Inhibitors of human neutrophil elastase

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DESCRIPTION

Inhibitors of human neutrophil elastase and human cathepsin G.

Field of the invention

The present invention relates to novel protease inhibitors and in particular to small engineered proteins that inhibit human neutrophil elastase (hNE) and to proteins that inhibit human cathepsin G (hCG).

Description of the background of the technique

Neutrophil elastase and cathepsin G

The active sites of serine proteases are very similar. Trypsin, chymotrypsin, neutrophil elastase, cathepsin G, and many other proteases share significant sequence homology. The so-called catalytic triad comprises (with the standard chymotrypsinogen numbering) aspartic acid-102, histidine57 and serine-195. The residues close to the catalytic triad determine the substrate specificity of each particular enzyme (compare CREI84, p. 366-7). The structure and function of the digestive enzymes trypsin, pancreatic elastase, and chymotrypsin have been studied in more detail than for neutrophil enzymes. The x-ray structures of hNE have been resolved into a complex with a substrate and the similarity of the active site of NE to that of trypsin is very high. The specificity of hNE is greater than that of trypsin and less than that of Factor Xa.

Serine proteases are ubiquitous in living beings and have crucial functions in processes such as: digestion, blood coagulation, fibrinolysis, immune response, fertilization, and the post-translational process of peptide hormones. Although the functions of these enzymes are crucial, uncontrolled or inappropriate proteolytic activity can be very damaging. Several serine proteases are directly implicated in serious diseases.

Human neutrophil elastase (hNE or HLE; EC 3.4.21.11) is a 29 kDa serum protease that has broad spectrum activity against extracellular matrix components (CAMP82, CAMP88, MCWH89 and references cited therein). The enzyme is one of the main neutral proteases of the azurophilic grains of polymorphonuclear leukocytes and is involved in the elimination of pathogens and the restructuring of connective tissue (TRAV88). In cases where there is an inherited reduction in the concentration of alpha-1-anti-protease inhibitor (α1 -PI), the main physiological inhibitor of hNE (HEID86) or inactivation of α1 -PI occurs by oxidation ("Smoker's emphysema"), considerable destruction of lung tissue can occur due to uncontrolled elastolytic activity of hNE (CANT89, BEIT86, HUBB86, HUBB89a, b, HUTC87, SOMM90, WEWE87). There are

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various human respiratory disorders, for example cystic fibrosis and emphysema, characterized by increased neutrophil load on the epithelial surface of the lungs (SNID91, MCEL91, GOLD86) and the release of hNE by neutrophils is has been associated with the progression of these disorders (MCEL91, WEIS89). HNE is involved as an essential ingredient in the pernicious cycle of:

seen in cystic fibrosis (CF) (NADE9O). Inappropriate hNE activity is very harmful and an inhibitor with a very high affinity is needed to stop the progression of emphysema or to alleviate the symptoms of CF. The inhibitor has to be very specific for hNE as it could inhibit other vital proteases or esterases. Nadel (NADE90) has suggested that the onset of excessive secretion is triggered with 10^-10 M hNE, so the inhibitor should reduce the free hNE concentration well below this level. So hNE is an enzyme for which you need an excellent inhibitor.

There are reports suggesting that proteinase 3 (also called p29 or PR-3) is as important or even more important than hNE; see NILE89, ARNA9O, KAOR88, CAMP90 and GUPT9O. Cathepsin G is another protease produced by neutrophils that can cause disease when present in excess; see FERR90, PETE89, SALV87 and S0MM90.

Cathepsin G is less stable than hNE and therefore more difficult to study in vitro. Powers and Harper (POWE86) indicate that cathepsin G is involved in inflammation, emphysema, acute adult breathlessness syndrome, and rheumatoid arthritis.

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Protein inhibitors of serine proteases

There are a large number of proteins that act as serine protease inhibitors as they serve as highly specific substrates but give very limited proteolysis in the enzymes they inhibit. In many cases, the reactive site peptide bond ("cleavable bond") is encompassed within at least one disulfide loop, ensuring that during conversion from intact to modified inhibitor the two peptide chains cannot dissociate.

A special nomenclature has been generated to describe the active site of the inhibitor. It begins at the residue on the amino side of the cleavable bond and, moving away from the bond, the residues are called P1, P2, P3, etc. (SCHE67). The residues that follow the cleavable bond are called P1 ', P2', P3 ', etc. It has been found that the main chain of protein inhibitors, with generally very diverse structures, are very similar in the region between P3 and p3 'with particularly marked similarities in P2, P1 and P1' (LASK8O and works cited there). It is generally accepted that each serine protease has S1, S2, etc. sites. that house the side groups of residues P1, P2, etc. of the substrate or inhibitor and sites S1 ', S2', etc. that house the side groups of P1 ', P2', etc. of the substrate or inhibitor (SCHE67). The interactions between the S sites and the P side groups are responsible for the specificity of proteases with respect to their substrates and of inhibitors with respect to proteases.

Serine protease inhibitors have been grouped into families based on similarities both in sequence and in the topological relationship of their active sites and their disulfide loops. Families include bovine pancreatic trypsin inhibitor (Kunitz), bovine pancreatic secretion trypsin inhibitor (Kazal), Bownian-Birk inhibitor, and soybean trypsin inhibitor (Kunitz). . Some inhibitors have multiple reactive sites on a single polypeptide chain, and those different domains can have different sequences, specificities, and even topologies.

One of the most unusual characteristics of these inhibitors is their ability to retain some of their inhibitory activity even when the P1 residue has been replaced. Furthermore, it has been found that amino acid substitution in the region of P₅ to P₅, and more particularly between P3 and P3 ', has a great influence on the specificity of an inhibitor. LASK80 has suggested that within the BPTI family (Kunitz), inhibitors with P1 Lys and Arg tend to inhibit trypsin, those with P1 = Tyr, Phe, Trp, Leu and Met tend to inhibit chymotrypsin and those with P1 = Ala or Ser are capable of inhibiting elastase. Among the Kazal inhibitors, they continue, those with P1 = Leu or Met are strong elastase inhibitors and in the Bowman-Kirk family elastase is inhibited by P1 Ala, but not P1 Leu.

Below we will discuss a particularly interesting set of anti elastase and anti cathepsin G protein inhibitors. Known inhibitors of hNE and cathepsin G include the UTI inhibitor of the Kunitz family (CEBH86), the eglin of the eglin / barley family of inhibitors (SCHN86b), and the serpin alpha-1- family inhibitors. antichymotrypsin and alpha-1-antitrypsin (BARR86).

A inhibitor₁-proteinase (al-antitrypsin)

A logical approach to the treatment of diseases attributable to excessive levels of hNE consists of treatment with the irreversible endogenous inhibitor, a1-PI. STON9O reports on studies of the efficacy of a1-PI in protecting hamster lungs from damage caused by hNE. They conclude that a1-PI has approximately only 16% effectiveness in vivo compared to what could be estimated by in vitro measurements. However, a1-PI has therapeutic effects . A preliminary study of aerosol administration of a1-PI to cystic fibrosis patients indicates that such treatment may be effective both in preventing damage to respiratory tissue and in increasing host antimicrobial defenses (MCEL91).

However, there are practical problems with its routine use as a pulmonary anti-elastolytic agent. Among them are: the relatively large size of the molecule (394 residues, 51 kDa), the absence of intramolecular disulfide bonds that contribute to stability, and post-translational modifications in the protein that involve specific glycosylation at three sites. HEIM91 reports the inhibition of PMN leukocyte-mediated endothelial cell shedding by the use of protease inhibitors. They compared the leukocyte secretion protease inhibitor (SLPI), the a1-protease inhibitor (a1-PI), and a chloromethyl ketone (CMK) inhibitor. While SLPI and CMK inhibited hNE-mediated cell shedding, a1-PI did not; the author suggests that the a1-PI cannot access the site where hNE acts because of its size. Since the inhibitors described in the present invention are smaller than SLPI, we expect them to move freely throughout the extracellular space.

Furthermore, both the hydrolyzed a1-PI and the a1-PI / hNE complex (BAND88a, b) can be neutrophil chemoattractants. This can be a serious disadvantage if you want to interrupt the cycle by which an excessive number of neutrophils migrate to the lung, release hNE, the hNE reacts with a1-PI which in turn generates a signal for more neutrophils to migrate to the lungs. lungs. Thus, a small, stable, non-toxic, and potent hNE inhibitor would have great therapeutic value.

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Trypsin inhibitor of human pancreatic secretion

This is an inhibitor from the Kazal family. Inhibitors of this family are stored in zymogen granules and are secreted with zymogens in pancreatic juice. In general, natural Kazal inhibitors are specific for trypsin. However, there are exceptions, such as some ovomucoid and ovoinhibitor domains that inhibit chymotrypsin, subtilisin, and elastase.

While wild-type hPSTI is completely inactive against hNE, Collins et al. (COLL90) report engineered variants of human pancreatic secretion trypsin inhibitors (hPSTI) that have high affinity for hNE. Three of the reported variants have Ki for hNE below 10 pM: PSTI-5D36 with 7.3 pM, PSTI-4A40 with 7 pM and PSTI-4F21 with 5.2 pM.

Pumpkin seed inhibitor

Pumpkin seed inhibitors are another family of serine protease inhibitors. Those that have been reported so far have lysine or arginine at the P1 residue, inhibit trypsin, and are completely inactive against hNE. McWherter et al. (1989) synthesized several homologues of the pumpkin seed inhibitor,

CMTI-III. CMTI-III has a Ki for trypsin of approximately 1.5 · 10^-12 M. McWherter et al. (MCWH89) suggested the substitution of "moderately bulky hydrophobic groups" in P1 to give it specificity towards HLE (it is the same as hNE). For cathepsin G they expected bulky side groups (especially aromatic ones) to have a strong preference. They found that PHE, LEU, MET and ALA had function according to their criteria, they did not test TRP, TYR or HIS. (Note that ALA has the second smallest amino acid side group) They found that a larger set of substituted residues (VAL, ILE, LEU, ALA, PHE, MET, and GLY) gave detectable hNE binding. In particular, CMTI-III (VAL5) has a Ki = 9 nM relative to hNE.

Protease inhibitors with "Kunitz" domain

Bovine pancreatic trypsin inhibitor (BPTI, also called aprotonin) is a 58 amino acid serine protease inhibitor of the BPTI domain family (Kunitz, KuDom). It is used under the trade name TRASYLOL to counteract the effects of trypsin released during pancreatitis. Not only is the sequence of its 58 amino acids known from BPTI, but its three-dimensional structure has been determined by means of high resolution X-ray diffraction (HUBE77, MARQ83, WLODB4, WLOD87a, WLOD87b), neutron diffraction (WL0D84) and NMR ( WAGN87). One of the X-ray structures is deposited with the Brookhaven Protein Data Bank as "6PTI" [sic]. The three-dimensional structure of several homologues of BPTI (EIGE9O, HYNE90) is also known. At least sixty homologues have been reported, the sequence of 39 of them is shown in Table 13 and the amino acid types that appear at each position are compiled in Table 15. Known human homologues include Inhibitor domains. Lipoprotein Associated Coagulation (LACI) (WUNT88, GIRA89), the Inter-a-trypsin Inhibitor (ALBR83a, ALBR83b, DIAR90, ENGH89, TRIB86, GEBH86, GEBH9O, KAUM86, ODOM90, SALI90) and Alzheimer's beta amyloid precursor protein . Circular BPTI and permuted circular have binding properties similar to BPTI (GOLD83). Some of the BPTI homologous proteins have more or fewer residues at each end.

In BPTI the P1 residue is at position 15. Tschesche et al. (TS0187) reported the binding to various proteases of several P1-modified BPTI derivatives:

Dissociation constants for BPTI derivatives P1, molar

<td>Residue N ° 15 P1</td><td>Trypsin (pancreas bovine)</td><td>Chymotrypsin (pancreas bovine)</td><td>Elastase (pancreas porcine)</td><td colspan="2">Elastase (leukocytes humans)</td>

<td>lysine</td><td> 6,0 · 10^-14</td><td> 9,0 · 10^-9</td><td> -</td><td> 3,5 · 10^-6</td><td>(natural)</td>

<td>wisteria</td><td> -</td><td> -</td><td> +</td><td> 7,0 · 10^-9</td><td></td>

<td>to the girl</td><td> +</td><td> -</td><td> 2,8 · 10^-8</td><td> 2,5 · 10^-9</td><td></td>

<td>valine</td><td> -</td><td> -</td><td> 5,7 · 10^-8</td><td> 1,1 · 10^-10</td><td></td>

<td>leucine</td><td> -</td><td> -</td><td> 1,9 · 10^-8</td><td> 2,9 · 10^-9</td><td></td>

From the report by Tschesche et al. we infer that pairs of molecules marked "+" have Kd =

3,5 · 10^-6 M and that the pairs marked "-" have Kd &gt;&gt; 3.5 · 10^-6 M. It is clear that wild type BPTI has a relatively low affinity for hNE, however, BPTI mutants with higher affinities are known. Although not shown in the Table, BPTI does not bind significantly to hCG. However, Brinkmann and Tschesche (BRIN9O) made a BPTI triple mutant (namely, K15F, R17F, M52E) that has a Ki of 5.010^-7M for hCG.

Among the works related to BPTI and its counterparts are: STAT87, SCHW87, GOLDB3, CHAZ83,

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CREI74, CREI77a, CREI77b, CREI80, SIEK87, SINH90, RUEH73, HUBE74, HUBE75, HUBE77, KIDO88, PONT88, KIDO90, AUER87, AUER90, SCOT87b, AUER88, AUER89, BECK88b, WACHFT85, WACHOR89, BECK88b, WACHFT85, WACHOR89, BECK88b, WACHFT85, WACHOR89 GOLD84, GOLD88, HOCH84, RITO83, NORR89a, NORR89b, OLTEB9, SWAI88 AND WAGN79.

The inter-a-trypsin inhibitor (ITI) is a large protease inhibitor (M_r approximately 240,000) and circulating found in the plasma of many mammalian species (for reviews see ODOM90, SALI90, GEBH90, GEBH86). Its affinity constant for hNE is between 60 and 150 nM and for cathepsin G is between 20 and 6000 nM. The intact inhibitor is a glycoprotein and is currently thought to consist of three glycosylated subunits that interact through a powerful glycosaminoglycan binding (ODOM90, SALI90, ENGH89, SELL87). The antitrypsin activity of ITI is located in the smallest subunit (ITI light chain, unglycosylated Mr approximately 15,000) which is identical in amino acid sequence to an acid-stable inhibitor found in urine (UTI) and serum (STI) (GEBH86, GEBH9O). The mature light chain consists of a 21 residue amino terminal sequence, glycosylated at SER10, followed by two tandem KuDoms, the first of which is glycosylated at ASN45 (ODOM90). In human protein, the second KuDom (ITI-D2 or HI-8t) has been shown to inhibit trypsin, chymotrypsin, and plasmin (ALBR83a, ALBR83b, SELL87, SWAI88). The first domain (ITI-D1 or HI-8e, comprising residues 22 to 76 of the UTI sequence shown in Figure 1 of GEBH86) lacks these activities (ALBR83a, b, SWAI88), but it has been reported which inhibits leukocyte elastase (10^-6 &lt;Ki &lt;10^-9) (ALBR83a, b, ODOM90) and cathepsin G (SWAI88, ODOM90). However, the affinity is too small to be of direct use.

