed to bind for 4 h at room temperature, with rocking. Unbound phage were removed by washing the kallilarein resin three times in 0.5 ml AB. Bound phage were eluted sequentially by successive 5 minute washes: 0.5 ml SOmM sodium citrate, pH 6.0, 150mM NaCI; 0.5 ml SOmM sodium citrate, pH 4.0, 154mM NaCI; and 0.5 ml SOmM glycine, pH 2.0, i50mM NaCI. Eluted phage were neutralized immediately and phagemids from the pH 2.0 elution were titered and amplified for reselection.
After three rounds of selection on kallikrein-Scpharose, phagemid DNA was isolated finm 22 individual colonies and subjected to DNA sequence analysis.
The most frequently occurring randomized KPI region encoded: Alai°-Alms-Ile~6-Phe~'. The phoA-KPI-geneBI region encoded by this class of selected KPI phage is shown in Figure 60. The KPI variant encoded by these phagemids is denoted ICPI
(155;
M15A, S17F).
L. Construction ojpDDl85 KPI (-4-X57; MISA, S17F) Figure 61 outlines the construction ofpDD185 KPI (-4-~57; M15A, S17F). The sequences encoding ICPI ( 155; M 15A, S 17F) were moved from one phagemid vector, pDWI (16-19) 185, to the yeast expression vector so that the KPI variant could be purified and tested.
Plasmid pTW113 encoding wild-type ICPI (-4-X57) was digested with AgeI and BamHI and the 135 by AgeI-BamHI fi~agment was discarded. The 135 by AgeI-BamHI
fragment of pDWI (16-19) 185 was isolated and Iigated into the yeast vector to yield plasmid pDD185, encoding a-factor firsed to KPI (-4-X57; M15A, S17F). See Figure 62.
M. Purification ojKPl ( 4-~57; Ml SA, SI7F) pDDl85 Transformation of yeast strain ABL115 with pDD185, induction of yeast cultures, and purification of KPI (-4-X57; M15A, S17~ pDD185 was accomplished as describai for the other KPI variants.
N. Construction ojKPl Library 6 MI SA, with residues 14, l6-18 random.
Library 6 was constructed to display KPI-genellI firsions in which amino acid positions Ala'4, Ile'6, Ser" and Arg'8 are randomized, but position 15 was held constant as Ala. For preparation of the library, plasmid pDWI #14 was used as the template in a PCR amplification of the KPI region extending from the AgeI site to the BamHI
site.
The oligonucleotide primers used were 551 and 1003.
1003: GCTGAGACCGGTCCGTGCCGTNNSGCA(NNS)3TGGTACTTTGACGTC
551: GGAATAGCGGATCCGCACACTGCCATGCAG
Oligonucleotide primer 1003 contained four randomized codons of the sequence NNS, where N represents equal mixtures of A/G/CrT and S an equal mixture of G
or C.
Each NNS codon thus encodes all 20 amino acids plus a single possible stop, in different DNA sequences. PCR amplification from the wild-type ICPI gene resulted in the production of a mixture of 135 by AgeI-BamHI fragments all containing different sequences in the randomized region. The PCR product was phenol extracted, ethanol precipitated, digested with BamHI and purified by preparative agarose gel electrophoresis. Plasmid pDWI 14-2 was digested with BamHI, phenol extracted and ethanol precipitated. The insert was ligated at high molar ratio to the vector, which was then digested with AgeI to remove the stuffer region. The vector containing the insert was purified by agarose gel electrophoresis and recircularized. The resulting library contains approximately Sx 1 O6 independent clones.
D. Construction ojKPl Library 7 with residues 14-18 random.
Library 7 was constructed to display ICPI-geneIll fusions in which amino acid positions Alai°, Met~s, Ile~6, Sere and Arg~$ are randomized. For preparation of the library, nlasmid pDWI #14 was used as template in a PCR amplification of the KPI
region extending from the AgeI site to the BamHI site. The oligonucleotide primers used were 551 and 1179.
1179: GCTGAGACCGGTCCGTGCCGT(NNS)STGGTACTTTGACGTC
551: GGAATAGCGGATCCGCACACTGCCATGCAG
Oligonucleotide primer 1179 contains five randomized codons of the sequence NNS, where N represents equal mixtures of A/G/C/T and S an equal mixture of G
or C.
Each NNS codon thus encoded ail 20 amino acids plus a single possible stop, in different DNA sequences. PCR amplification from the wild-type KPI gene resulted in the production of a mixture of 135 by AgeI BamHI fiagments all containing different sequences in the randomized region. The PCR product was phenol extracted, ethaaol precipitated, digested with BamHI and purified by preparative agarose gel electrophoresis. Plasntid pDWI 14-2 was digested with BamHI, phenol extracted and ethanol precipitated. The insert was ligated at high molar ratio to the vector, which was then digested with AgeI to remove the stuffer.region. The vector containing the insert was purified by agarose gel electrophoresis and recircularized. The resulting library contains approximately 1x10' independent clones.

P. Selection ofLioraries 6 & 7 with human factorXlla KPI phage were prepared and amplified by infecting transformed cells with M13K07 helper phage (Matthews and Wells, 1993). Human factor 3~Ia (Enzyme Research Laboratories, South Bend, IN), was biotinylated as follows. Factor XIIa (0.5 mg) in SmM sodium acetate pH 8.3 was incubated with Biotin Ester (Zymed) at room temperature for 1.5 h, then buffer-exchanged into assay buffer (AB).
Approximately 1x10'° phage particles of each amplified Library 6 or 7 in PBS, pH 7.5, containing 300mM NaCI and 0.1% gelatin, were incubated with SO pmoles of active biotinylated human factor XIIa in a total volume of 200 Icl. Phage were allowed to bind for 2 h at room temperature, with rocking. Following the binding period, 100 ul Strepavidin Magnetic Particles (Boehringer Mannheim) were added to the mixture and incubated at room temperature for 30 minutes. Separation of magnetic particles from the supernatant and wash/elution buffers was carried out using MPC-E-I Neodymium-iron-boron permanent magnets (Dynal). Unbound phage were removed by washing the magnetically bound biotinylated 3QIa-phage complexes three times with 0.5 ml AB.
Bound phage were eluted sequentially by successive 5 minute washes: 0.5 ml SOmM
sodium citrate, pH 6.0, 150mM NaCI; 0.5 ml SOmM sodium citrate, pH 4.0, 150mM
NaCI; and 0.5 ml SOmM glycine, pH 2.0, 150mM NaCI. Eluted phage were neutralized immediately and phagemids fiom the pH 2.0 elution were titered and amplified for reselection. After 3 or 4 rounds of selection with factor 3HIa, phagemid DNA
was isolated fiom individual colonies and subjected to DNA sequence analysis.
Sequences in the randomized regions were compared with one another to identify consensus sequences appearing more than once. From Library 6 a phagemid was identified which encoded M15L, S17Y, RIBH. Fmm Library 7 a phagemid was identified which encoded M15A, S17Y, R18H.
Q. Construction of KPI Library P48 with residue 48 random.
Library P48 was constructed to for expression of KPI (M15A, S17Y, R18H) IN
WHICH AMINO ACID n which amino acid position Tyr4g is randomized. Construction of Library P48 is detailed in Figure 55. For preparation of the library, plasmid pDWI
L6-16, encoding the pBG022 KPI peptide as a fusion with the m13 gI)1 protein, was used as template in a PCR amplification of the KPI region extending from the RsrII site to the BamHI site. The oligonucleotide primers used were 1663 and 1945.