Sinha et al. (S1NH91) report the transformation of the Alzheimer's ^ -amyloid precursor KuDom into an hNE inhibitor with a Ki = 800 pM when they substitute arginine for valine at the P1 site (residue 13). They made a second protein with three mutations (namely, R13V (P1), A14S (P1 '), M15I (P2')). The changes in P1 'and P2' correspond to the amino acids present in the active site of the a₁ -PI. This protein is completely inactive with respect to hNE. These researchers state: “Extrapolation of site-directed mutagenesis results should be done with caution when it comes to inhibitors that have no related mechanisms. Furthermore, unpredictable results can be obtained even within the KuDom family, as illustrated by our experience with chymotrypsin and kallikrein ”

Non-protein elastase inhibitors

Compounds ICI 200,355 (SOMM91) and ICI 200,880 show a marked preference for hNE over other proteases such as trypsin. These compounds are peptide analogs in which the amide nitrogen of the cleavable bond has been replaced by a CF3 group. Each of these compounds has an isopropyl group (such as valine) at the P1 position and a proline residue at P2. Neither compound extends in the direction of P1 '. Imperiali and Abeles (IMPE86) describe protease inhibitors formed by acetyl peptidyl methyl ethyl ketones in which the terminal methyl group has between zero and three fluorine atoms; there is no P1 'residue in any of its compounds. In PEET9O (and references cited there) the synthesis of peptidyl fluoromethyl ketones and peptidyl αketo esters and the inhibitory properties of these compounds towards porcine pancreatic elastase (PPE), hNE, rat cathepsin G and human cathepsin G are reported; these compounds do not extend in the direction of P1 '. Mehdi et al. (MEHD90) report inhibition of HNE and human cathepsin by methyl esters of peptidyl α-keto carboxylic acids; none of these compounds contain P1 'residues. Angelastro et al. (ANGE90) report protease inhibitors with dikete groups; none of those compounds extend beyond P1.

Govhardan and Abeles (GOVH90) describe compounds in which the -NH- of the amide has been replaced with a -CF₂-CH₂- followed by a methyl ester of an amino acid linked by the α-amino in order to provide a residue

P1 '.

Imperiali and Abeles (IMPR87) describe chymotrypsin inhibitors that extend up to P3 '. The works cited by these authors indicate that the inhibition constant, Ki, can be made smaller if residues are used that specifically adjust to the S1 ', S2', S3 ', ... binding sites of the protease. These authors do not discuss HNE inhibition. Also, their inhibitors do not come from high-affinity protease inhibitors, but rather the side groups at P1 ', P2', and P3 'are determined by trial and error. Furthermore, between P1 and P1 ', they insert a -CO-CF2-CH2- group in place of the -CO-NH- group so that the distal portion of the chain is displaced. We prefer to replace the -CO-NH- group with -CO-CF2- or -CO-CFH- so that the rest of the residues can acquire conformations very similar to those found in EpiNE proteins.

Another class of protease inhibitors is one in which the cleavable peptide carbonyl carbon is replaced with boron. These compounds inhibit serine proteases but are not very specific.

Another class of elastase inhibitors are the chloromethyl ketones described by Robert et al. (US 4,665,053). These compounds have a chlorine atom adjacent to the keto group. The serine of the active site of the protease acts as a nucleophile and displaces chloride, which generates an enzyme-inhibitor covalent adduct that is irreversibly inactivated. An-Zhi et al. (FEBS Lett, 234 (2) 367-373 (1988)) describe the X-ray structure of HNE with a peptidyl chloromethyl ketone. Tsuda et al. (Chem Pharm Bull, 35 (9) 3576-84 (1987)) describe the synthesis of peptide chloromethyl ketones and their activity against proteases, including HNE. Ganu and Shaw (Thrombosis Research, 45: 1-6 (1987)) describe peptidyl chloromethyl ketones which are improved plasmin inhibitors. Since chloromethyl ketones for5

ES 2 124 203 T3 man irreversible adducts are less attractive as drugs. Among other classes of inhibitors that form irreversible complexes are: a) peptide enol lactones (J Biol Chem 266 (1) 13-21 (1991) and Biochemistry 29: 4305-11 (1990)), isocoumarins (Krantz et al., US 4,657,893, Powers et al., US 4,845,242, and Kobuko et al., US 4,980287), and peptidyl (α-aminoalkyl) phosphonate diphenyl esters (Biochemistry 30: 485-93 (1991)).

One class of compounds, related to chloromethyl ketones, that reversibly bind proteases with some degree of specificity comprises peptidyl methyl ketones. Peters and Fittkau (Biomed Biochim Acta 49 (4) 173-178 (1990) and references cited there) report that peptidyl methyl ketones bind to serine and cysteine ​​proteases reversibly and that the binding depends on the sequence of the peptidyl group. . If peptidyl methyl ketones are considered as peptide analogs in which the carbonyl group of an amino acid was replaced with a methyl group, Peters and Fittkau only discuss compounds that extend toward the amino terminus. So they provide P1, P2, etc., but not P1 ', P2', etc.

Various information on elastase inhibition

PADR91 reports that elastin (the natural substrate for all elastases) significantly reduces the efficacy of various reversible and irreversible hNE inhibitors compared to the efficacy determined with small, soluble artificial substrates. These researchers found that both classes of inhibitors are 20 to more than 100 times less effective. They suggest that elastin slows the ignition rate, but they claim they have no explanation for this phenomenon. One possibility is that synthetic inhibitors (all of which are rather hydrophobic) bind to elastin (which is also hydrophobic). These investigators tested a reversible protein inhibitor, the mucus protease inhibitor, which has a Ki = 30 nM without elastin or 900 nM with elastin. If our inhibitors experienced a 30-fold loss of efficacy, they would still be able to reduce the hNE concentration to below 10^-10 M.

None of the cited references is admitted to constitute prior art or relevant prior art, and the dates provided are those that appear in the reference and may not be identical to the actual publication date. All bibliography cited in this specification is therefore incorporated by reference.

Compendium of the invention

The present invention relates to serine protease inhibitor mutants with Kunitz domains, such as BPTI and ITI-D1, with substantially increased affinity for elastase. Thus, the invention relates to an artificial protein that inhibits human neutrophil elastase and which is a protein that contains at least the central sequence of an artificial Kunitz domain, a Kunitz domain that is characterized by having cysteines in the corresponding positions to positions 5, 30, 51 and 55 of the bovine pancreatic trypsin inhibitor (BPTI), glycine in a position corresponding to position 12 of the BPTI, Asn in a position corresponding to 43 of the BPTI and Phe in a position corresponding to 33 of the BPTI, provided that when the positions corresponding to 14 and 38 of the BPTI are cysteine, the position corresponding to 37 of the BPTI is glycine; the central sequence is that of the residues corresponding to positions 5 to 55 of the BPTI; where, in said artificial Kunitz domain, the residue corresponding to position 18 of the BPTI is Phe and where the residues corresponding to positions 39 to 42 of the BPTI are all uncharged amino acids.

These muteins have an affinity for elastase estimated to be at least one order of magnitude greater than the natural domain and, in some cases, at least three orders of magnitude (1,000 times) greater. For some of the proteins, the inhibition kinetic data show that the binding affinity is in the range of 1.0 x 10^-12 M and 3.0 x 10^-12 M. Other proteins are displayed on a fusion phage and the affinity for hNE or hCG is estimated by the pH elution profile from active immobilized proteases (hNE or hCG). A set of proteins has been produced in quantities useful as secretory proteins in yeast.

The present invention also relates to linear or cyclic oligopeptide analogs of aprotonin and related polypeptides that specifically bind to human neutrophil elastase or cathepsin G or both. It relates in particular to analogs of the novel elastase-binding polypeptides (EpiNE) and cathepsin G-binding polypeptides described herein.

These analogs differ from aprotonin and related inhibitors in several respects. In the first instance, they are smaller molecules, preferably with molecular weights less than 1,500 Daltons and that include only the P residues.₅ to P₅, (or their analogs) or a subset thereof. Second, the peptide bond (-CO-NH-) cleavable between the P residues₁ And p_r it is replaced with a substantially non-hydrolyzable bond that substantially maintains the distance between the alpha carbons of those two residues.

Brief description of the drawings

Figure 1 illustrates the fractionation of the Mini PEPI library into hNE spheres. The abscissa shows the pH of the buffer. The ordinate shows the amount of phage (as a fraction of the aggregated phage) that is obtained at a given pH.

The ordinates are multiplied by 10³.

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Figure 2 illustrates the fractionation of the MYMUT PEPI library into hNE spheres. The abscissa shows the pH of the buffer. The ordinate shows the amount of phage (as a fraction of the aggregated phage) that is obtained at a given pH. The ordinates are multiplied by 10³ .

Figure 3 shows the elution profiles of EpiNE clones 1, 3 and 7. Each profile is scaled such that the peak value is 1.0 in order to emphasize the shape of the curve.

Figure 4 shows the pH profile for the binding of BPTI-III MK and EpiNE1 on cathepsin G spheres. The abscissa shows the pH of the buffer. The ordinate shows the amount of phage (as a fraction of the aggregated phage) that is obtained at a given pH. The ordinates are multiplied by 10³ .

Figure 5 shows the pH profile for the fractionation of the MYMUT library on cathepsin spheres.

G. The abscissa shows the pH of the buffer. The ordinate shows the amount of phage (as a fraction of the aggregated phage) that is obtained at a given pH. The ordinates are multiplied by 10³ .

Figure 6 shows a second fractionation of the MYMUT library on cathepsin G.

Figure 7 shows elution profiles on immobilized cathepsin G for phage selected for binding to cathepsin G.

Figure 8 shows the shape of a group of preferred HNE inhibitors, hereinafter Class inhibitors.

I. The carbons labeled 7, 8, 9, and 10 are chiral centers.

Figure 9 shows the form of a second group of preferred HNE inhibitors, hereinafter Class II inhibitors. The carbons labeled 7, 8, 9, and 10 are chiral centers.

Figure 10 shows 2-carboxymethyl-6-aminomethyl anthraquinone as a linker. Other relatively rigid molecules of similar dimensions can be used.

Figure 11 shows compounds I through XVIII used to prepare the VAL-ALA dipeptide analogs in which -NH- has been replaced by -CF2 -, -C- or -CHF- for Class I and Class inhibitors. II.

Figure 12 shows the form of a third group of preferred HNE inhibitors, hereinafter Class III inhibitors. The carbons labeled 8, 9, and 10 are chiral centers.

Figure 13 shows compounds XXXI to XXXV that were used in the synthesis of the boron-containing dipeptide analog used in Class I and Class II inhibitors.

Figure 14 shows the compounds XLI to XLIV that were used in the synthesis of a part of the molecule shown in Figure 5.

Detailed description of preferred embodiments

Small proteins with high affinity for elastase or cathepsin G

The present invention relates to muteins of BPTI, ITI-D1 and other inhibitors of the Kunitz domain type that have a high affinity towards elastase and cathepsin G. Some of the described inhibitors are derived from BPTI and some from ITI-D1 . However, hybrids of the identified muteins and other inhibitors of the Kunitz domain type could be constructed.

In order to simultaneously assess the affinity of a large number of different BPTI and ITI-D1 muteins, the DNA sequences encoding BPTI or ITI-D1 were incorporated into the genome of bacteriophage M13. KuDom occurs on the surface of M13 as an amino terminal fusion with the gene III coat protein. Alterations were introduced in the amino acid sequence of the KuDom. Each pure population of phages displaying a particular KuDom was characterized for its interactions with immobilized hNE or hCG. Based on the comparison of the pH elution profiles of phage displaying KuDom of known affinities for protease in studies, KuDom mutants with high affinity for the target protease were identified. The sequences of these mutant KuDom were then determined (usually by sequencing the corresponding DNA).

Some aprotonin-like protease inhibitors were shown to have a high affinity for hNE O 10¹²/ M). These 58 amino acid polypeptides were biologically selected from a library of aprotinin mutants produced by synthetic diversity. The positions P1, P1 ', P2', P3 'and P4' were varied. In P1 only VAL and ILE were selected, although LEU, PHE and MET were allowed by the synthesis conditions. ALA and GLY were allowed in P1 ', and both were found in high affinity proteins. (Although not explored in the library, many inhibitors of the Kazal family of serine proteases have glutamic or aspartic acid at P1 '). All selected proteins contained PHE or MET at P2 '; LEU, ILE and VAL, which are amino acids with branched aliphatic side groups were in the library, but apparently prevent binding to HNE. The P3 'position of all proteins selected for their high affinity for HNE is, surprisingly, occupied

ES 2 124 203 T3 for phenylalanine. It had never been suggested that P3 'was a crucial position for determining specificity relative to HNE. In P4 'SER, PRO, THR, LYS and GLN were allowed and all of them except THR were observed. In the derivatives with the highest affinity, PRO and SER were found.

As already mentioned, BPTI is a 58 amino acid protein. The sequence of the BPTI is presented in the first column of Table 13. The invention is not limited to proteins of 58 amino acids, since it is expected that there are homologues with greater or lesser number of amino acids that are active.

Natural BPTI is not a good HNE inhibitor. BPTI with a single K15L mutation has a moderate affinity for HNE (Kd = 2.9 · 10^-9 M) (BECK88b). However, the Kunitz domain (BI-8e) of the amino terminus of the bovine inter-a-trypsin inhibitor light chain has been generated by proteolysis and has been shown to be a potent inhibitor of HNE (Kd = 4, 4 10^-11 M) (ALBRB3).

The P1 residue has been proposed to be the main determinant of potency in BPTI-like molecules (SINH91, BECK88b, LASK80 and references cited there). Although both BI-8e and BPTI (K15L) have a LEU at their respective P1 positions, there is a 66-fold difference in the affinities of these molecules for HNE. We therefore hypothesize that there are other structural features that should contribute to the affinity of BPTI-like molecules for HNE.

A comparison of the structures of BI-8e and BPTI (K15L) reveals the presence of three positively charged residues at positions 9, 41, and 42 of BPTI that are absent in BI-8e. These hydrophilic and highly charged residues of BPTI occur in a loop that underlies the loop containing the P1 residue and is connected to it by a disulfide bridge. The residues underlying the loop (particularly residue 39) participate in the interaction of BPTI with the surface of trypsin (BLOW72) and may contribute significantly to the stubborn binding of BPTI to trypsin. However, these hydrophilic residues could interfere with the coupling of BPTI variants with HNE. In favor of this hypothesis, BI-8e has a high affinity for HNE and does not contain charged residues between positions 39 and 42. Therefore, residues 39 to 42 of the wild type BPTI were replaced with the corresponding residues (MGNG) to the human homologue of BI-8e. As we anticipated, a BPTI derivative (K15L) containing an MCNG substitution at 39-42 exhibited a higher affinity for HNE than the BPTI single substitution mutant (K15L). BPTI mutants with Met at position 39 are known, but positions 40 to 42 were not mutated simultaneously.

Tables 207 and 208 present the sequences of the rest of the new BPTI mutants with high affinity for HNE. We think that these mutants have an affinity for hNE that is approximately an order of magnitude greater than that of BPTI (K15V, R17L). All of these mutants contain, in addition to the active site mutations shown in the tables, the MGNG mutations at positions 39 to 42. Similarly, Table 209 presents the sequences of the new BPTI mutants that have high affinity for cathepsin G. The P1 residue in EpiC mutants is predominantly MET, with an example of PHE, while in BPTI P1 is LYS and in EpiNE variants, P1 is VAL or ILE. In EpiC mutants, P1 '(residue 16) is predominantly ALA with an example of GLY and P2' (residue 17) is PHE, ILE, or LEU. Interestingly, residues 16 and 17 appear to pair by complementary size, at least in this small sample. The small GLY residue pairs with the bulky PHE, while the ALA residue, which is relatively larger, pairs with LEU and ILE that are less bulky. Alternatively, the formation of pairs could be given by the flexibility in P1 '; glycine at P1 'would allow the phenylalanine side group to reach a gap that is not accessible when P1' is alanine. When P1 'is alanine, leucine or isoleucine seem to be the best options.

Although BPTI has been used in humans with very few adverse effects, a KuDom that bears much greater similarity to a human KuDom has a much lower risk of eliciting an immune response. So we transferred the active site changes found in EpiNE7 to the first KuDom of the inter-a-trypsin inhibitor (Example IV). For the purposes of this application, the nucleic acid sequence numbering of the ITI light chain gene is that of TRAB86 and that of the amino acid sequence is that presented for the UTI in Fig. 1 of GEBH86 . The sequence required to encode ITI-D1 consists of the 168 bases between positions 750 and 917 in the cDNA sequence that occurs in TRAB86. The amino acid sequence of human ITID1 is 56 amino acids long and extends from Lys-22 to Arg-77 of the complete ITI light chain sequence. The P1 site of ITI-D1 is Met-36. Tables 220 and 221 present certain ITI mutants; note that the residues are numbered according to the homologous Kunitz domain of the BPTI, that is, the P1 residue has the number 15. It should be noted that it is probably acceptable to truncate the amino terminus of ITI-D1, at least to the first residue homologous to BPTI.

The EpiNE7-inspired mutation (BPTI region 15-19) of ITI-D1 significantly increased its affinity for hNE. We also found that mutation of a different part of the molecule (BPTI region 1-4) provided a similar increase in affinity. When these two patterns of mutations were combined, a synergistic increase in affinity was observed. Additional mutations in nearby amino acids (BPTI 26, 31, and 34) led to further improvements in affinity.

The projected elastase-binding ITI-D1 muteins herein preferably differ from the natural domain in one or more of the following positions (numbered according to BPTI): 1, 2, 4, 15, 16, 18, 19, 31, and 34. More preferably, they have one or more of the following mutations: Lys1 -&gt; Arg; Glu2 -&gt; Pro; Ser4 -&gt; Phe *; Met15 -&gt; Val *,

IS 2 124 203 T3

Ile; Gly16 -&gt; Ala; Thr18 -&gt; Phe *; Ser19 -&gt; Pro; Thr26 -&gt; Ala; Glu31 -&gt; Gln; Gln34 -&gt; Val *. The introduction of one or more of the asterisked mutations is especially desirable and, in a preferred embodiment, at least all of the asterisked mutations are found.

Those of average training in the art will note that identified HNE and HCG inhibitors can be modified such that the change does not greatly decrease the affinity, specificity, or stability of the inhibitor. The proposed changes can be evaluated on various bases. We first ask whether a particular amino acid can fit into the KuDom structure at a given location; a change that disturbs the structure is very likely to impair binding and decrease specificity. The probability that an amino acid conforms to the KuDom structure can be judged in several ways; 1) does the amino acid appear in any known KuDom?

2) Do the KuDom structural models indicate the compatibility between the structure and the proposed substitution? and 3) do dynamic computational models suggest that the proposed mutant protein will be stable? The sequence variability of the KuDom found in nature proves that certain amino acids are acceptable in certain locations, but the absence of examples does not prove that the amino acid does not fit.

If the proposed change is considered to be acceptable in terms of structure we then have to ask what effect it is likely to have on binding to the target and other substances. Generally, a mutant protein that has a changed residue at the interface between the KuDom and the target will have to be tested, usually by binding studies of a phage displaying the mutant protein. Most changes in the binding interface reduce binding, but some increase affinity. Changes in residues away from the binding interface do not normally reduce binding unless the protein is destabilized.

Table 61 presents the variability of 39 Kunitz domains that exist in nature. All of these proteins have 51 residues in the C5 to C55 region; the total number of residues varies since proteins have more or less residues at the ends. Table 62 presents the names of the proteins included in Table 61. Table 64 cites the works in which these sequences were recorded. Table 63 presents a histogram of how many loci show particular variability as a function of variability. "Central" refers to residues between 5 and 55 that exhibit greater similarity in sequence and structure than residues outside the central region.

There are ten positions in which a single type of amino acid is observed in the 42 cases, they are C3, G12, C14, C30, F33, G37, C38, N43, C51 and C55. Although there are reports that each of these positions can be replaced without complete loss of structure, only G12, C14, G37, and C38 are close enough to the junction interface to offer any incentive to make changes. G12 is in a conformation that only glycine can adopt; it is best to leave this residue as is. Marks et al. (MARK67) replaced C14 and C38 with two alanines or two threonines. The C14 / C38 cystine bridge that Marks et al. eliminated is the one that is close to the cleavable link of the BPTI; It is surprising that both mutant molecules functioned as trypsin inhibitors. Both BPTI (C14A, C38A) and BPTI (C14T, C38T) are stable and inhibit trypsin. Alteration of these residues can result in a useful inhibitor that retains useful stability, and phage display of a diverse population is the best way to obtain and test mutants incorporating alterations at 14 or 38. Only if the C14 disulfide is removed / C38 the strict preservation of the G37 may be removed.

At seven positions (viz. 23, 35, 36, 40, 41, 45, and 47) only two types of amino acid were found. At position 23 only Y and F are observed; the para position of the phenyl ring is solvent accessible and is away from the binding site. Changes in this position are likely to have subtle influences on the union and do not represent a high diversification priority. Similarly, position 35 has only the aromatic residues Y and W; phenylalanine probably works well here too. At position 36 glycine predominates, but serine is also observed. Other amino acids should be included, especially {N, D, A, R}, and are likely to affect the binding properties. Position 40 has only C or A; structural models suggest that other amino acids may be tolerated, particularly those of the {S, D, N, E, K, R, L, M, Q and T} set. Position 40 is close enough to the binding site that alterations in it affect binding. Only N and K are observed at position 41, but any other amino acid except proline can be allowed. The side group is exposed, so hydrophilic groups are preferred, especially {D, S, T, E, R, Q and A}. This residue is far enough away from the binding site that changes in it are not expected to have a great effect on binding. At position 45 F it is highly preferred, but Y is observed once. Since one edge of the phenyl ring is exposed, substitution with other aromatics (W or H) is likely to generate molecules of similar structure, although it is difficult to predict how stability will be affected. Aliphatics such as leucine or methionine (which do not have branches in the C_and) might work here too. At position 47 only S and T have been observed but other amino acids, especially {N, D, G and A}, should generate stable proteins.

At position (44) only three types of amino acids have been observed. Here asparagine predominates and can form internal hydrogen bonds. Other amino acids should be allowed, perhaps with the exception of proline.

In the remaining 40 positions four or more amino acids have been observed and in 28 positions eight or more types of amino acids are seen. Position 25 has 13 different types and there are 5 positions (1, 6, 17, 26 and 34) that have 12 types. Proline (the most rigid amino acid) has been observed in fourteen positions: 1, 2, 8, 9, 11, 13, 19, 25, 32, 34, 39, 49, 57 and 58. The angles Φ and Ψ of the BPTI (CREI84, Table 6-3, p. 222) indicate that proline must be allowed in positions 1, 2, 3, 7, 8, 9, 11, 13, 16, 19, 23, 25, 26, 32, 35, 36, 40, 42, 43, 48, 49, 50, 52, 53, 54, 56 and 58. The

ES 2 124 203 T3 proline is present in four positions (34, 39, 57 and 58) where the angles Φ and Ψ of the BPTI indicate that it should not be acceptable. We conclude that the main chain is reordered locally in those cases.

Based on these data and excluding the six cysteines, we evaluated that the structure of the KuDom will allow the substitutions shown in Table 65. The class indicates whether the substitutions are expected to: A) have a high probability of producing stable proteins with essentially the same binding to hNE, hCG or some other serine protease as the source sequence, B) have similar junctions as the original, or C) are likely to generate proteins that retain the KuDom structure but whose binding has been modified. Class C mutants must be tested for affinity, which is relatively easy using a phage display system such as that proposed in WO / 02809. The affinity of hNE and hCG inhibitors is more sensitive to substitutions at positions 15, 16, 17, 18, 34, 39, 19, 13, 11, 20, and 36 of the BPTI, if the inhibitor is an ITI mutant. -D1, these positions must be converted to the ITI-D1 equivalents by aligning the BPTI and ITI-D1 cysteines.

Some of our hNE inhibitors will be useful as PR-3 inhibitors. We have modeled the interaction of our inhibitors with hNE by reference to the BPTI-trypsin complex. We first generate a list of the trypsin residues that are in contact with the BPTI. Next we consider the corresponding sets of hNE and PR-3 residues. These sets differ by eleven residues. Only one of the differences occurs at the S1 site of specificity, namely, V190 of hNE versus I190 of PR-3. Therefore we think that our hNE inhibitors have the potential to be PR-3 inhibitors as well. In particular, inhibitors having valine in P1 are likely to inhibit PR3. PR3 has an extra methyl in this region so inhibitors with less methyl have a better chance of binding tightly.

The BPTI is very small, if this were to cause a pharmacological problem, such as excessively rapid clearance from the circulation, two or more BPTI-derived domains could be joined by a linker. This linker is preferably a sequence of one or more amino acids. A preferred linker is the one found between repeating domains of a human protein, especially the linkers found in human BPTI homologues, one of which has two domains (BALD85, ALBR83b) and another of which has three (WUNT88 ). Peptide linkers have the advantage that the entire protein can be expressed by recombinant DNA techniques. It is also possible to use a non-peptide linker, such as those commonly used to form immunogenic conjugates. For example, a BPTI-like KuDom attached to polyethylene glycol, the so-called PEGylation (DAVI79).

Another possible drug problem is immunogenicity. BPTI has been used in humans with very few adverse effects. Siekmann et al. (SIEK89) have studied the immunological characteristics of BPTI and some homologues. Also, the likelihood of an immune response can be reduced by starting with a human protein. So changing non-essential residues can change the protein to look more like a human protein. Other modifications, such as PEGylation, have been shown to reduce the immune response (LVAD79).

Reference example

Affinity measures

The affinity of a protein for another molecule can be measured in many ways. Scatchard (Ann NY Acad Sci (1949) 51: 660-669) described a classical method for measuring and analyzing binding that has been applied to protein binding. This method requires a relatively pure protein and the ability to distinguish bound from free protein.

A second method that is suitable for measuring the affinity of inhibitors for enzymes is to measure the ability of the inhibitor to slow the action of the enzyme. This method requires, depending on the rate at which the enzyme hydrolyzes the substrate and the availability of chromogenic or fluorogenic substrates, between tens of micrograms and milligrams of a relatively pure inhibitor.

A third method of determining the affinity of a protein for another material is to have the protein present in a genetic package, such as M13, and to measure the ability of the protein to adhere to immobilized "other material." This method is very sensitive since genetic packages can be amplified. This approach is not entirely new. Makela, O; H Sarvas and I Seppala (“Immunological Methods Based on Antigen-Coupled Bacteriophages”, J. Immunol Methods (1960), 12: 213-223) discuss methods for using haptens chemically conjugated to bacteriophage to measure the concentration of antibodies that have affinity for haptens. The present invention uses a novel approach, since the binding protein is genetically encoded on phage. Likewise, we obtain an at least semi-quantitative value of the binding constants by using a stepped pH gradient. Inhibitors of known affinity for immobilized protease are used to establish standard profiles against which inhibitors displayed on phage can be evaluated. Table 203 shows the profile of phage-BPTI and phage-BPTI (K15L) when these phages are eluted from immobilized hNE. The profiles can vary from one lot of immobilized protease to another and with the time the preparation is immobilized. However, the relative shapes of the profiles allow us to identify the best inhibitors.

Ascenzi et al. (ASCE9O) studied the thermodynamics of binding of BPTI to human coagulation factor Xa and

ES 2 124 203 T3 bovine. They found that KA decreases more than 30 times when the pH is lowered from 9 to 5. Those changes in KA with pH are probably general to the binding of serine proteases to KuDom (and other inhibitors) due to histidine that it is in the active site. The pH at which these changes occur is characteristic of the particular protease and inhibitor. It can be seen that the protonation of histidine from the active site when an inhibitor is attached involves leaving a charge on the inside, which generally represents an unfavorable energy. The reciprocal effect is that a very tightly binding inhibitor effectively lowers the pKa of imidazole protonation.

Throughout this specification, shake incubations used Labquake shakers.

Preparation of Immobilized Human Neutrophil Elastase

One ml of Reacti-Gel 6 x CDI activated agarose (Pierce Chemical Co.) in acetone (200 µl packed spheres) was loaded onto a Select-D empty centrifuge column (5Prime-3Prime). The acetone was drained and the spheres were washed twice rapidly with 1.0 ml of ice water and 1.0 ml of ice cold boric acid, pH 8.5, 0.9% NaCl. To the spheres, 200 µL of human neutrophil elastase (hNE), 2.0 mg / ml (Calbiochem, San Diego, CA) in borate buffer was added. The column was sealed and mixed over the ends on a Labquake shaker at 4 ° C for 36 hours. The hNE solution was removed and the spheres were washed with ice cold 2.0 M Tris, pH 8.0 for a period of 2 hours at 4 ° C to block the remaining reactive groups. A 50% wet suspension was prepared with the spheres in TBS / BSA. To that was added an equal volume of sterile 100% glycerol and the spheres were stored as a 25% suspension at -20 ° C. Before use, the spheres were washed 3 times with TBS / BSA and a 50% suspension was made in TBS / BSA.

Example I

Characterization and fractionation of populations of pure phage clones, each of which presents a single chimeric protein homologous to aprotinin / M13 gene III

This Example demonstrates that chimeric phage proteins displaying a binding target domain can be eluted from the immobilized target by a decrease in pH and that the pH at which the protein elutes indicates the binding affinity of the domain towards the target.