WO 99/63090 PCT/tJS99/12276 1663: GCTTTACTGTTTACCCCGGTGACCAAAGCCGAGGTGTGC
1945: ATTAGCGGATCCGCACACTGCCATGCASNNCTCTTCAGTGTCAAAG
Oligonucleotide primer 1945 contains a single randomized codon of the sequence SNN, where N represents equal mixtures of AIG/CrI' and S an equal mixture of G or C. Following the procedure delineated supra, PCR amplification from the wild-type ICPI gene resulted in the production of a mixture of RsrII-BamHI
fiagments all containing different sequences in the randomized region. The PCR product was phenol extracted, ethanol precipitated, digested with RsrII and BamHI and purified by preparative agarose gel electrophoresis. Plasmid pBG022 was digested with RsrII and BamHI, phenol extracted and ethanol precipitated. The insert was ligated at high molar ratio to the vector. The vector containing the insert was purified by agarose gel electrophoresis and recircularized.
R. Construction ojpBG015 KPI (-4-~57; MISL, S17Y, RIBH), pBG012 (-4--~57; MI SA, SI7Y, RI BH, having spontaneous mutation Y48H) The sequences encoding KPI (155; MISL, S17Y, Ri8H) and KPI (155; M17A, S 17Y, R18H) were moved from the pragemid vectors to the yeast expression vector so that the ICPI variant could be purified and tested. Plasmid pTWlI3 encoding wild-type KPI (-4-~57) was digested with AgeI and BamHI and the 135 by AgeI-BamHI
fi~agment was discarded. The 135 by AgeI-BamHI fi-agment of the phagemid vectors were isolated and Iigated into the yeast vector to yield plasmids pBG015 and pBG022, encoding yeast x-factor fused to KPI (-4-~57; M15L, S17Y, R18I~, and KPI (-4-X57; M15A, SI7Y, RI8H, having spontaneous mutation Y48H), respectively. Figure 20 shows the synthetic gene for the x-factor fusion with KPI variant (-4-~57; M15A, S17Y, RIBFi, having spontaneous mutation Y48H). Figure 37 shows the amino acid sequence of KPI
variant (-4-X57; M15A, S17Y, RI8H, having spontaner~us mutation Y48H).
S. Construction of pBG033 KPI ( 4--57; T~9V, M15A, S17Y, R18H) Plasmid pBG022 was digested with XbaI and RsrII, and the larger of the two resulting fi-agments was isolated. An oligonucleotide pair ( 1593 + 1642) was phosphorylated, annealed and gel-purified as described previously. The annealed oligonucieotides were ligated into the.YbaI and RsrII-digested pBG022, and the Iigation product was used to transform E. cell strain MC1061 to ampicillin resistance.
The resulting plasmid pBG033, encodes the 445 by synthetic gene for the yeast x-factor-KPI
(-4-X57; T9V, M15A, SI7F, R18H) fusion. Figure 21 shows the synthetic gene for the x-factor fusion with KPI variant (-4-X57; T9V, M15A, S17Y, R18H, Y48H). ).
Figure 38 shows the amino acid sequence of KPI variant (-4-~57; T9V, M15A, SI7Y, R18H, Y48H).
T. Construction of pBG048 KPI ( 4-~57; Y48H) Figure 52 outlines the construction of pBG048 KPI (-4-X57; Y48H). Plasmid pTW113 encoding wild-type KPI (-4-i57) was digested with AatII and BamHI and the 92 by AatII-BamHI fragment was discarded. Plasmid pBG022 encoding KPI (-4-X57;
M15L, S 17Y, R18H, Y48H) was digested with AatII and BamHI. The resulting 92 by AatII-BamHI fragment was isolated and ligated into the yeast vector to yield plasmid pBG048, encoding yeast x-factor fused to KPI (-4-~57; Y48H). Figure 22 shows the synthetic gene for the x-factor fusion with KPI variant (-4-~57; Y48I-~.
Figure 39 shows the amino acid sequence of KPI variant (-4--~57; 48H).
U. Constraction ofpBG049KP1 (-4-X57; MISA, SI7Y, RI8H) Figure 53 outlines the construction of pBG049 KPI (-4-X57; M15A, S17Y, R18H). Plasmid pBG022 encoding KPI (-4-~57; M15A, S17Y, R18H, Y48H) was digested with AatII and BamHI and the 92 by AatII-BarnHI fragment was discarded.
Plasmid pTW 113 encoding wild-type ICPI (-4-X57) was digested with AatII and BamHI.
The resulting 92 by AatII-BamHI fragment was isolated and ligated into the yeast vector to yield piasmid pBG048, encoding yeast x-factor fused to KPI (-4-X57; M15A, S17Y, R18H). ). Figure 23 shows the synthetic gene for the x-factor fusion with KPI
variant (-4-X57; M15A, S17Y, R18H). Figure 40 shows the amino acid sequence ofKPI
variaat (-4-~57; M15A, S17Y, R18H).
V. Construction oJpBG050 KPI ( 4-~57; ?'9V, Ml SA, SI7Y, R18H) Figure 54 outlines the construction of pBG050 KPI (-4-i57; T9V, M15A, S17Y, RIBH). Plasmid pBG033 encoding KPI (-4-~57; T9V, M15A, R18H, Y48H) was digested with AatII and BamHI and the 92 by AatII-BamHI fragment was discarded Plasmid pTW113 encoding wild-type KPI (-4-i57) was digested with.4atII and BamHL
The resulting 92 by AatII-BamHI 6ragrnent was isolated and ligated into the yeast vector to yield plasmid pBG050, encoding yeast x-factor fined to KPI (-4-~57; T9V;
M15A, S17Y, R18H). ). Figure 24 shows the synthetic gene for the x-factor fusion with KPI
variant (-4-~57; T9V, MISA, S17Y, RIBH). Figure 41 shows the amino acid sequence ofKPI variant (-4-X57; T9V, M15A, S17Y, R18H).
W. Construction of pBG019 KPI (-4-X57, ?'9V, MISL, SI7Y, R18H) Plasmid pBG015 was digested with XbaI and RsrII, and the larger of the two resulting fi~agments was isolated. An oligonucleotide pair (1593 + 1642) was phosphorylated, annealed and gel-purified as described previously.
1593:
CTAGATAAAAGAGAGGTTGTTAGAGAGGTGTGCTCTGAACAAGCTG
AGGTTG
1642:
GACCAACCTCAGCTTGTTCAGAGCACACCTCTCTAACAA
CCTCTCIT)'TAT
The annealed oligonucleotides were ligated into the XbaI and RsrII-digested pBG015, and the ligation product was used to transform E. colt strain MC1061 to ampicillin resistance. The resulting piasmid pBG029, encodes the 445 by synthetic gene for the yeast x-factor-KPI (-4-~57; T9V, M15L, S17F, R18H) fission.
X. Selection ofLibrary l6-19 with human factorXa KPI phage were prepared and amplified by infecting transformed cells with M13K07 helper phage (Matthews and Wells, 1993). Human factor Xa (Haematologic Technologies, Inc., Essex function, VT) was coupled to Sepharose 6B resin.
Prior to phage binding, the immobilized Xa resin was washed three times with 0.5 ml assay buffer (AB = 100mM Tris-HCI, pH 7.5, O.SM NaCI, SmM each of KCI, CaCl2, MgCl2, 0.1% gelatin, and 0.05% Triton X-100). Approximately 4x10'° phage particles of the amplified Library 16-19 in PBS, pH 7.5, containing 300mM NaCI and 0.1%
gelatin, were bound to 50 pl Xa resin in a total volume of 250 pl. Phage were allowed to bind for 4 h at room temperaturz, with rocking. Unbound phage were removed by washing the Xa resin three times in 0.5 ml AB. Bound phage were eluted sequentially by successive 5 minute washes: 0.5 ml SOmM sodium citrate, pH 6.0, 150mM NaCI;
0.5 ml SOmM sodium citrate, pH 4.0 150mM NaCI; and 0.5 ml 50mM glycine, pH 2.0, 150mM
NaCI. Eluted phage were neutralized immediately and phagemids from the pH 2.0 elution were titered and amplified for reselection. After three rounds of selection on Xa-Sepharose, phagemid DNA was isolated and subjected to DNA sequence analysis.
Sequences in the randomized Alai°-Sere region were compared with one another to identify consensus sequences appearing more than once. A phagemid was identified which encoded ICPI (155; M15L, I16F, S17K).
Y. Construction ofpDDl31 KPI (-4-X57; MISL,116F, S17K) The sequences encoding KPI (155; M15L, I16F, S17K) were moved from the phagemid vector to the yeast expression vector so that the ICPI variant could be purified and tested.
Plasmid pTW113 encoding wild-type KPI (-4-X57) was digested with AgeI and BamHI and the 135 by AgeI-BamHI fragment was discarded. The 135 by AgeI-BamHI
fragment of the phagemid vector was isolated and ligated into the yeast vector to yield plasmid pDDl3l, encoding yeast x-factor fused to ICPI {-4-~57; M15L, I16F, S17K).
Z. Constraction of pDDl34 KPI ( 4-~57; MI SL, I16F, S17K, G37~
Plasmid pDD 131 was digested with AatI and BamHI, and the larger of the two resulting fragments was isolated. An oligonucleotide pair (738 + 739) was phosphorylated, annealed and gel-purified as described previously.
738:
CACTGAAGGTAAGTGCGCTCCATTCTTTTACGGCGGTTGCTACGGCA
ACCGT
AACAACTT.TGACACTGAAGAGTACTGCATGGCAGTGTGCG
739:

GATCCGCACACTGCCATGCAGTACTCTTCAGTGTCAAAGTTGTTACG
GTTGC
CGTAGCAACCGCCGTAAAAGAATGGAGCGCACTTACCTTCAGTGAC
GT
The annealed oligonucleotides were ligated into the AatI and BamHI-digested pDD131, and the ligation product was used to transform E. coli strain MC1061 to ampicillin resistance. The resulting plasmid pDD134 encodes the 445 by synthetic gene for the yeast x-factor-KPI (-4-~57; M1SL, I16F, S17K, G37I~ fusion.
AA. Corrstraction of pDD135 KPI ( 4-~57; MI SL,116F, SI7K, G37L) Plasmid pDD131 was digested with AatII and BamHI, and the larger of the two resulting fragments was isolated. An oligonucleotide pair (738 + 739) was phosphorylated, annealed and gel-purified as described previously.
738:
CACTGAAGGTAAGTGCGCTCCATTCTTTZ'ACGGCGGTTGCTACGGCA
ACCGT
AACAACTTTGACACTGAAGAGTACTGCATGGCAGTGTGCG
739:
GATCCGCACACTGCCATGCAGTACTCTTCAGTGTCAAAGTTGTTACG
GTTGC
CGTAGCAACCGCCGTAAAAGAATGGAGCGCACTTACCTTCAGTGAC
GT
The annealed oligonucleoddes were ligated into the AatII and BamHI-digested pDD131, and the ligation product was used to transform E. coli strain MC1061 to ampicillin resistance. The resulting plasmid pDD135 encodes the 445 by synthetic gene for the yeast x-factor-KPI (-4-~57; M 15L, I16F, S 17K, G37L) fusion.
Example 4. Kinetic analysis of Kpi(-4-~5'n variants The concentrations of active human plasma kallikrein, factor 3QIa, and trypsin were determined by titration with p-nitrophenyl p'-guanidinobenzoate as described by Bender et ai., supra, and Chase et al., Biochem. Biophys. Res. Commun. 29:508 (1967).
Accurate concentrations of active KPI(-4--~S7) inhibitors were determined by titration of the activity of a known amount of active-site-titrated trypsin. For testing against kallikrein and trypsin, each KPI(-4-rS7) variant (0.5 to 100nM) was incubated with S protease in low-binding 96-well microtiter plates at 30°C for 1 S-2S
min, in 100mM Tris-HCI, pH 7.5, with SOOmM NaCI, SmM KCI, SmM CaCl2, SmM MgCl2, 0.1% Difco gelatin, and 0.05% Triton X-100. Chromogenic synthetic substrate was then be added, and initial rates at 30°C recorded by the SOFTmax kinetics program via a T'1-iERMOmax microplate reader (Molecular Devices Corp., Menlo Park, CA). The substrates used were N-a-benzoyl-L-Arg p-nitroanilide (lrriM) for trypsin (20nM), and N-benzoyl-Pro-Phe-Arg p-nitroanilide (0.3rnM) for plasma kallikrein (1nM). The Enzfitter (Elsevier) program was used both to plot fractional activity (i.e., activity with inhibitor, divided by activity without inhibitor), a, versus total concentration of inhibitor, I,, and to calculate the dissociation constant of the inhibitor (K;) by fitting the curve to 1 S the following equarion:
a=I-!E>~+!I J,+K.- flElr+!I J.+Kil'-4IEJ.lII~
The K;s determined for purified KPI variants are shown in Figures 63 and 69.
The most potent variant, KPI (-4-~57; M1SA, S 17F) DD185 is 11 S-fold more potent as a human kallikrein inhibitor than wild-type KPI (-4-~S7). The least potent variant, KPI
(-4--~S7; I16H, S 17V1~ TW618S is still 3S-fold more potent than wild-type KPI.
Replacement of arginine at position 18 of the native KPI peptide with histidine (R18H) in combination with one or more additional substitutions at residues 9, 15 and 17 of the native KPI peptide also exhibited potent and specific serine protease inhibition toward selected serine proteases of interest than the native KPI peptide. In particular, the 2S specific substitutions T9V, M1SA, S17Y and M1SA ,S17Y in the context of the substitution exhibited such potent serine protease inhibition. See Figures 63 and 64.
Substituting the native tyrosine at position 48 in these R18H substituted peptides with histidine (pBG022, SOD4, SOB6; Y48H), glutamine (SOB6, SOL1, SOM1; Y48~, alanine (SOPS, SOC4; Y48A) or aspartic acid (SON1; Y48D) produced a significant SS