Standard procedure

Unless stated otherwise, all manipulations were carried out at room temperature. Unless otherwise stated, all cells are XL1-Blue (TM) (Stratagene, La Jolla, CA).

1) Demonstration of BPTI-III MK phage binding to active trypsin spheres

We show that phage displaying BPTI-III binds to immobilized active trypsin. Demonstration of display phage binding to immobilized active protease and subsequent retrieval of infectious phage with a characteristic pH elution profile facilitates evaluation of particular mutants, since tens of micrograms of each protein do not need to be produced and purified. mutant.

Phage MK is derived from M13 with the insertion of a kan gene^R in the intergenic region. The BPTI-III MK phages are derived from MK by insertion into gene III, between the codons that specify the signal sequence and those that specify the mature protein, of the DNA that encodes the BPTI. Phage MA is derived from M13 by insertion of an amp gene^R in the intergenic region; phage BPTI-III MA is derived from phage MA by inserting bpti at iii, between the regions encoding the signal peptide and the mature III. BPTI-III MK and BPTI-III MA have BPTI fused to the amino end of the gene III protein, approximately five copies per virion.

50 μl of phage BPTI-IIIMK (3.7 10¹¹ pfu / ml) in 50 mM Tris buffer, pH 7.5; 150 mM NaCl, 1.0 mg / ml BSA (TBS / BSA) or in buffer 50 mM sodium citrate, pH 6.5, 150 mM NaCl, 1.0 mg / ml BSA (CBS / BSA) at 10 μl of 25% immobilized trypsin suspension (Pierce Chemical Co., Rockford, IL) also in TBS / BSA or CBS / BSA. As a control, 50 μl of phage MK (9.310¹² pfu / ml) to 10 μl of a 25% suspension of trypsin immobilized in TBS / BSA or CBS / BSA buffer. The infectivity of phage BPTI-III MK is 25 times lower than that of phage MK; so that the conditions chosen above ensure that an equivalent number of phage particles have been added to the trypsin spheres. After mixing for 3 hours on a Labquake shaker (Labindustries Inc., Berkeley, CA) 0.5 ml of TBS / BSA or CBS / BSA was added to the samples as appropriate. The spheres were washed for 5 min and recovered by centrifugation for 30 s. The supernatant was removed and 0.5 ml of TBS / 0.1% Tween-20 was added. The spheres were mixed for 5 minutes on the shaker and recovered by centrifugation. The supernatant was removed and the spheres were washed five more times with TBS / 0.1% Tween-20 as already described. Finally, the spheres were resuspended in 0.5 ml of elution buffer (0.1 M HCl with 1.0 mg / ml of BSA adjusted to pH 2.2 with glycine), mixed for 5 minutes and recovered by centrifugation. . The supernatant fraction was separated and neutralized by the addition of 130 µl of 1 M Tris, pH 8.0. Aliquots of the neutralized eluate were diluted in LB medium and titrated to count plaque-forming units (pfu).

A significant percentage of the starter phage BPTI-III MK bound to immobilized alatrypsin was recovered in the lava11

ES 2 124 203 T3 two with elution buffer. The amount of fusion phage that bound to the spheres was greater in the TBS buffer (pH 7.5) than in the CBS buffer (pH 6.5). This is consistent with the observation that BPTI's affinity for trypsin is higher at pH 7.5 than at pH 6.5 (VINC72, VINC74). A much smaller percentage of the control MK phage (does not display BPTI) bound to the immobilized trypsin and this binding was independent of pH. At pH 6.5, a 1675 times greater amount of the phage BPTI-III MK than phage MK bound to the trypsin beads, while at pH 7.5 a difference of 2103 times was observed. Therefore the fusion phage displaying BPTI adheres to the active trypsin spheres and can be recovered as an infectious phage.

Generation of BPTI P1 mutants

To demonstrate the specificity of the interaction of the BPTI-III fusion phage with the immobilized serine proteases, one amino acid substitutions were introduced at the P1 position (BPTI residue 15) of the BPTI-III fusion protein. The K15L alteration is desirable since BPTI (K15L) is a moderately good inhibitor of human neutrophil elastase (HNE) (Kd = 2.9 · 10^-9 M) (BECK88b) and a poor trypsin inhibitor. The fusion phage displaying BPTI (K15L) binds to immobilized HNE but not to immobilized trypsin. The BPTI-III MK fusion phage exhibits the opposite phenotype (binds trypsin but not HNE). These observations illustrate the binding specificity of the BPTI-III fusion phage with immobilized serine proteases.

Characterization of the affinity of the phages BPTI-III MK and BPTI (K15L) -III MA towards immobilized human neutrophil trypsin and elastase

30 μl of phage BPTI-III MA in TBS / BSA (1.7 10¹¹ pfu / ml) to 5 µl of a 50% suspension of immobilized human neutrophil elastase or immobilized trypsin (Pierce Chemical Co.) also in TBS / BSA. Similarly, 30 μl of phage BPTI (K15L) -III ML in TBS / BSA (3.2 10¹⁰ pfu / ml) to immobilized HNE or trypsin. The samples were mixed on a Labquake shaker for 3 hours. The spheres were washed with 0.5 ml of TBS / BSA for 5 minutes and recovered by centrifugation. The supernatant was removed and the spheres were washed 5 times with 0.5 ml TBS / 0.1% Tween-20. Finally, the spheres were resuspended in 0.5 ml of elution buffer (0.1 M HCl with 1.0 mg / ml of BSA adjusted to pH 2.2 with glycine), mixed for 5 minutes and recovered by centrifugation. . The supernatant fraction was discarded, neutralized with 130 µl of 1 M Tris, pH 8.0, diluted in LB medium and titrated to count plaque-forming units.

Effect of pH on the dissociation of phages BPTI-III MK and BPTI (K15L) -III MA bound to immobilized neutrophil elastase

The affinity of a given fusion phage for an immobilized serine protease can be characterized based on the amount of bound fusion phage that elutes from the spheres when washed with a step gradient ranging, for example, from about pH 7, 0 to about pH 2.2 in steps of 1 or 0.5 pH units. Since the affinity of the BPTI variants already mentioned is not high (Kd&gt; 1 · 10^-9 M), we predict that fusion phage displaying these variants will dissociate from hNE spheres at pH above 2.2. Likewise, the fusion phage could dissociate from the hNE spheres at a specific pH characteristic and characteristic of the particular BPTI variant it exhibits. Low pH buffers that provide stringent wash conditions may be needed to dissociate a fusion phage displaying a BPTI variant with high affinity for HNE, while they can achieve neutral pH conditions to shed a fusion phage exhibiting a BPTI variant with low affinity for HNE.

30 μl of phage BPTI (K15L) -III MA (1.710¹⁰ pfu / ml in TBS / BSA) to 5 µl of a 50% suspension of HNE spheres also in TBS / BSA. Similarly, 30 μl of phage BPTI-III MA (8.610¹⁰pfu / ml in TBS / BSA) to 5 µl of hNE beads. In this way an approximately equivalent number of phage particles were added to the spheres. The samples were incubated for 3 hours with shaking. The spheres were washed with 0.5 ml of TBS / BSA for 5 min with shaking, recovered by centrifugation and the supernatant was removed. The spheres were washed with 0.5 ml of TBS / 0.1% Tween-20 for 5 minutes and recovered by centrifugation. Four additional washes were performed with TBS / 0.1% Tween-20. The spheres were washed with 0.5 ml of 100 mM sodium citrate, pH 7.0 containing 1.0 mg / ml BSA. The spheres were recovered by centrifugation and the supernatant was removed. The hNE spheres were washed sequentially with a series of 100 mM sodium citrate buffers and 1.0 mg / ml BSA at pH 6.0; 5.0; 4.0 and 3.0, and finally with the elution buffer at pH 2.2. The pH washes were neutralized with the addition of 1 M Tris, pH 8.0, diluted in LB medium and titrated to count plaque-forming units.

Table 203 shows that a low percentage of the BPTI-III MK fusion phage aggregated and adhered to the HNE spheres was recovered and recovered predominantly in the washes at pH 7.0 and 6.0. For the phage BPTI (K15L) III MA bound to the HNE spheres, a significantly higher percentage was recovered and predominantly in the washes of pH 5.0 and 4.0. Therefore lower pH (ie, more stringent) conditions are needed to dissociate phage BPTI (K15L) -III MA than for BPTI-MK from immobilized HNE. The affinity of BPTI (K15L) is more than 1,000 times greater than that of BPTI towards HNE (according to the Kd values ​​of the literature (BECK88b)). This therefore suggests that lower pH conditions are needed to dissociate fusion phage displaying a BPTI variant with higher affinity for HNE.

IS 2 124 203 T3

Effect of the mutation of residues 39 to 42 of BPTI (K15L) on their affinity for immobilized HNE

30 μl of phage BPTI (K15L, MGNG) -III MA (9.210¹⁰ pfu / ml in TBS / BSA) to 5 µl of a 50% suspension of HNE also immobilized in TBS / BSA. Similarly, 30 μl of phage BPTI (k15L) -III MA (1,210¹⁰ pfu / ml in TBS / BSA) to immobilized HNE. The samples were incubated for 3 hours with shaking. The spheres were washed for 5 min with 0.5 ml of TBS / BSA and precipitated by centrifugation. The spheres were washed 5 times with 0.5 ml of TBS / 0.1% Tween-20. Finally, the spheres were washed sequentially with a series of 100 mM sodium citrate pH 7.0 buffers; 6.0; 5.5; 5.0; 4.75; 4.5; 4.25; 4.0 and 3.5. The different pH washes were neutralized, diluted in LB medium and titrated to count plaque-forming units.

Almost twice as many BPTI (K15L, MGNG) -III MA phage than BPTI (K15L) -III MA bind to the HNE spheres. In both cases, the pH 4.75 fraction contained the highest proportion of recovered phage, confirming that replacement of residues 39 to 42 of wild-type BPTI with BI-8e M39GNG increases the binding of the BPTI variant (K15L ) to the HNE.

Construction of the BPTI (K15V. R17L) -III MA

BPTI (K15V, R17L) has the highest affinity for HNE of all BPTI variants described so far (Kd = 6 · 10^-11 M) (AUER89). To test the elution system, a phage displaying BPTI (K15V, R17L) was generated and used as a reference phage to characterize the affinity for immobilized HNE of fusion phage displaying a BPTI variant of known affinity for the free HNE.

Affinity of BPTI (K15V, R17L) -IIII MA phage towards immobilized HNE

40 μl of phage BPTI (K15L, R17L) -III MA (9.8 · 10¹⁰ pfu / ml in TBS / BSA) to 10 µl of a 50% suspension of HNE also immobilized in TBS / BSA. Similarly, 40 μl of phage BPTI (K15L, MGNG) -III MA (5.13 · 10⁹ pfu / ml) in TBS / BSA to immobilized HNE. The samples were shaken for 1.5 hours. The spheres were washed once for 5 min with 0.5 ml of TBS / BSA and then 5 times with 0.5 ml of TBS / 1.0% Tween20. The spheres were then washed sequentially with a series of 50 mM sodium citrate buffers with 150 mM NaCl, 1.0 mg / ml BSA pH 7.0, 6.0; 5.0; 4.5; 4.0, 3.75, 3.5 and 3.0. In the case of BPTI (K15L, MGNG) III MA washes at pH 3.75 and 3.0 were omitted. Two washes were done at each pH and the supernatants were mixed, neutralized with 1 M Tris pH 8.0, diluted in LB medium and titrated to count plaque-forming units.

The pH4.5 and 4.0 fractions contained the largest proportion of the recovered BPTI (K15V, R17L) -III MA phage. Phage BPTI (K15L, MGNG) -III MA as well as BPTI (K15L) -III MA were recovered mainly in the pH 5.0 and 4.5 fractions, as before. The affinity of BPTI (K15V, R17L) is 48 times greater than that of BPTI (K15L) towards HNE (based on the values ​​of Kd, AUER89 for BPTI (K15V, R17L) and BECK88b for BPTI (K15L)). The fact that the pH elution profile of phage BPTI (K15V, R17L) -III MA shows a peak at pH 4.0 while the phage profile BPTI (K15L) -III MA shows a peak at pH 4.5 supports the conclusion that lower pH conditions are required to dissociate, from immobilized HNE, the fusion phage displaying the BPTI variant that has the highest affinity for free HNE.

Example II

Derivatives of BPTI with high affinity towards hNE

We made BPTI mutants appear on the surface of M13-derived phage as fusion proteins with gene III protein (gIIIp); M13 has approximately five copies of gIIIp per virion. Our phage library theoretically included 1,728 BPTI mutants with PHE, LEU, ILE, VAL or MET at positions 15 and 17, GLY or ALA at position 16, PHE, SER, THR or ILE at position 18 and SER, PRO , THR, LYS or GLN at position 19, as a result of the expression of a BPTI gene (encoding the aforementioned MGNG mutation) subjected to controlled random mutagenesis and selected for its hNE-binding activity by incubation of the phage carrying the mutants with immobilized hNE and elution of the phage with buffers of increasing acidity. Twenty mutants were selected for sequencing (see clone identifiers in Tables 207 and 208) and presented eight unique sequences. Tables 207 and 208 show the sequences of nine (all eight plus one other identified in a pilot study) derived from BPTI with high affinity for hNE. EpiNE1, EpiNE3, EpiNE5, EpiNE6 and EpiNE7 eluted at pH 3.5; EpiNE2, EpiNE4 and EpiNE8 between pH 3.5 and 4.

The fact that conditions of pH less than 4.0 were required to elute the phage displaying EpiNE1, EpiNE3 and EpiNE7 suggests that they possess variants that have an affinity for HNE greater than that of BPTI (K15V, R17L).

EpiNE1, EpiNE3 and EpiNE7 were expressed as soluble proteins and analyzed for their inhibition of HNE activity by the fluorimetric assay of Castillo et al. (CAST79); the data were analyzed by the method of Green and Work (GREE53). EpiNE1, EpiNE3, and EpiNE7 have been produced as free proteins, both in E. coli and in yeast. The ability of these proteins to inhibit hNE was measured by hydrolysis of a fluorogenic substrate. The Ki for these compounds is 1 pM, 3 pM, and 3 pM. EpiNE1-displaying phages are used to

ES 2 124 203 T3 establish the reference pH elution profile to allow rapid characterization of other phage displayed KuDom inhibitors. All EpiNE in the list have Kd values ​​less than BPTI (K15V, R17L) (60 pM).

An examination of the sequences of the EpiNE clones is revealing. A strong preference for VAL or ILE at position P1 (residue 15) is indicated with VAL favored over ILE at ratio 14 to 6. No examples of LEU, PHE or MET were observed at position P1 although the selected library should theoretically include these amino acids in P1. This is consistent with the observation that BPTI variants with a single amino acid substitution of LEU, PHE or MET for LYS15 have significantly lower affinity for HNE than the corresponding variants containing VAL or ILE (BECK88b)

PHE is highly favored at position 17 and appears in 12 of the 20 clones. MET is the second most important residue at this position, but only appears when VAL is at position 15. At position 18 PHE was seen in all 20 clones although the library should have included other residues at this position. This result is very surprising and could not be predicted from previous BPTI mutant analyzes or model building or any other theoretical background. We infer that the presence of PHE at position 18 significantly increases the ability of each of the EpiNE to bind to the HNE. Finally, at position 19; PRO appears in 10 of the 20 codons while SER, the second most important residue, appears in 6 of the 20 codons. Of the residues that were targeted for mutagenesis in this study, 19 is the closest to the edge of an inhibitor's interaction surface with HNE. However, a preponderance of PRO is observed and may indicate that PRO at position 19, like PHE at 18, increases the binding of these proteins to HNE. Interestingly, EpiNE5 appears only once and differs from EpiNE1 only at position 19; similarly, EpiNE6 differs from EpiNE3 only at position 19. These alterations may have only a minor effect on the ability of these proteins to interact with HNE. This is supported by the fact that the pH elution profiles of EpiNE5 and EpiNE6 are very similar to those of EpiNE1 and EpiNE3 respectively.

Only EpiNE2 and EpiNE8 have different pH profiles from the other selected clones. Both clones contain LYS at position 19 which can restrict the interaction of BPTI with HNE. However, we cannot exclude the possibility that other alterations within EpiNE2 and EpiNE8 (R15L and Y21S respectively) influence their affinity for HNE.

Position 18 had not previously been identified as a key position in determining the specificity or affinity of aprotinin homologs or derivatives for particular serine proteases. No one had reported or suggested that phenylalanine at position 18 confers specificity and high affinity for HNE.

Example III

BPTI derivatives with high affinity for hCG

The same phage library displaying BPTI mutants was screened for cathepsin G binding activity. Figure 7 presents the binding and pH profiles of individual Cat G binding clones (designated EpiC variants). All clones exhibited minor peaks, superimposed on a gradual decrease in bound phage, in elutions at pH 5 (clones 1, 8, 10 and 11) or pH 4.5 (clone 7). Table 209 presents the clones that bind to the Cat G spheres.

Comparison of the pH profiles generated in the EpiC variants with Cat G and the EpiNE variants with hNE indicates that the EpiNE variants have a high affinity for hNE while the EpiC variants have a moderate affinity for Cat G.

The P1 residue in EpiC mutants is predominantly MET, with an example PHE, while in BPTI P1 it is LYS and in EpiNE variants it is VAL or ILE. In EpiC mutants residue 16 is predominantly ALA with an example of GLY and residue 17 is PHE, ILE or LEU. Interestingly, residues 16 and 17 appear to pair by complementary size, at least in this small sample. The small GLY residue pairs with the bulky PHE, while the ALA residue, which is relatively larger, pairs with LEU and ILE which are less bulky. Most of the residues available in the MYMUT library for positions 18 and 19 are represented in the EpiC variants.

Example IV

Derivatives of ITI: D1 with high affinity towards hNE

Construction of the presentation vector

We use the numbering for the nucleic acid of the ITI-light-chain gene found in TRAB86 and the amino acid numbering that is presented for the UTI in Fig. 1 of GEBH8G. We manipulate the DNA according to standard methods as described in SAMB89 and AUSU87.

The protein sequence of human ITI-D1 is composed of 56 amino acid residues that extend from

IS 2 124 203 T3

LYS22 to ARG77 of the complete ITI light chain sequence. This sequence is encoded by the 168 bases between positions 750 and 917 of the sequence of the cDNA presented in TRAB86. DNA encoding this amino acid sequence was introduced into M13 gene iii with standard methods. Phage isolates containing the ITI-D1-III fusion gene are called MA-ITI and have an amp gene^R. The expression of the ITI-D1 :: III fusion protein and its display on the phage surface were demonstrated by Western blot analysis and anti-rabbit serum titration and neutralization experiments of the phage (hITI).

Fractionation of phage MA-ITI bound to protease spheres immobilized on agarose

To test whether phage displaying the ITI-D1-III fusion protein strongly interact with human neutrophil elastase (hNE) proteases or cathepsin G, aliquots of the display phage were incubated with beads containing immobilized hNE or cathepsin G ( hNE spheres or Cat-G spheres respectively). The spheres were washed and bound phage eluted by pH fractionation. The decrease in pH was: pH 7.0; 6.0; 5.5; 5.0; 4.5; 4.0; 3.5; 3.0; 2.5 and 2.0. After elution and neutralization, the different aggregate, wash and eluate fractions were titrated by pH.

The results of various splittings are summarized in Table 212 (EpiNE-7 or MA-ITI phage bound to hNE spheres) and 213 (EpiC-10 or MA-ITI phage bound to Cat-G spheres). For the two types of spheres (hNE or Cat-G), the pH elution profiles obtained with the control display phage (EpiNE-7 or EpiC-10, respectively) were similar to those already observed. Approximately 0.3% of the phage displaying EpiNE-7 applied to the hNE spheres was eluted during the fractionation process and the elution profile had a maximum elution at approximately pH 4.0. From the EpiC-10 phage applied to the Cat-G spheres, a smaller fraction eluted, 0.02%, and the elution profile showed a maximum near pH 5.5.

The MA-ITI phage does not show evidence of high affinity for hNE or cathepsin G immobilized on agarose beads. The pH elution profiles for MA-ITI phage bound to hNE or Cat-G spheres show essentially a monotonous decrease in recovered phage with decreasing pH. Furthermore, the total fractions of phage applied to the spheres recovered during the fractionation process were very low: 0.002% of hNE spheres and 0.003% of Cat-G spheres.

Published Ki values ​​for inhibition of neutrophil elastase by intact and large protein ITI (Mr = 240,000) are between 60 and 150 nM and values ​​between 20 and 6000 nM have been reported for inhibition of cathepsin G by ITI (SWAI88 , ODOM90). Our own measurements of the pH fraction of phage bound to hNE spheres show that phages displaying proteins with low affinity for hNE (&gt; μΜ) do not bind to the spheres whereas phages displaying proteins with higher affinity ( nM) bind to the spheres and elute at approximately pH 5. If the first KuDom of the ITI light chain is entirely responsible for the inhibitory activity of ITI against hNE and if this domain is correctly displayed on the MA-ITI phage, it appears that the minimal affinity of an inhibitor for hNE that allows the Binding and fractionation of the display phage on hNE spheres is between 50 and 100 nM.

Alterations in the P1 region of the ITI-D1

If ITI-D1 and EpiNE-7 assume the same configuration in solution as BPTI, then these two polypeptides have identical amino acid sequences in the primary and secondary binding loops except for four residues close to the P1 position and for the positions 11 and 34. For ITI-D1 the sequence for positions 15 to 20 is (position 15 in ITI-D1 corresponds to position 36 in the sequence of the GEBH86 UTI):

position number in

BPTI Affinity

<td>Domain</td><td> 11</td><td> 15</td><td> 16</td><td> 17</td><td> 18</td><td> 19</td><td> 20</td><td> 31</td><td> 34</td><td>towards hNE</td>

<td>EpiNE7</td><td>T</td><td>V</td><td>TO</td><td>M</td><td>F</td><td>P</td><td>R</td><td>Q</td><td>V</td><td>very high</td>

<td>ITI-D1</td><td>TO</td><td>M</td><td>G</td><td>M</td><td>T</td><td>S</td><td>R</td><td>AND</td><td>Q</td><td>limited</td>

<td></td><td> 32</td><td> 36</td><td> 37</td><td> 38</td><td> 39</td><td> 40</td><td> 41</td><td> 52</td><td> 55</td><td>positions</td>

of the ITI

These two proteins differ greatly in their affinities for hNE. To improve the affinity of ITI-D1 for hNE, the EpiNE-7 sequence was incorporated by cassette mutagenesis into the ITI-D1 sequence at positions 15 to 20. Phage containing the ITI-D1-III fusion gene with EpiNE-7 changes around the P1 position are called MA-ITI-E7.

MA-ITI-E7 phage fractionation

To test whether changes in positions 15, 16, 18 and 19 of the ITI-D1-III fusion protein influence phage binding to hNE spheres, abbreviated pH elution profiles were measured. Aliquots of EpiNE-7, MA-ITI and MA-ITI-E7 phages were incubated with hNE spheres for three hours at room temperature. The spheres were washed

ES 2 124 203 T3 and phages were eluted as described above, except that only three elutions were performed: pH 7.0; 3.5 and 2.0. The results of these elutions are presented in Table 214.

The binding and elution of phage EpiNE-7 and MA-III was found to be the same as described. The total fraction of the aggregated phage that was recovered was high (0.4% for the EpiNE-7 phage and low (0.001% for the MA-ITI phage. Furthermore, the EpiNE-7 phage showed a maximum elution in the pH fraction 3.5 whereas the MA-ITI phage showed only a monotonous decrease in phage yield as the pH was lowered, as already mentioned.

The MA-ITI-E7 phage exhibits higher levels of binding to hNE spheres compared to the MA-ITI phage. The total fraction of aggregated phage that eluted from the spheres is 10 times higher for both MA-ITIE7 phage strains than for the MA-ITI phage (although it is still 40 times lower than for the EpiNE-7 phage). Furthermore, the pH elution profiles of the MA-ITI-E7 phage strains show elution peaks in the pH 3.5 fractions similar to those of the EpiNE-7 phage. To better define the binding properties of the MA-ITI-E7 phage, the extended pH fractionation procedure described above was performed with the phage bound to the hNE spheres, as presented in Table 215. The pH elution profile of the EpiNE-7 phage is the one already described. In this better resolution pH profile, the MA-ITI-E7 phage shows a broad elution maximum centered around pH 5. Again, the total fraction of MA-ITI-E7 phage obtained from the pH elution of the hNE spheres was about 40 times lower than that obtained with the EpiNE-7 phage.

The pH elution behavior of phage MA-ITI-E7 bound to hNE spheres is qualitatively similar to that observed with phage BPTI [K15L] -III-MA. BPTI with the K15L mutation has an affinity for hNE of »3 · 10^-9 M. Assuming that the other factors are constant, the pH elution profile of MA-ITI-E7 suggests that the affinity of the free ITI-D1-E7 domain for hNE is in the nM order. Thus, the substitution of the EpiNE-7 sequence in place of the ITI-D1 sequence around the P1 region has produced an apparent 20-50-fold increase in affinity for hNE (it is assumed that Ki = 60-150 nM for ITI-D1).

If EpiNE-7 and ITI-D1-E7 have the same structure in solution, these proteins have identical amino acid sequences to hNE on the interaction surface. Despite this similarity, EpiNE-7 exhibits an approximately 1,000-fold higher affinity for hNE than ITI-D1-E7. This observation highlights the importance of non-contacting secondary residues for modulation of interaction forces.

The ITI light chain is glycosylated at SER10 and ASN45 (GEBH86). Removal of glycosaminoglycan chains has been shown to decrease the affinity of the inhibitor for hNE by about 5-fold (SELL87). Another potentially important difference between EpiNE-7 and ITI-D1-E7 is that of net charge. The BPTI has a charge of +6 while the EpiNE7 has a charge of +1 and ITI-D1 has a charge of -1. Furthermore, the change in charge on these two molecules arises from differences in the central portions of the molecule that adjoin the bonding surface. Position 26 is LYS in EpiNE-7 and it is THR in ITI-D1-E7, while at position 31 the residues are GLN and GLU, respectively. These sequence changes not only alter the net charge of the molecule but also place the negative charge closer to the interaction surface on the ITI-D1-E7. It may be that the existence of a negative charge at position 31 (not found in any of the other hNE inhibitors described here) destabilizes the inhibitor-protease interaction.

Preparation of the BITI-E7 phage

We replaced K1EDS from ITI-D1 with R1PDF from EpiNE7 to make the MA-BITI-E7 phage. The Phe4 of BPTI is part of the hydrophobic core of the protein and its replacement with serine may adversely alter the stability or dynamic character of ITI-E7. ITI-E7 has a negatively charged Glu at position 2 while EpiNE7 has Pro.

We made the same changes to the putative amino terminus of the ITI-III fusion protein displayed on the MA-ITI phage. These phages are called MA-BITI.

We compared the properties of the ITI-III fusion proteins displayed on the MA-ITI and MA-BITI phages by Western blot analysis. We did not find significant differences in the apparent size or in the relative abundance of the fusion proteins produced by each phage strain. So there are no major differences in the processed forms of any of the fusion proteins displayed by phage. By extension, there are also no large differences in the processed forms of the gene III fusion proteins exhibited by MA-ITI-E7 and MA-EpiNE7. Therefore, it is unlikely that there are large changes in protein conformation due to a highly altered process and are responsible for the large differences in binding to hNE spheres exhibited by the MA-ITI-E7 and MA phages. -EpiNE7.

We characterize the hNE bead-binding properties of the MA-BITI and MA-BITI-E7 phages by the pH-extended fractionation process already described, see Table 216. The pH elution profile of the MA-EpiNE7 phage bound to hNE spheres is similar to that already described. The pH elution profiles of MA-BITI and MABITI-E7 show significant differences with respect to the profiles shown by MA-ITI and MA-ITI-E7 (compare Tables 212 and 215). In both cases, alterations in the putative amino terminus of the displayed fusion protein produce a multiple-fold increase in the fraction of aggregated phage eluting from the hNE spheres.

IS 2 124 203 T3

The ability of phage to bind to hNE spheres varies between different spheres preparations and with the time of each preparation. Thus, the relative shapes of the profiles that were obtained with spheres of practically the time and from the same batch must be compared. For example, the fraction of the phage MAEpiNE7 that is recovered from hNE spheres varies in a two-fold interval between the experiments shown in Tables 212, 215 and 216 and the results given in the remainder of this specification. However, the shapes of the pH elution profiles are very similar. Variations in the binding capacity of hNE spheres can be roughly corrected by normalizing the phage yields to the total yields of MA-EpiNE7 phage recovered from the spheres in a coincident elution. When the data in Tables 212, 215 and 216 are normalized in this way, the phage recoveries relative to the recovered MA-EpiNE7 are:

<td>phage strain</td><td>normalized fraction of aggregate</td>

<td>MA-ITI</td><td> 0,0067</td>

<td>MA-BITI</td><td> 0,027</td>

<td>MA-ITI-E7</td><td> 0,027</td>

<td>MA-BITI-E7</td><td> 0,13</td>

Thus, alterations in the amino-terminal sequence of the displayed fusion protein produce a three- to five-fold increase in the fraction of the phage that elutes from the hNE spheres. While MA-ITI-E7 eluted with a broad pH maximum centered at approximately pH 5.0, the pH elution profile of MA-BITI-E7 phage has a maximum approximately between pH 4.75 and pH 4.5.

The maximum elution by pH of phage MA-BITI-E7 is located between the maximums exhibited by phages BPTI (K15L) and BPTI (K15V, R17L) (pH 4.75 and pH 4.5 at pH 4.0, respectively) already described (Example III). From the pH of the maximum that the phage presents, we estimate that the free BITI-E7 protein in solution has an affinity for hNE in the order of 10^-10 M. This would represent an approximately ten-fold increase in affinity for hNE above that previously estimated for ITI-E7.