increase in their level of expression in comparison to the R18H substituted peptides without the position 48 substitution. See Figures 69B, E and F.
For testing against factor XIIa, essentially the same reaction conditibns were used, except that the substrate was N-benzoyl-Ile-GIu-Gly-Arg p-nitroaniline hydrochloride and its methyl ester (obtained from Pharmacia Hepar, Franklin, OH), and corn trypsin inhibitor (Enzyme Research Laboratories, South Bend, IN) was used as a control inhibitor. Factor XTIa was also obtained from Enzyme Research Laboratories.
Various data for inhibition of the serine proteases of interest kallikrein, plasmin, and factors Xa, XIa, and XIIa by a series of ICPI variants are given in Figure 64. The results indicate that KPI variants can be produced that can bind to and preferably inhibit the activity ~f serine proteases. The results also indicate that the peptides of the invention may exhibit the preferable more potent and specific inhibition of one or morn serene proteases of interest.
Example 5. Effect of KPI variant KPI185-1 on postoperative bleeding A randomized, double-blinded study using an acute porcine cardiopulmonary bypass (CPB) model was used to investigate the effect of ICPI185-1 on postoperative bleeding. Sixteen pigs (55-65 kg) underwent 60 minutes of hypothermic (28°C) open-chest CPB with 30 minutes of cardioplegic cardiac arrest. Pigs were randomized against a control solution of physiological saline (NS; n=8) or ICPI-I85 (n=8) groups.
Duriag aortic cross-clamping, the tricuspid valve was inspected through an atriotomy which was subsequently repaired. Following reversal of heparin with protamine, dilateral thoracostomy tubes were placed and shed blood collected for 3 hours. Shed blood volume and hemoglobin (Hgb) loss were calculated from total chest tube output and residual intrathoracic blood at time of sacrifice.
Total blood loss was significantly reduced in the ICPI185-1 group (245.75 ~
66.24 ml vs. 344.25 ~ 63.97 ml, p~.009). In addition, there was a marked reduction in total Hgb loss in the treatment group (13.59 ~ 4.26 gm vs. 23.61 ~ 4.69 gm, p~.0005).
Thoracostomy drainage Hgb was significantly increased at 30 and 60 minutes in the control group [6.89 + 1.44 vs. 4.41 + 1.45 gm/dl (p~.004) and 7.6 ~ 1.03 vs.
5.26 ~
1.04 gm/dl (p=0.0002), respectively]. Preoperative and post-CPB hematocrits were not statistically different between the groups. These results are shown in graphical form in Figures 65-68.

Example 6. Effect of KPI Variant hPI-BG022 on Transplant Rejection ICPI-BG022 was tested for its ability to delay transplant rejection in a tat model of acute xenograft rejection. Xenotransplantation of vascularized organs between discordant species results in hyperacute graft rejection within minutes to hours after graft reperfusion. Cardiac xenografts from male Hartley guinea pigs were hetemtopically grafted into male rats that were complement deficient.
Experimental animals received 5 mg/kg ICPI-BG022 N prior to reperfusion, and control animals received saline placebo. The data in Figure 70 demonstrate that a single ICPI-dose significantly prolongs survival of guinea pig hearts grafted into complement-deficient rats.
Example 7. Effect of KPI Variant KPI-BG022 on Ulcerative Colitis KPI-BG022 was tested in a rat model of TNBS (trinitrobenzene sulfonic acid) induced colitis. Animals were subjected to intracolonic instillation of TNBS
to induce inflammation and ulceration. Tail-vein injection of ICPI or vehicle was begun at the time of TNBS infusion and continued with three different dosing regimens:
twice daily injections for 7 days; once daily injections for 7 days; and, two injections only in the day following injury. In each treatment group, half of the animals were sacrificed and scored for colonic injury 8 days following injury, and the remaining animals were sacrificed at 14 days. There were no significant differences in damage scores between saline or ICPI treated animals sacrificed 8 days following injury. As shown in Figure 71, in all three dosing groups there was a significant reduction in damage in ICPI-treated animals at 14 days after injury. Even the animals receiving only three doses of ICPI in the 24 hours following injury showed significant reduction in colonic damage two weeks after the TNBS instillation.
Example $. Effect of KPI Variant KPI-BG022 on Postoperative Bleeding ICPI-BG022 will be tested in an ovine model of cardiopulmonary bypass-associated pulmonary pathophysiology and blood loss and conducted as described in Friedman, M., Sellke, F.W., Wang, S.Y., Weintraub, R.M., and Johnson, R.G.
(1994) Circulation 90: II262-II268; Friedman, M., Wang, S.Y., Sellke, F.W., Cohn, W.E., Weintraub, R.M., and Johnson, R.G. ( 1996) J. Thorac. Cardiovasc. Surg. lll:

468) with modifications as follows:

Surgical procedures:
Dorset-Rambouillet sheep (n=10 in each group) weighing 25-30 kg each will be anesthetized with intravenous 80 mg/kg alpha-chlorarose and S00 mg/kg urethane.
Animals will be intubated and mechanically ventilated (Harvard Apparatus).
Arterial blood gas and pH measurements will be performed during the procedure (pH blood gas analyzer 1306, Instruments Lab, Lexington, MA) and alpha-stat pH
management will be used.during CPB. Systemic arterial pressure will be continuously monitored afrer direct cannulation of the femoral artery, and a separate femoral artery cannula will be used for blood collection. A jugular vein cannula will be used for drug administration. Lymph fluid will be collected from the lungs as follows: the efferent duct of the caudal mediastinal lymph node will be cannulated through a right thoracotomy in the fiRh intercostal space using a silicone, heparin-coated catheter.
CPB preparation:
A midline sternotomy will be performed and the pulmonary artery (PA) isolated and surrounded with an ultrasonic flowmeter (Transonic System, Ithaca, N~.
Animals will be heparinized to achieve an activated clotting time (ACT) > 750 seconds as monitored using a Hemochron device. At the end of CPB the heparin will be reversed with protamine sulphate to baseline ACT. A catheter will be inserted into the left atrium (LA) for blood withdrawl and pressure recording, and the PA
will be cannulated for continuous pressure monitoring. Venous drainage will be provided by a cannula in the right atrium (R.A) and an aortic perfusion catheter will be placed in the aorta. The extracorporeal circuit will consist of a roller pump (Cardiovascular Instruments, Wakef eld, MA) and bubble oxygenator (Bently Bio-2, Baxter Health Care). The circuit will be primed with 1 l lactated Ringer's solution.
Myocardial protection will be provided by antegrade cold blood cardioplegia at 4°C using a 4:1 ratio of autologous blood to crystalloid cardioplegia (KCI 60 meq, mannitoi 12.5 g, citrate-phosphate-dextrose solution 50 mL, THAM 10 meq, 5%
dextrose and saline 0.225% QS). Iced slush will be used for topicial cooling to augment the cardioplegia. Immediately after application of the aortic cross-clamp cardioplegia will be given until arrest of the heart and then reinfused every minutes. With institution of CPB all animals will be cooled to a core temperature of 27°C. After a mean time of SC minutes, rewarming will be commenced approximately 10 minutes before removal of aortic cross-clamp to achieve a core temperature of 37°C at the termination of bypass. Flow will be maintained to keep aortic mean pressure not less than 40 mm/Iig. Norepinephrine bitartrate injection will be given through the CVP line to all animals after termination of CPB with an incrementaily decreasing infusion rate until the infusion is stopped one hour post-CPB.
Physiologic and biochemical determinations:
Hemodyamic measurements will be made before institution of CPB (baseline), every 30 minutes during bypass and every 15 minutes for the first hour after termination of CPB. Thereafter measurements will be made every 30 minutes for hours. Cardiac output will be determined as pulmonary artery flow (Qpa in L/min) or, during CPB, as pump flow. Cardiac index (CI), systemic vascular index (SVRI), pulmonary vascular resistance index (PVRI), will be calculated by standard equations.
Simultaneous with the hemodynamic measurements, 2 ml blood samples will be collected from left and right artria and placed into ice-cooled EDTA tubes.
Hematocrit, blood gases, and oxygen content will be measwed for each sample.
After blood is centrifuged, supernatant platelet. counts and white blood cell counts will be performed.
Lymph collection and measurements:
Lymph volume will be measured and the protein content detemtined. ~ Lymph protein clearance will be calculated as milliliters lung lymph flow per 30 minutes x lymph:plasma protein ratio. Protein clearance is considered reflective of the degree to which larger molecules leak into the lymph, as an indication of damage greater than that seen with lymph fluid flow alone.
Study protocol:
A double-blind study will be performed. Sheep will be randomized to 3 groups of 10 animals each: Group 1 = saline control; Group 2 = ICPI-BG022 dose 1;
and, Group 3 = ICPI-BG022 dose 2. Vehicle and KPI-BG022 will be formulated and aliquoted into coded tubes such that after anesthesia each animal wiii receive a loading dose of 100 ml, a 100 ml pump prime and a 25 ml/hr infusion during the course of CPB. We propose to test two total doses of the KPI-BG022 variant: 5 mg/kg, and 0.5 mg/kg. Therefore, Group 2 will receive a 70 mg loading dose of ICPI-BG022, a 70 mg pump prime and 18 mg/hr infusion. Group 3 will receive a 7 mg loading dose, a 7 mg pump prime and 1.8 mglhr infusion..
Total, non-pulsatile hypothermic CPB will be continued for 90 minutes with a cross-clamp time of 1 hour. Rewarming will start 10 minutes before removal of the cross-clamp and will be continued until a core temperature of 37°C is attained. CPB
will be terminated when core temperature has stabilized at 37°C. Post-CPB
monitoring will continue for 3 hours. Protamine will be given in the first 30 minutes post-CPB, and when ACT has been reduced to baseline levels the chest will be closed with a large-bore thoracostomy tube left in place for drainage.
Blood and hemoglobin loss measurements:
The thoracostomy tube will be connected to a drainage system and suction applied at a force of 10 lcPa. Drain losses will be collected for a total of two hours post-CPB, and then the sternotomy wound wilt be reopened and all shed blood will be aspirated from the thorax and pericardium. The volume of blood loss and hemoglobin will be measured and used to calculate the total hemoglobin loss in grams.
Based on previous experience with this (Friedman et al., 1994; Friedman et al., 1996) model the control group should demonstrate several parameters of pulmonary injury, including increases in: pulmonary vascular resistance (PVR) (170% increase reported), pulmonary lymph flow (233% reported), and lung water (15% reported). An increase in sequestration of WBCs and platelets in the lung should be seen in the control group. Arterial oxygenation (PaOZ) should fall significantly upon cessation of CPB with a gradual recovery in the post-bypass period.
With respect to blood and hemoglobin loss in the post-bypass period, our experience with KPI-wt in another sheep model of CPB (Ohri et al, Ann. Thorac.
Surg. 1996 Apr;61(4):1223-1230) leads us to anticipate collection of 200-400 mI
blood in chest drains in the control group, containing 10-20 g hemoglobin. In that study, recombinant ICPI was assessed in an ovine model of CPB as a hemostatic agent after CPB. Sheep (n = 22) underwent CPB for 90 minutes. Two thoracic drains were sited and drain losses collected for a period of 3 hours after CPB. Wounds were subjectively assessed before closure for "dryness" using a visual analogue scale.
Sheep were randorr~ized to control (n = 8), aprotinin (n = 8), and rKPI (n =
6) groups.
Control animals had a drain loss of 409.4 +/- 39.4 mL/3 h, compared with 131.3 +/-20.3 ml,/3 h for the aprotinin group and 163.7 +/_ 34.3 mL3 h for the rICPI
group (p =
0.16). Hemoglobin loss was 11.6 +/- 3.6, 6.02 +/- 2.1, and 4.6 +.~- 1.2 g/3 h for the control, rKPI, and aprotinin groups respectively (p = 0.25). The subjective analysis of the wounds at the end of CPB found aprotinin (1.25 +/- 0.16; p < 0.05) and rKPI (I.17 +/- 0.17; p < 0.05) animals to score significantly lower than control animals (2.63 +/-0.42).
Indeed, significant reductions in blood and hemoglobin Ioss in the KPI-BG022 treated groups are expected. Measureable reductions in one or more of the other parameters of post-bypass pulmonary injury are also expected in the KPI-BG022 treated group. Positive results in this regard would include smaller increases or no increases in PVR, pulmonary lymph flow or lung water content in the KPI-treated groups, as well as reduced WBCs and platelets sequestered in the lungs. One significant indication of improved pulmonary function in the KPI groups would be improved arterial oxygenation in the immediate post-bypass period.
The invention has been disclosed broadly and illustrated in reference to representative embodiments described above. Those skilled in the art will recognize that various modifications can be made to the present invention without departing from the spirit and scope thereof.