As described above, Western blot analysis of the phage proteins shows that there are no major changes in the gene III fusion proteins due to the alteration in the amino terminus sequence. Thus, it is unlikely that changes in phage affinity for hNE spheres could be attributed to large-scale alterations in protein folding as a result of altered ("correct") processing of the fusion protein in mutants. from the amino end. Improvements in binding may be due in part to: 1) the decrease in the net negative charge (-1 to 0) of the protein as a result of the switch from GLU to PRO at position 2, or

2) an increase in protein stability as a result of the replacement of SER by PHE at residue 4 in the hydrophobic core of the protein, or 3) the combined effect of both substitutions.

Production and properties of MA-BITI-E7-l222 and MA-BITI-E7-l4l

Within the supposed KuDom: hNE interface, BITI-E7 and EpiNE7 are only distinguished in two positions: 11 and 34. In EpiNE7 these residues are THR and VAL, respectively. In BITI-E7 they are ALA and GLN. Also, BITIE7 has GLU at position 31 while EpiNE7 has GLN. This negative charge can influence the bond even though the residue is not directly at the interface. We employ oligonucleotide-directed mutagenesis to investigate the effects of substitutions at positions 11, 31, and 34 on protease: inhibitor interaction.

The MA-BITI-E7-1222 phage is the same as BITI-E7 with the A11T mutation. The phage MA-BITI-E7-141 is the same as BITI-E7 with the E31Q and Q34V mutations.

We determined the hNE sphere-binding properties of the phages MA-BITI-E7-1222 and MA-BITI-E7-141 using the extended pH fractionation protocol, as presented in Tables 217 (for MA-BITI-E7 and MA-BITI-E7-1222) and 218 (for MA-EpiNE7 and MA-BITI-E7-141).

Thus, the substitution of ALA for THR at position 11 of the displayed ITI derivative has no appreciable effect on phage binding to hNE spheres.

In contrast, changes at positions 31 and 34 profoundly affect the hNE-binding properties of phage (Table 218). The maximum of the pH elution profile of phage MA-BITI-E7-141 is shifted to a lower pH relative to the parent phage MA-BITI-E7. Furthermore, the position of the maximum (between pH 4.5 and pH 4.0) is identical to that shown by the MA-EpiNE7 phage in this experiment. Finally, the phage MA-BITI-E7-141 shows a ten-fold increase, relative to its predecessor MA-BITI-E7, in the total fraction of aggregated phage that is eluted from the hNE spheres (0.3% against 0 , 03%). In fact, the total fraction of MA-BITI-E7-141 phage that eluted from the hNE spheres is almost double that of the corresponding MA-EpiNE7 phage.

The results discussed above show that the binding of the phage MA-BITI-E7-141 to the hNE spheres is com17

ES 2 124 203 T3 parable to that of the MA-EpiNE7 phage. So BITI-E7-141 can have a KD &lt;1 pM. That affinity is approximately 100 times higher than previously estimated for the original protein (BITI-E7) and is 10⁵ to 10⁶ times greater than the affinity for hNE reported for intact ITI protein.

BITI-E7-141 mutagenesis

BITI-E7-141 differs from ITI-D1 in nine positions (1, 2, 4, 15, 16, 18, 19, 31, and 34). To obtain the protein that has the least number of changes with respect to ITI-D1 while retaining a high specific affinity towards hNE, we have investigated the effects of reversing the changes in positions 1, 2, 4, 16, 19, 31 and 34. The changes that we have introduced in the BITI-E7-141 protein are presented schematically below.

residue

<td colspan="5">Protein</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td> 2</td><td> 3</td><td> 3</td>

<td colspan="2">presentable</td><td> 2</td><td> 3</td><td> 4 .</td><td> . . . 1 .</td><td> ... 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9 .</td><td> . 6 .</td><td> . 1 .</td><td> . 4</td>

<td>ITI-D1</td><td>K</td><td>AND</td><td>D</td><td>S.</td><td>. . . TO .</td><td>. . . M</td><td>G</td><td>M</td><td>T</td><td>S.</td><td>.T.</td><td>. E.</td><td>• Q</td>

<td> 141</td><td>R</td><td>P</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . V</td><td>TO</td><td>M</td><td>F</td><td>P.</td><td>. T.</td><td>.Q.</td><td>. V</td>

<td>MUT1619</td><td>R</td><td>P</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . V</td><td>G</td><td>M</td><td>F</td><td>S.</td><td>. T.</td><td>• Q ·</td><td>. V</td>

<td>MUTP1</td><td>R</td><td>P</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . I</td><td>G</td><td>M</td><td>F</td><td>s.</td><td>.T.</td><td>.Q.</td><td>. V</td>

<td>AMIN01</td><td>K</td><td>AND</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . V</td><td>TO</td><td>M</td><td>F</td><td>P.</td><td>. T.</td><td>• Q.</td><td>. V</td>

<td>AMIN02</td><td>K</td><td>AND</td><td>D</td><td>S.</td><td>. . . TO .</td><td>. . . V</td><td>TO</td><td>M</td><td>F</td><td>P.</td><td>. T.</td><td>.Q</td><td>.V</td>

<td>MUTQE</td><td>R</td><td>P</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . V</td><td>TO</td><td>M</td><td>F</td><td>P.</td><td>. T.</td><td>E.</td><td>. V</td>

<td>MUTT26A</td><td>R</td><td>P</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . V</td><td>TO</td><td>M</td><td>F</td><td>P.</td><td>.TO .</td><td>• Q.</td><td>. V</td>

<td>MUT200</td><td>K</td><td>P</td><td>D</td><td>F.</td><td>. . . TO .</td><td>. . . V</td><td>G</td><td>M</td><td>F</td><td>s.</td><td>.TO .</td><td>• E.</td><td>. V</td>

ITI-D1 residues appear in bold and non-ITI-D1 and non-BITI-E7-141 residues are underlined in bold. In MUT1619, ITI-D1 residues at positions 16 and 19 are restored. MET at position 17 and PHE at 18 are likely to be optimal for high hNE binding affinity, but F17F18 are also effective. LaGLY at position 16 and SER at position 19 appeared frequently in BPTI variants that bind to hNE with high affinity obtained from fractionation of a library of BPTI variants against hNE (ROBE91). So it seems likely that the ITI-D1 sequence at these positions can be restored while maintaining the high specific affinity for hNE. The sequence designated MUT200 is hypothetical, but it is very likely that it has a high affinity for hNE.

BITI phages were produced by substituting R1PDF from EpiNE7 for K1EDS from phage ITI.

Two changes had been made to the BITI-E7 sequence to produce BITI-E7-141: from GLU to GLN at position 31 and from GLN to VAL at position 34.

The protein sequence present in BITI-E7-141, ASN24-GLY25-THR26, coincides with the general ASNX-THR / SER recognition sequence for N-glycosylation in eukaryotic organisms. In intact ITI molecule isolated from human serum, the light chain polypeptide is glycosylated at this site (ASN45, ODOM90). ASN24 is likely to become glycosylated if the BITI-E7-141 protein is produced by expression in a eukaryote. Such glycosylation can make the protein difficult to purify to homogeneity and become immunogenic when used in long-term treatment. We change T26 to A since alanine is frequently found at this location in the KuDom.

HNE-binding properties of phage MA-BITI-E7-141 subject to mutagenesis

The hNE bead binding properties of individual phage populations were determined by the abbreviated and extended pH elution protocols described above. The results of these elutions are presented in Table 219.

Table 219 shows the pH elution data for the various phages that were eluted from hNE spheres. The total pfu applied to the spheres are shown in the second column. The fractions of this pfu aggregate that are

ES 2 124 203 T3 recovered in each pH fraction in the abbreviated pH elution protocol (pH 7.0, pH 3.5 and pH 2.0) are presented in the following three columns. For the data obtained with the extended pH elution protocol, the list at pH 3.5 represents the sum of the aggregate fractions that were recovered in the samples that eluted at pH 6.0, pH 5.5, pH 5, 0, pH 4.5, pH 4.0 and pH 3.5. Similarly, the pH 2.0 list is the sum of the aggregate fractions obtained in the elution samples at pH 3.0, pH 2.5, and pH 2.0. The total fraction of the added pfu that was obtained in the entire pH elution protocol is recorded in the sixth column of Table 219. The final column of the table shows the total fraction of the added pfu that were recovered normalized with respect to the value. obtained for phage MA-BITIE7-141.

Two factors have to be considered when comparing between the data presented in Table 219. The first is that, due to the kinetic nature of phage release from hNE spheres and the longer time involved in the extended elution protocol , the fraction of the added pfu that is recovered in the pH 3.5 fraction will be enriched at the expense of that of pH 2.0 in the extended protocol relative to the values ​​obtained in the abbreviated protocol. The magnitude of this effect can be appreciated when comparing the results obtained when the phage MA-BITI-E7-141 was eluted from the hNE spheres with the two protocols. The second factor is that, for the range of added pfu shown in Table 219, the added pfu influence recovery. The more pfu added, the greater the total fraction of the aggregate that was recovered in the elution. This effect is evident when the aggregated pfu differ by more than a factor of 3 or 4. The effect can lead to an overestimation of the affinity of the phage towards the hNE spheres when the data from the phage applied in larger quantities is compared to those obtained from smaller quantities.

We interpret Table 219 attentive to these warnings. The effects of mutations introduced in the phage MA-BITI-E7-141 ("original") on the binding of the phage to the hNE spheres can be grouped into three categories: changes that have little or no effects, those that have moderate effects (2 to 3 times) and those with large effects (more than 5 times).

The MUTT26A and MUTQE changes appear to have little effect on phage binding to hNE spheres. In terms of the total pfu recovered, the phage containing these alterations binds as well as the original to the hNE spheres. In fact, the pH elution profiles obtained for the original and MUTT26A phages with the extended pH elution protocol are indistinguishable. The binding of the MUTQE phage appears to be slightly reduced relative to the original and, in view of the applied pfu, it is likely that this binding is somewhat overestimated.

The sequence alterations that were introduced by oligonucleotides MUTP1 and MUT1619 appear to reduce binding of phage to hNE spheres by 2 to 3 times. In light of the aggregated amounts and the distributions of the recovered pfu among the various elution fractions it seems likely that 1) the two phages have lower affinities towards hNE spheres than the MA-EpiNE7 phage and 2) the MUT1619 phage has a higher affinity for hNE spheres than MUTP1 phage.

Sequence alterations at the amino terminus of BITI-E7-141 appear to reduce phage binding to hNE spheres by at least tenfold. AMINO2 changes are likely to reduce phage binding to a much greater extent than AMINO1 changes.

Based on the previous interpretations of the data in Table 219, we can conclude that:

1. ) The substitution of ALA for THR at position 26 in ITI-D1 and its derivatives has no effect on the interaction of the inhibitor with hNE. Therefore, the possibility of glycosylation of ASN24 from the inhibitory protein produced in a culture of eukaryotic cells can be avoided without reducing the affinity for hNE.

two. ) The increase in the affinity of the phage towards the hNE spheres that produce the changes from GLU to GLN at position 31 and GLN to VAL at 34 are the result, mainly, of the substitution of VAL at position 34.

3. ) The three changes introduced in the region of the amino terminus of ITI-D1 (positions 1, 2 and 4) influence the binding of the phage to the hNE spheres with different intensity. The change in position 4 (SER to PHE) appears to have a much larger effect than the change in position 2. The change in position 1 may have little or no effect.

4.) Changes in the region near the P1 residue of BITI-E7-141 (position 15) influence the binding of phage to hNE. Changes from ALA to GLY at position 16 and from PRO to SER at position 19 appear to somewhat (perhaps 3-fold) reduce the affinity of the inhibitor. Substituting ILE for VAL at position 15 further reduces binding.

The BITI-E7-141 differs from the ITI-D1 in nine positions. Based on the above discussion it seems likely that a high affinity inhibitor for hNE based on ITI-D1 can be constructed and that it will differ from the ITI-D1 sequence in only four or five positions. These differences would be PHE at position 4, VAL at position 15, PHE at position 18, VAL at position 34 and ALA at position 26. If the glycosylation of ASN24 were not considered, THR could be conserved at position 26.

IS 2 124 203 T3

10. Compendium of Estimated Affinities of Isolated ITI-D1 Derivatives

Based on the binding of the phages and their subsequent elution of hNE spheres, the affinities for hNE exhibited by the various free ITI-D1 derivatives in solution can be estimated. These estimates are summarized below and in Table 220.

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(Table goes to next page)

IS 2 124 203 T3

TABLE 13

BPTI counterparts (1-19)

1234567891111111111

0123456789

<td> -2 -1 1 2 3</td><td>R P D</td><td>R P D</td><td>R P D</td><td>Q T P P D</td><td>T AND R P D</td><td>R P D</td><td>R P D</td><td>R P D</td><td>R P D</td><td>R P D</td><td>R P D</td><td>Q P L R K</td><td>TO TO K</td><td>R P D</td><td>R P R</td><td>H D R P T</td><td>G D K R D</td><td>Z G R P S</td><td>TO TO K</td>

<td> 4</td><td>F</td><td>F</td><td>F</td><td>L</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>L</td><td>Y</td><td>F</td><td>F</td><td>F</td><td>I</td><td>F</td><td>Y</td>

<td> 5</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td><td>C</td><td>C</td>

<td> 6</td><td>L</td><td>L</td><td>L</td><td>Q</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>I</td><td>K</td><td>AND</td><td>AND</td><td>N</td><td>R</td><td>N</td><td>K</td>

<td> 7</td><td>AND</td><td>AND</td><td>AND</td><td>L</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td>

<td> 8</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>H</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td>

<td> 9</td><td>P</td><td>P</td><td>P</td><td>P</td><td>Q</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>R</td><td>L</td><td>TO</td><td>TO</td><td>P</td><td>P</td><td>TO</td><td>V</td>

<td> 10</td><td>Y</td><td>Y</td><td>Y</td><td>TO</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>R</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>R</td>

<td> 11</td><td>T</td><td>T</td><td>T</td><td>R</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>P</td><td>I</td><td>T</td><td>T</td><td>S</td><td>Q</td><td>T</td><td>Y</td>

<td> 12</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 13</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>R</td><td>P</td><td>L</td><td>L</td><td>R</td><td>P</td><td>P</td><td>P</td>

<td> 14</td><td>C</td><td>T</td><td>TO</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td><td>c</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 15</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>V</td><td>G</td><td>TO</td><td>L</td><td>I</td><td>K</td><td>Y</td><td>K</td><td>K</td><td>K</td><td>R</td><td>K</td><td>K</td><td>K</td>

<td> 16</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>Q</td><td>R</td><td>TO</td><td>TO</td><td>G</td><td>G</td><td>TO</td><td>K</td>

<td> 17</td><td>R</td><td>R</td><td>R</td><td>TO</td><td>TO</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>K</td><td>K</td><td>Y</td><td>R</td><td>H</td><td>R</td><td>S</td><td>K</td>