Claims (126)

What Is Claimed Is:

1. A protease inhibitor comprising the sequence:
X1-Val-Cys-Ser-Glu-Gln-Ala-Glu-X2-Gly-X3-Cys-Arg-Ala-X4-X5-X6-X7-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala Pro-Phe-X4-Tyr-Gly-Gly-Cy8-X9-X10-X11-X12-Aan-Asn-Phe-Asp-Thr-Glu-Glu-X13-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile, wherein:
X1 is selected from Glu-Val-Val-Arg-Glu-, Asp-Yal-Val-Arg-Glu-, Asp, and Glu;
X2 is selected from Thr, Val, Ile and Ser;
X3 is selected from Pro and Ala;
X4 is selected from Arg, Ala, Leu, Gly, and Met;
X5 is selected from Ile, His, Leu, Lys, Ala, and Phe;
X6 is selected from Ser, Ile, Pro, Phe, Tyr, Trp, Asn, Leu, His, Lys, and Glu;
X7 is selected from Arg, His, and Ala;
X8 is selected froth Phe, Val, Leu, and Gly;
X9 is selected from Gly, Ala, Lys, Pro, Arg, Leu, Met, and Tyr;
X10 is selected from Ala, Arg, and Gly;
X11 is selected from Lys, Ala, and Asn;
X12 is selected from Ser, Ala, and Arg;
X13 is selected from His, Gln, Ala, and Asp.

2. A protease inhibitor according to claim 1, wherein X1 is Asp-Val-Val-Arg-Glu-, X2 is Thr, Val, or Ser, X3 is Pro, X4 is Ala or Met, X5 is Ile, X6 is Ser or Tyr, X7 is His, X8 is Phe, X9 is Gly, X10 is Gly, X11 is Asn, and X12 is Arg.

3. A protease inhibitor according to claim 1, wherein X1 is Asp-YaI-Val-Arg-Glu-, X2 is Thr, X3 is Pro, X4 is Ala, X5 is Ile, X6 is Phe, X7 is Arg, X8 is Phe, X9 is Gly, X10 is Gly, X11 is Asn, and X12 is Arg.

4. A protease inhibitor according to claim 2, wherein X2 is Thr or Val.

5. A protease inhibitor according to claim 4, wherein X2 is Thr.

6. A protease inhibitor according to claim 4, whGrean X2 is Val.

7. A protease inhibitor according to claim 2, wherein X2 is Thr or Val, and X4 is Ala.

8. A protease inhibitor according to claim 2, wherein X2 is Thr or Val, and X4 is Met.

9. A protease inhibitor according to claim 2, wherein X2 is Thr, X4 is Ala, X6 is Tyr, and X1~ is His.

10. A protease inhibitor according to claim 2, wherein X2 is Thr, X4 is Ala, X6 is Tyr, and X1~ is Gln.

11. A protease inhibitor according to claim 2, wherein X2 is Thr, X4 is Ala, X6 is Tyr, and X1~ is Ala.

12. A protease inhibitor according to claim 2, wherein X2 is Thr, X4 is Ala, X6 is Tyr, and X1~ is Asp.

13. A protease inhibitor according to claim 2, wherein X2 is Thr, X4 is Met, X6 is Ser, and X1~ is selected from His, Ala, or Gln.

14. A protease inhibitor according to claim 2, wherein X2 is Val, X4 is Ala, X6 is Tyr, and X1~ is selected from His, Ala, or Gln.

15. A protease inhibitor according to claim 2, wherein X is Thr, X4 is Ala, X6 is Tyr, and X1~ is selected from His, Ala, or Gln.

16. A protease inhibitor according to claim 14, wherein X13 is selected from His or Ala.

17. A protease inhibitor according to claim 15, wherein X13 is selected from His or Ala.

18. A protease inhibitor according to claim 16, wherein X13 is His.

19. A protease inhibitor according to claim 16, wherein X13 is Ala.

20. An isolated DNA molecule comprising a DNA sequence encoding a protease inhibitor according to claim 1.

21. An isolated DNA molecule according to claim 20, operably linked to a regulatory sequence that controls expression of the coding sequence in a host cell.

22. An isolated DNA molecule according to claim 21, further comprising a DNA sequence encoding a secretory signal peptide.

23. An isolated DNA molecule according to claim 22, wherein said secretory signal peptide comprises the signal sequence of yeast ~-mating factor.

24. A host cell transformed with a DNA molecule according to claim 20.

25. A host cell according to claim 24, wherein said host cell is E. coli or a yeast cell.

26. A host cell according to claim 25, wherein said host cell is a yeast cell.

27. A host cell according to claim 26, wherein said yeast cell is Saccharomyces cerevisiae.

28. A host cell according to claim 26, wherein said yeast cell is Pichia pastoris,

29. A method for producing a protease inhibitor, comprising the steps of culturing a host cell according to claim 24 and isolating and purifying said protease inhibitor.

30. A pharmaceutical composition, comprising a protease inhibitor according to claim 1, together with a pharmaceutically acceptable sterile vehicle.

31. A method of treatment of a clinical condition associated with increased activity of one or more serine proteases, comprising administering to a patient suffering from said clinical condition an effective amount of a pharmaceutical composition according to claim 30.

32. The method of treatment of claim 31, Wherein said clinical condition is blood loss during surgery.

33. A method for inhibiting the activity of serine proteases of interest in a mammal comprising administering a therapeutically effective dose of a pharmaceutical composition according to claim 30.

34. The method of claim 33, wherein said serine proteases are selected from the group consisting of: kallikrein; chymotrypsins A and B; trypsin; elastase;
subtilisin;
coagulants and procoagulants, particularly those in active form, including coagulation factors such as factors VIIa, IXa, Xa, XIa, and XIIa; plasmin; thrombin;
proteinase-3;
enterokinase; acrosin; cathepsin; urokinase; and tissue plasminogen activator.

35. A protease inhibitor comprising the sequence:

X1-Val-Cys-Ser-Glu-Gln-Ala-Glu-X2-Gly-Pro-Cys-Arg-Ala-Ala-Ile-Tyr-His-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-Gly-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-X~-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile, wherein:

X1 is selected from Glu-Val-Val-Arg-Glu-, Asp-Val-Val-Arg-Glu-, Asp, or Glu;
X2 is selected from Thr and Val;
X3 is selected from His, Gln, Ala, or Asp.

36. A protease inhibitor according to claim 35, wherein X1 is Glu-Val-Val-Arg-Glu.

37. A protease inhibitor according to claim 36, wherein X2 is Thr.

38. A protease inhibitor according to claim 36, wherein X2 is Val.

39. A protease inhibitor according to claim 38, wherein X~ is His,

40. A protease inhibitor according to claim 38, wherein X~ is Gln.

41. A protease inhibitor according to claim 38, wherein X~ is Ala.

42. A protease inhibitor according to claim 38, wherein X~ is Asp.

43. A protease inhibitor according to claim 35, wherein X1 is Asp-Val-Val-Arg-Glu.

44. A protease inhibitor according to claim 43, wherein X2 is Thr.

45. A protease inhibitor according to claim 43, wherein X2 is Val.

46. A protease inhibitor according to claim 45, wherein X~ is His.

47. A protease inhibitor according to claim 45, wherein X~ is Gln.

48. A protease inhibitor according to claim 45, wherein X~ is Ala.

49. A protease inhibitor according to claim 45, wherein X~ is Asp.

50. A protease inhibitor according to claim 35, wherein X1 is Glu.

51. A protease inhibitor according to claim 50, wherein X2 is Thr.

52. A protease inhibitor according to claim 50, wherein X2 is Val.

53. A protease inhibitor according to claim 52, wherein X3 is His.

54. A protease inhibitor according to claim 52, wherein X~ is Gln.

55. A protease inhibitor according to claim 52, wherein X~ is Ala.

56. A protease inhibitor according to claim 52, wherein X~ is Asp.

57. A protease inhibitor according to claim 35, wherein X~ is Asp.

58. A protease inhibitor according to claim 57, wherein X2 is Thr.

59. A protease inhibitor according to claim 57, wherein X2 is Val.

60. A protease inhibitor according to claim 59, wherein X~ is His,

61. A protease inhibitor according to clam 59, wherein X~ is Gln.

52. A protease inhibitor according to claim 59, wherein X~ is Ala.

63. A protease inhibitor according to claim 59, wherein X~ is Asp.

64. A protease inhibitor according to claim 1, wherein X1 is Glu-Val-Val-Arg-Glu-, X2 is Thr, Val, or Ser, X~ is Pro, X4 is Ala or Met, X5 is Ile, X6 is Ser or Tyr, X7 is His, X~ is Phe, X9 is Gly, X10 is Gly, X11 is Asn, and X12 is Arg.

65. A protease inhibitor according to claim 64, wherein X2 is Thr or Val.

66. A protease inhibitor according to claim 65, wherein X2 is Thr.

67. A protease inhibitor according to claim 65, wherein X2 is Val.

68. A protease inhibitor according to claim 64, wherein X2 is Thr or Val, and is Ala.

69. A protease inhibitor according to claim 64, wherein X2 is Thr or Val, and is Met.

70. A protease inhibitor according to claim 64, wherein X2 is Thr, X4 is Ala, X~
is Tyr, and X1~ is His.

71. A protease inhibitor according to claim 64, wherein X2 is Thr, X4 is Ala, is Tyr, and X1~ is Gln.

72. A protease inhibitor according to claim 64, wherein X2 is Thr, X4 is Ala, is Tyr, and X1~ is Ala.

73. A protease inhibitor according to claim 64, wherein X2 is Thr, X4 is Ala, is Tyr, and X1~ is Asp.

74. A protease inhibitor according to claim 64, wherein X2 is Thr, X4 is Met, is Ser, and X1~ is selected from His, Ala, or Gln.

75. A protease inhibitor according to claim 64, wherein X2 is Val, X4 is Ala, is Tyr, and X1~ is selected from His, Ala, or Gln.

76. A protease inhibitor according to claim 64, wherein X2 is Thr, X4 is Ala, is Tyr, and X13 is selected from His, Ala, or Gln.