<td> 18</td><td>I</td><td>I</td><td>I</td><td>L</td><td>M</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>L</td><td>I</td><td>F</td>

<td> 19</td><td>I</td><td>I</td><td>I</td><td>L</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>P</td><td>P</td><td>R</td><td>R</td><td>R</td><td>P</td><td>R</td><td>P</td>

<td> 20</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>TO</td><td>S</td><td>S</td><td>S</td><td>R</td><td>R</td><td>Q</td><td>S</td>

<td> 21</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>F</td><td>F</td><td>F</td><td>F</td><td>I</td><td>Y</td><td>Y</td><td>F</td>

<td> 22</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>Y</td><td>Y</td><td>H</td><td>H</td><td>Y</td><td>F</td><td>Y</td><td>Y</td>

<td> 23</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td>

<td> 24</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>K</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td>

<td> 25</td><td>TO</td><td>TO</td><td>TO</td><td>S</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>Q</td><td>W</td><td>L</td><td>R</td><td>L</td><td>P</td><td>S</td><td>W</td>

<td> 26</td><td>K</td><td>K</td><td>K</td><td>T</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>TO</td><td>TO</td><td>AND</td><td>TO</td><td>K</td><td>K</td>

<td> 27</td><td>TO</td><td>TO</td><td>TO</td><td>s</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>K</td><td>TO</td><td>TO</td><td>TO</td><td>S</td><td>S</td><td>S</td><td>TO</td>

<td> 28</td><td>G</td><td>G</td><td>G</td><td>N</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>K</td><td>K</td><td>Q</td><td>Q</td><td>N</td><td>R</td><td>G</td><td>K</td>

<td> 29</td><td>L</td><td>L</td><td>L</td><td>TO</td><td>F</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>K</td><td>M</td><td>G</td><td>Q</td>

<td> 30</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td><td>c</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td>

IS 2 124 203 T3

<td>R No.</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 1 0</td><td> 1 1</td><td> 1 2</td><td> 1 3</td><td> 1 4</td><td> 1 5</td><td> 1 6</td><td> 1 7</td><td> 1 8</td><td> 1 9</td>

<td> 31</td><td>Q</td><td>Q</td><td>Q</td><td>AND</td><td>AND</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>L</td><td>L</td><td>L</td><td>K</td><td>AND</td><td>Q</td><td>L</td>

<td> 32</td><td>T</td><td>T</td><td>T</td><td>P</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>G</td><td>P</td><td>Q</td><td>R</td><td>V</td><td>S</td><td>Q</td><td>P</td>

<td> 33</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td>

<td> 34</td><td>V</td><td>V</td><td>V</td><td>T</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>T</td><td>D</td><td>I</td><td>I</td><td>F</td><td>I</td><td>I</td><td>N</td>

<td> 35</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>W</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td>

<td> 36</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>s</td><td>S</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>S</td>

<td> 37</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 38</td><td>C</td><td>T</td><td>TO</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 39</td><td>R</td><td>R</td><td>R</td><td>Q</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>K</td><td>R</td><td>G</td>

<td> 40</td><td>TO</td><td>TO</td><td>TO</td><td>G</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 41</td><td>K</td><td>K</td><td>K</td><td>N</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td>

<td> 42</td><td>R</td><td>R</td><td>R</td><td>N</td><td>s</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>S</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>K</td><td>Q</td><td>TO</td>

<td> 43</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td>

<td> 44</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>R</td><td>R</td><td>R</td><td>R</td><td>N</td><td>N</td><td>R</td><td>R</td>

<td> 45</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td>

<td> 46</td><td>K</td><td>K</td><td>K</td><td>AND</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>AND</td><td>K</td><td>D</td><td>K</td>

<td> 47</td><td>S</td><td>S</td><td>s</td><td>T</td><td>s</td><td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td>s</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td>

<td> 48</td><td>TO</td><td>TO</td><td>TO</td><td>T</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>I</td><td>I</td><td>I</td><td>I</td><td>R</td><td>K</td><td>T</td><td>I</td>

<td> 49</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>D</td><td>D</td><td>D</td><td>TO</td><td>Q</td><td>AND</td>

<td> 50</td><td>D</td><td>D</td><td>D</td><td>M</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>Q</td><td>AND</td>

<td> 51</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td><td>C</td>

<td> 52</td><td>M</td><td>M</td><td>M</td><td>L</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>AND</td><td>R</td><td>R</td><td>R</td><td>H</td><td>R</td><td>V</td><td>Q</td><td>R</td>

<td> 53</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>AND</td><td>R</td><td>G</td><td>R</td>

<td> 54</td><td>T</td><td>T</td><td>T</td><td>I</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>TO</td><td>V</td><td>T</td>

<td> 55</td><td>C</td><td>c</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 56</td><td>G</td><td>G</td><td>G</td><td>AND</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>I</td><td>V</td><td>V</td><td>V</td><td>G</td><td>R</td><td>V</td><td>V</td>

<td> 57</td><td>G</td><td>G</td><td>G</td><td>P</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>R</td><td>G</td><td>G</td><td>G</td><td>G</td><td>P</td><td> -</td><td>G</td>

<td> 58</td><td>TO</td><td>TO</td><td>TO</td><td>P</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>K</td><td> -</td><td> -</td><td> -</td><td>K</td><td>P</td><td> -</td><td> -</td>

<td> 59</td><td></td><td></td><td></td><td>Q</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>AND</td><td> -</td><td> -</td>

<td> 60</td><td></td><td></td><td></td><td>Q</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>R</td><td></td><td></td>

<td> 61</td><td></td><td></td><td></td><td>T</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>P</td><td> -</td><td> -</td>

<td> 62</td><td></td><td></td><td></td><td>D</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 63</td><td></td><td></td><td></td><td>K</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 64</td><td></td><td></td><td></td><td>S</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

IS 2 124 203 T3

TABLE 13 (continued) (BPTI 20-35 homologues)

RN ° 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

<td> -2 -1 1</td><td>Z P R</td><td>R</td><td>L Q H</td><td>Z D H</td><td>R D R</td><td>K N R</td><td>I</td><td>K</td><td>T</td><td>R Q R</td><td>R K R</td><td>R</td><td>AND R G</td><td colspan="2">T - T - DK</td><td>T</td>

<td> 2</td><td>R</td><td>P</td><td>R</td><td>P</td><td>P</td><td>P</td><td>N</td><td>AND</td><td>V</td><td>H</td><td>H</td><td>P</td><td>F</td><td>L</td><td>TO</td><td>V</td>

<td> 3</td><td>K</td><td>Y</td><td>T</td><td>K</td><td>K</td><td>T</td><td>G</td><td>D</td><td>TO</td><td>R</td><td>P</td><td>D</td><td>L</td><td>P</td><td>D</td><td>AND</td>

<td> 4</td><td>L</td><td>TO</td><td>F</td><td>F</td><td>F</td><td>F</td><td>D</td><td>S</td><td>TO</td><td>D</td><td>D</td><td>F</td><td>D</td><td>I</td><td>S</td><td>TO</td>

<td> 5</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 6</td><td>I</td><td>AND</td><td>K</td><td>Y</td><td>Y</td><td>N</td><td>AND</td><td>Q</td><td>N</td><td>D</td><td>D</td><td>L</td><td>T</td><td>AND</td><td>Q</td><td>N</td>

<td> 7</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>K</td><td>K</td><td>AND</td><td>s</td><td>Q</td><td>L</td><td>L</td>

<td> 8</td><td>H</td><td>I</td><td>P</td><td>P</td><td>P</td><td>L</td><td>P</td><td>G</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>TO</td><td>D</td><td>F</td>

<td> 9</td><td>R</td><td>V</td><td>TO</td><td>TO</td><td>TO</td><td>P</td><td>K</td><td>Y</td><td>V</td><td>P</td><td>P</td><td>P</td><td>P</td><td colspan="2">FG Y</td><td>I</td>

<td> 10</td><td>N</td><td>TO</td><td>AND</td><td>D</td><td>D</td><td>AND</td><td>V</td><td>S</td><td>I</td><td>D</td><td>D</td><td>Y</td><td>V</td><td>D</td><td>S</td><td>V</td>

<td> 11</td><td>P</td><td>TO</td><td>P</td><td>P</td><td>P</td><td>T</td><td>V</td><td>TO</td><td>R</td><td>K</td><td>T</td><td>T</td><td>T</td><td>TO</td><td>Q</td><td>Q</td>

<td> 12</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>K</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 13</td><td>R</td><td>P</td><td>P</td><td>R</td><td>R</td><td>R</td><td>P</td><td>P</td><td>P</td><td>N</td><td>I</td><td>P</td><td>P</td><td>L</td><td>P</td><td>P</td>

<td> 14</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td><td>C</td><td>C</td><td>c</td><td>C</td><td>c</td><td>C</td><td>C</td><td>C</td>

<td> 15</td><td>Y</td><td>M</td><td>K</td><td>K</td><td>L</td><td>N</td><td>R</td><td>M</td><td>R</td><td> -</td><td> -</td><td>K</td><td>R</td><td>F</td><td>L</td><td>R</td>

<td> 16</td><td>D</td><td>F</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>G</td><td>TO</td><td>G</td><td>Q</td><td>TO</td><td>TO</td><td>G</td><td>G</td><td>TO</td>

<td> 17</td><td>K</td><td>F</td><td>S</td><td>H</td><td>Y</td><td>L</td><td>R</td><td>M</td><td>F</td><td>P</td><td>T</td><td>K</td><td>G</td><td>Y</td><td>L</td><td>F</td>

<td> 18</td><td>I</td><td>I</td><td>I</td><td>I</td><td>M</td><td>I</td><td>. F</td><td>T</td><td>I</td><td>V</td><td>V</td><td>M</td><td>F</td><td>M</td><td>F</td><td>I</td>

<td> 19</td><td>P</td><td>S</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>s</td><td>Q</td><td>R</td><td>R</td><td>I</td><td>K</td><td>K</td><td>K</td><td>Q</td>

<td> 20</td><td>TO</td><td>TO</td><td>TO</td><td>R</td><td>R</td><td>TO</td><td>R</td><td>R</td><td>L</td><td>TO</td><td>TO</td><td>R</td><td>R</td><td>L</td><td>R</td><td>L</td>

<td> 21</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>Y</td><td>Y</td><td>W</td><td>F</td><td>F</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>W</td>

<td> 22</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>F</td><td>TO</td><td>Y</td><td>Y</td><td>F</td><td>N</td><td>S</td><td>F</td><td>TO</td>

<td> 23</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>F</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>F</td>

<td> 24</td><td>N</td><td>S</td><td>N</td><td>D</td><td>N</td><td>N</td><td>N</td><td>N</td><td>D</td><td>D</td><td>K</td><td>N</td><td>N</td><td>N</td><td>N</td><td>D</td>

<td> 25</td><td>Q</td><td>K</td><td>W</td><td>S</td><td>P</td><td>S</td><td>S</td><td>G</td><td>TO</td><td>T</td><td>P</td><td>TO</td><td>T</td><td>Q</td><td>G</td><td>TO</td>

<td> 26</td><td>K</td><td>G</td><td>TO</td><td>TO</td><td>TO</td><td>H</td><td>S</td><td>T</td><td>V</td><td>R</td><td>s</td><td>K</td><td>R</td><td>AND</td><td>T</td><td>V</td>

<td> 27</td><td>K</td><td>TO</td><td>TO</td><td>S</td><td>S</td><td>L</td><td>s</td><td>S</td><td>K</td><td>L</td><td>TO</td><td>TO</td><td>T</td><td>T</td><td>S</td><td>K</td>

<td> 28</td><td>K</td><td>N</td><td>K</td><td>N</td><td>N</td><td>H</td><td>K</td><td>M</td><td>G</td><td>K</td><td>K</td><td>G</td><td>K</td><td>K</td><td>M</td><td>G</td>

IS 2 124 203 T3

<td>Ν °</td><td> 20</td><td> 21</td><td> 22</td><td> 23</td><td> 24</td><td> 25</td><td> 26</td><td> 27</td><td> 28</td><td> 29</td><td> 30</td><td> 31</td><td> 32</td><td> 33</td><td> 34</td><td> 35</td>

<td> 29</td><td>Q</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>R</td><td>TO</td><td>K</td><td>T</td><td>R</td><td>F</td><td>Q</td><td>N</td><td>TO</td><td>K</td>

<td> 30</td><td>C</td><td>c</td><td>c</td><td>C</td><td>C</td><td>c</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>c</td>

<td> 31</td><td>AND</td><td>Y</td><td>Q</td><td>N</td><td>AND</td><td>Q</td><td>AND</td><td>AND</td><td>V</td><td>K</td><td>V</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>V</td>

<td> 32</td><td>R</td><td>P</td><td>L</td><td>K</td><td>K</td><td>K</td><td>K</td><td>T</td><td>L</td><td>TO</td><td>Q</td><td>T</td><td>P</td><td>AND</td><td>T</td><td>R</td>

<td> 33</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td>

<td> 34</td><td>D</td><td>T</td><td>H</td><td>I</td><td>I</td><td>N</td><td>I</td><td>Q</td><td>P</td><td>Q</td><td>R</td><td>V</td><td>K</td><td>I</td><td>L</td><td>S</td>

<td> 35</td><td>W</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td>

<td></td><td></td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td> —</td><td></td><td> —-</td><td> —</td><td> —</td>

<td> 36</td><td>S</td><td>s</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>R</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 37</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 38</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 39</td><td>G</td><td>R</td><td>K</td><td>P</td><td>R</td><td>G</td><td>G</td><td>M</td><td>Q</td><td>D</td><td>D</td><td>K</td><td>K</td><td>Q</td><td>M</td><td>K</td>

<td> 40</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>TO</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 41</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>D</td><td>D</td><td>K</td><td>N</td><td>N</td><td>N</td><td>N</td>

<td> 42</td><td>S</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>G</td><td>G</td><td>H</td><td>H</td><td>s</td><td>G</td><td>D</td><td>L</td><td>G</td>

<td> 43</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>G</td><td>G</td><td></td><td></td><td>N</td><td>N</td><td>N</td>

<td></td><td> —</td><td></td><td> —</td><td> —</td><td> —</td><td></td><td> —</td><td> —</td><td> —</td><td></td><td></td><td></td><td></td><td> —</td><td></td><td> —</td>

<td> 44</td><td>R</td><td>R</td><td>R</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>K</td><td>N</td><td>N</td><td>N</td><td>R</td><td>R</td><td>N</td><td>K</td>

<td> 45</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td>Y</td><td>F</td><td>F</td><td>F</td>

<td> 46</td><td>K</td><td>K</td><td>S</td><td>K</td><td>K</td><td>K</td><td>H</td><td>V</td><td>Y</td><td>K</td><td>K</td><td>R</td><td>K</td><td>S</td><td>L</td><td>Y</td>

<td> 47</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>s</td><td>T</td><td>s</td><td>S</td><td>s</td><td>T</td><td>S</td><td>s</td>