77. A protease inhibitor according to claim 75, wherein X1~ is selected from His or Ala.

78. A protease inhibitor according to claim 76, wherein X1~ is selected from His or Ala.

79. A protease inhibitor according to claim 77, wherein X1~ is His.

80. A protease inhibitor according to claim 77, wherein X1~ is Ala.

81. An isolated DNA, molecule comprising a DNA sequence encoding a protease inhibitor according to claim 18.

82. An isolated DNA molecule according to claim 81, operably linked to a regulatory sequence that controls expression of the coding sequence in a host cell.

83. An isolated DNA molecule according to claim 82, further comprising a DNA sequence encoding a secretory signal peptide.

84. An isolated DNA molecule according to claim 83, wherein said secretory signal peptide comprises the signal sequence of yeast ~-mating factor.

85. A host cell transformed with a DNA mole-u1c according to claim 81.

86. A host cell according to claim 85, wherein said host cell is E. coli or a yeast cell.

87. A host cell according to claim 86, wherein said host cell is a yeast cell.

88. A host cell according to claim 87, wherein said yeast cell is Saccharomyces cerevisiae.

89. A host cell according to claim 87, wherein said yeast cell is Pichia pastoris.

9a. A method for producing a protease inhibitor, comprising the steps of culturing a host cell according to claim 85 and isolating and purifying said protease inhibitor.

91. A pharmaceutical composition, comprising a protease inhibitor according to claim 18, together with a pharmaceutically acceptable sterile vehicle.

92. A method of treatment of a clinical condition associated with increased activity of one or more serine proteases, comprising administering to a patient suffering from said clinical condition an effective amount of a pharmaceutical composition according to claim 91.

93. The method of treatment of claim 92, wherein said clinical condition is blood loss during surgery.

94. A method for inhibiting the activity of serine proteases of interest in a mammal comprising administering a therapeutically effective dose of a pharmaceutical composition according to claim 91.

95. The method of claim 94, wherein said serine proteases are selected from the group consisting of: kallikrein; chymotrypsins A and B; trypsin; elastase;
subtilisin;
coagulants and procoagulants, particularly those in active form, including coagulation factors such as factors VIIa, IXa, Xa, XIa, and XIIa; plasmin; thrombin;
proteinase-3;
enterokinase; acrosin; cathepsin; urokinase; and tissue plasminogen activator.

96. A method for increasing the expression levels of recombinant protease inhibitors comprising the step of culturing a host call transformed with an isolated DNA
molecule comprising a DNA sequence encoding a protease inhibitor according to claim 1.

97. The method according to claim 96, wherein said host cell is E. coli or a yeast cell.

98. The method according to claim 97, wherein said host cell is a yeast cell.

99. The method according to claim 98, wherein said yeast cell is Saccharomyces cerevisiae.

140. The method according to claim 98, wherein said yeast cell is Pichia pastoris.

101. A method for increasing the yield of recombinant protease inhibitors comprising the step of culturing a host cell transformed with an isolated DNA
molecule comprising a DNA sequence encoding a protease inhibitor according to claim 1, wherein X1 is Asp-Val-Val-Arg-Glu-, and isolating and purifying said protease inhibitor.

102. The method according to claim 101, wherein said host cell is a yeast cell.

103. The method according to claim 102, wherein said yeast cell is Saccharomyces cerevisiae.

144. The method according to claim 102, wherein said yeast cell is Pichia pastoris.

105. A protease inhibitor comprising the sequence:

X1-Val-Cys-Ser-Glu-Gln-Ala-Glu-X2-Gly-Pro-Cys-Arg-Ala-X~-Ile-X4-X5-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-Gly-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-X6-Cys-Met-Ala-Val-Cys-Gly-Ser-Ala-Ile, wherein;
X1 is selected from Glu-Val-Val-Arg-Glu-, Asp-Val-Val-Arg-Glu-, Asp, or Glu;
X2 is selected from Thr or Val;
X3 is selected from Arg and Met;
X4 is selected from Ser and Tyr;
X5 is selected from Arg, His, or Ala; and X6 is selected from His, Gln, Ala or Asp.

106. A protease inhibitor comprising the sequence;
X1-Val-Cys-Ser-Glu-Gln-Ala-Glu-Thr-Gly-Pro-Cys-Arg-Ala-Leu-Phe-Lys-Arg-Trp-Tyr-Phe-Asp-Val-Thr-Glu-Gly-Lys-Cys-Ala-Pro-Phe-Phe-Tyr-Gly-Gly-Cys-Leu-Gly-Asn-Arg-Asn-Asn-Phe-Asp-Thr-Glu-Glu-X2-Cys-Met-Ala-Val-cys-Gly-Ser-Ala-Ile, wherein:
X1 is selected from Glu-Val-Val-Arg-Glu-, Asp-Val-Val-Arg-Glu-, Asp, and Glu;
X2 is selected from His, Gln, Ala, and Asp.

147. A protease inhibitor according to claim 106, wherein X1 is Asp-Val-Val-Arg-Glu.

108. A protease inhibitor according to claim 107, wherein X2 is His.

109. A protease inhibitor according to claim 107, wherein X2 is Gln.

110. A protease inhibitor according to claim 107, wherein X2 is Ala.

111. A protease inhibitor according to claim 107, wherein X2 is Asp.

112. A protease inhibitor according to claim 106, wherein X1 is Glu-Val-Val-Arg-Glu.

113. A protease inhibitor according to claim 112, wherein X2 is His.

114. A protease inhibitor according to claim 112, wherein X2 is Gln.

115. A protease inhibitor according to claim 112, wherein X2 is Ala.

116. A protease inhibitor according to claim 112, wherein X2 is Asp.

117. A protease inhibitor according to claim 106, wherein X1 is Asp.

118. A protease inhibitor according to claim 117, wherein X2 is His.

119. A protease inhibitor according to claim 117, wherein X2 is Gln.

120. A protease inhibitor according to claim 117, wherein X2 is Ala.

121. A protease inhibitor according to claim 117, wherein X2 is Asp.

122. A protease inhibitor according to claim 106, wherein X1 is Glu.

123. A protease inhibitor according to claim 122, wherein X2 is His.

124. A protease inhibitor according to claim 122, wherein X2 is Gln.

125. A protease inhibitor according to claim 122, wherein X2 is Ala.

126. A protease inhibitor according to claim 122, wherein X2 is Asp.