<td> 48</td><td>I</td><td>I</td><td>I</td><td>w</td><td>w</td><td>I</td><td>L</td><td>AND</td><td>AND</td><td>AND</td><td>D</td><td>TO</td><td>AND</td><td>L</td><td>Q</td><td>Q</td>

<td> 49</td><td>AND</td><td>AND</td><td>AND</td><td>D</td><td>D</td><td>D</td><td>AND</td><td>K</td><td>K</td><td>T</td><td>H</td><td>AND</td><td>Q</td><td>TO</td><td>K</td><td>K</td>

<td> 50</td><td>AND</td><td>AND</td><td>K</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>L</td><td>L</td><td>D</td><td>D</td><td>AND</td><td>AND</td><td>AND</td>

<td> 51</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 52</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>Q</td><td>AND</td><td>L</td><td>R</td><td>R</td><td>R</td><td>M</td><td>L</td><td>AND</td><td>L</td><td>K</td>

<td> 53</td><td>R</td><td>R</td><td>H</td><td>Q</td><td>H</td><td>R</td><td>K</td><td>Q</td><td>AND</td><td>C</td><td>C</td><td>R</td><td>D</td><td>Q</td><td>Q</td><td>AND</td>

<td> 54</td><td>T</td><td>T</td><td>TO</td><td>T</td><td>T</td><td>T</td><td>V</td><td>T</td><td>Y</td><td>AND</td><td>AND</td><td>T</td><td>TO</td><td>K</td><td>T</td><td>Y</td>

<td> 55</td><td>c</td><td>C</td><td>C</td><td>c</td><td>C</td><td>C</td><td>C</td><td>c</td><td>C</td><td>C</td><td>C</td><td>c</td><td>C</td><td>c</td><td>c</td><td>C</td>

<td> 56</td><td>I</td><td>V</td><td>V</td><td>G</td><td>V</td><td>TO</td><td>G</td><td>R</td><td>G</td><td>L</td><td>AND</td><td>G</td><td>S</td><td>I</td><td>R</td><td>G</td>

<td> 57</td><td>G</td><td>V</td><td>G</td><td>TO</td><td>TO</td><td>TO</td><td>V</td><td> -</td><td>V</td><td>V</td><td>L</td><td>G</td><td>G</td><td>N</td><td> -</td><td>I</td>

<td> 58</td><td> -</td><td> -</td><td> -</td><td>S</td><td>S</td><td>K</td><td>R</td><td> -</td><td>P</td><td>Y</td><td>Y</td><td>TO</td><td>F</td><td> -</td><td> -</td><td>P</td>

<td> 59</td><td> -</td><td> -</td><td> -</td><td>TO</td><td>G</td><td>Y</td><td>S</td><td> -</td><td>G</td><td>P</td><td>R</td><td> -</td><td> -</td><td> -</td><td> -</td><td>G</td>

<td> 60</td><td> -</td><td> -</td><td> -</td><td> -</td><td>I</td><td>G</td><td> -</td><td> -</td><td>D</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td>AND</td>

<td> 61</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>AND</td><td></td><td></td><td></td><td></td><td></td><td></td><td>TO</td>

IS 2 124 203 T3

RN ° -5 -4 -3 -2 -1 1 2

9

21 22

27

TABLE 13 (continued) (Homologues 36-40)

37 38 39 40

- Z - - RRRRRPPPPPDDDDDFFFFF ccccc LLLLLEEBBBPPPPPPPPPP

YYYYYTTTTTGGGGGPPPPPC CCCCRKKKKAAAAARRRRKI Μ I Μ MIIIIIRRRRR

YYYYYFFFFF

YYYYYNNNNNAAAAAKKKKKA AAAA

IS 2 124 203 T3

<td rowspan="2"> 28 29 30</td><td colspan="2">GG</td><td rowspan="2">G L C</td><td colspan="2">GG</td>

<td>L C</td><td>L C</td><td>L C</td><td>F C</td>

<td> 31</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>AND</td>

<td> 32</td><td>T</td><td>P</td><td>P</td><td>P</td><td>T</td>

<td> 33</td><td>F</td><td>F</td><td>F</td><td>F</td><td>F</td>

<td> 34</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td>

<td> 35</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td>

<td> 36</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 37</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td>

<td> 38</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td>

<td> 39</td><td>R</td><td>R</td><td>R</td><td>R</td><td>K</td>

<td> 40</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td>

<td> 41</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td>

<td> 42</td><td>R</td><td>s</td><td>R</td><td>R</td><td>s</td>

<td> 43</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td>

TABLE 13 (continued)

RN ° 36 37 38 39 40

NNNNN ⁴⁵ Σ I f F Σ

KKKKR

SSSSS

AASAA

EEEEE

DDDDD ⁵¹ 2 CCCC

Ε Μ Μ Μ M

RRRRR

TTTTT ⁵⁵ 2 CCCC

GGGGG

GGGGG

AAAAA

59----60----61 - - - - 30

IS 2 124 203 T3

Legend to Table 13

BPTI

Recombinant BPTI from MARK87

Recombinant BPTI from MARK87

Bovine Colostrum (DUFT85)

Bovine Serum (DUFT85)

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Recombinant BPTI, AUER87

Venom I of Dendroaspis polyiepis polyiepis (black mamba) (DUFT85)

Dendroaspis polyiepis polyiepis (black mamba) poison K (DUFT85)

Hemachatus hemachates (Ringhal cobra) HHV II (DUFT85)

NNV II of Naja nivea (Cape cobra) (DUFT85)

Vipera russelli's RVV II (Russel's Viper) (TAKA74)

Red Sea Turtle Egg White (DUFT85)

Snail mucus (Helix pomatia) (WAGN78)

Dendroaspis angusticeps (eastern green mamba) toxin C13 S1 C3 (DUFT85)

Dendroaspis angusticeps (eastern green mamba) toxin C13 S2 C3 (DUFT85)

Dendroaspis polyiepis polylepes (black mamba) toxin B (DUFT85)

Dendroaspis polyiepis polylepes (black mamba) toxin E (DUFT85)

Vipera ammodytes IT toxin (DUFT85)

Vipera ammodytes CTI toxin (DUFT85)

Bungarus fasciatus toxin VIII B (DUFT85)

5 Anemonia sulcata II (Sea Anemone) (DUFT85)

Homo sapiens HI-8e, domain "inactive" (DUFT85)

HI-8t from Homo sapiens, "active" domain (DUFT85)

Bungarotoxin beta B1 (DUFT85)

Bungarotoxin beta B2 (DUFT85)

Bovine spleen TI II (FIOR85)

Tachypleus tridentatus (horseshoe crab) hemocyte inhibitor (NAKA87)

Bombyx mori SCI-III (silkworm) (SASA84)

IS 2 124 203 T3

Bos taurus BI-14 (inactive)

Bos taurus BI-8 (active)

Recombinant BPTI (KR15, ME52): Auerswald '88, Biol Chem Hoppe-Seyler, 369 Supplement, pp 27-35.

Isoaprotinin G-1: Siekmann, Wenzel, Schroder, and Tschesche '88, Biol Chem Hoppe-Seyler, 369: 157-163.

Isoaprotinin 2: @Siekmann, Wenzel, Schroder, and Tschesche '88, Biol Chem Hoppe-Seyler, 369: 157-163.

Isoaprotinin G-2: Siekmann, Wenzel, Schroder, and Tschesche '88, Biol Chem Hoppe-Seyler, 369: 157-163.

Isoaprotinin 1: Siekmann, Wenzel, Schroder, and Tschesche '88, Biol Chem Hoppe-Seyler, 369: 157-163.

Notes:

a) the two beta bungarotoxins have residue 15 deleted .

b) B. mori has an extra residue between C5 and C14; we have assigned F and G to remainder 9.

c) all natural proteins have C in positions 5, 14, 30, 38, 50 and 55.

d) all homologues have F33 and G37.

e) Extra C residues in bungarotoxins form interchain cystine bridges.

(Table goes to next page)

IS 2 124 203 T3

TABLE 15

Frequency of amino acids at each position in the BPTI and 58 homologues

<td>ID of</td><td colspan="3">amino acids</td><td></td><td></td><td></td>

<td>beef.</td><td colspan="2">different</td><td colspan="2">Contents</td><td></td><td>First</td>

<td> -5</td><td> 2</td><td> -58</td><td>D</td><td></td><td></td><td></td>

<td> -4</td><td> 2</td><td> -58</td><td>AND</td><td></td><td></td><td></td>

<td> -3</td><td> 5</td><td> -55</td><td>PTZF</td><td></td><td></td><td></td>

<td> -2</td><td> 10</td><td> -43</td><td>R3 Z3 Q3</td><td>T2</td><td>EG Η KL</td><td></td>

<td> -1</td><td> 11</td><td> -41</td><td>D4 P3 R2</td><td>T2</td><td>Q2 GKNZE</td><td></td>

<td> 1</td><td> 13</td><td>R35</td><td>K6 T4 A3</td><td>H2</td><td>G2LHNPID-</td><td>R</td>

<td> 2</td><td> 10</td><td>Q35</td><td>R6 A4 V4</td><td>H3</td><td>E3 NFIL</td><td>P</td>

<td> 3</td><td> 11</td><td>D32</td><td>K8 S4 A3</td><td>T3</td><td>R2 E2 P2 GLY</td><td>D</td>

<td> 4</td><td> 9</td><td>F34</td><td>A6 D4 L4</td><td>S4</td><td>Y3 12 WV</td><td>F</td>

<td> 5</td><td> 1</td><td>C59</td><td></td><td></td><td></td><td>C</td>

<td> 6</td><td> 13</td><td>L25</td><td>N7 E6 K4</td><td>Q4</td><td>13 D2 S2 Y2 RFTA</td><td>L</td>

<td> 7</td><td> 7</td><td>L28</td><td>E25 K2 F</td><td>Q 5</td><td>; T</td><td>AND</td>

<td> 8</td><td> 10</td><td>Q46</td><td>H3 D2 G2</td><td>EI</td><td>: KLAQ</td><td>P</td>

<td> 9</td><td> 12</td><td>P30</td><td>A9 14 V4</td><td>R3</td><td>Y3 LFQ Η EK</td><td>P</td>

<td>9a</td><td> 2</td><td> -58</td><td>G</td><td></td><td></td><td></td>

<td> 10</td><td> 9</td><td>Y24</td><td>E8 D8 V6</td><td>R3</td><td>S3 A3 N3 I</td><td>Y</td>

<td> 11</td><td> 11</td><td>T31</td><td>Q8 P7 R3</td><td>A3</td><td>Y2 KSDVI</td><td>T</td>

<td> 12</td><td> 2</td><td>G58</td><td>K</td><td></td><td></td><td>K</td>

<td> 13</td><td> 5</td><td>Q45</td><td>R7 L4 12</td><td>N</td><td></td><td>P</td>

<td> 14</td><td> 3</td><td>C57</td><td>AT</td><td></td><td></td><td>c</td>

<td> 15</td><td> 12</td><td>K22</td><td colspan="2">R12 L7 V6 Y3</td><td>M2 -2 NIAFG</td><td>K</td>

<td> 16</td><td> 7</td><td>A41</td><td>G9 F2 D2</td><td>K2</td><td>Q2 R</td><td>TO</td>

<td> 17</td><td> 14</td><td>R19</td><td>L8 K7 F5</td><td>M4</td><td>Y4 H2 A2 S2 G2 INT</td><td>PR</td>

<td> 18</td><td> 8</td><td> 141</td><td>M7 F4 L2</td><td>V2</td><td>ETA</td><td>I</td>

<td> 19</td><td> 10</td><td> 124</td><td>P12 R8 K5</td><td>, S4</td><td>Q2 LNET</td><td>I</td>

<td> 20</td><td> 5</td><td>R39</td><td>A8 L6 S5</td><td>Q</td><td></td><td>R</td>

<td> 21</td><td> 5</td><td>Y35</td><td>F17 W5 I</td><td>L</td><td></td><td>Y</td>

<td> 22</td><td> 6</td><td></td><td>F32 Y18</td><td>TO 5</td><td>H2 SN</td><td>F</td>

<td> 23</td><td> 2</td><td>Y52</td><td>F7</td><td></td><td></td><td>Y</td>

<td> 24</td><td> 4</td><td>N47</td><td>D8 K3 S</td><td></td><td></td><td>N</td>

<td> 25</td><td> 13</td><td>A29</td><td>S6 Q4 G4</td><td>W4</td><td>P3 T2 L2 RNKVI</td><td>TO</td>

<td> 26</td><td> 11</td><td>K31</td><td>A9 T5 S3</td><td>V3</td><td>R2 E2 GHFQ</td><td>K</td>

<td> 27</td><td> 8</td><td>A32</td><td>Sil K5 T4</td><td>Q3</td><td>L2 IE</td><td>TO</td>

<td> 28</td><td> 7</td><td>G32</td><td>K13 N5 M4</td><td>Q2</td><td>R2 H</td><td>G</td>

<td> 29</td><td> 10</td><td>L22</td><td colspan="3">K13 Qll A5 F2 R2 NG Μ T</td><td>L</td>

<td> 30</td><td> 2</td><td>C58</td><td>TO</td><td></td><td></td><td>C</td>

<td> 31</td><td> 10</td><td>Q25</td><td>E17 L5 V5</td><td>K2</td><td>NARIY</td><td>Q</td>

<td> 32</td><td> 11</td><td>T25</td><td>Pll K4 Q4</td><td>L4</td><td>R3 E3 G2 SAV</td><td>T</td>

<td> 33</td><td> 1</td><td>F59</td><td></td><td></td><td></td><td>F</td>

<td> 34</td><td> 13</td><td>V24</td><td>110 T5 N3</td><td>Q3</td><td>D3 K3 F2 H2 RSPL</td><td>V</td>

<td> 35</td><td> 2</td><td>Y56</td><td>W3</td><td></td><td></td><td>Y</td>

<td> 36</td><td> 3</td><td>G50</td><td>S8 R</td><td></td><td></td><td>G</td>

<td> 37</td><td> 1</td><td>G59</td><td></td><td></td><td></td><td>G</td>

<td> 38</td><td> 3</td><td>C57</td><td>AT</td><td></td><td></td><td>C</td>

IS 2 124 203 T3

TABLE 15 (continued)

Id. Amino acids first

<td>of</td><td>different res</td><td colspan="2">I contained</td><td>do</td><td></td><td></td><td></td><td></td>

<td> 39</td><td> 9</td><td></td><td>R2</td><td>5 G13 K6</td><td>Q4</td><td colspan="2">E3 M3</td><td>L2 D2 PR</td>

<td> 40</td><td> 2</td><td>G35</td><td>A24</td><td></td><td></td><td></td><td></td><td>TO</td>

<td> 41</td><td> 3</td><td>N33</td><td>K24</td><td>D2</td><td></td><td></td><td></td><td>K</td>

<td> 42</td><td> 12</td><td>R22</td><td>A12</td><td>G8 S6 Q2</td><td>! H2</td><td>N2</td><td>M</td><td>DEKLR</td>

<td> 43</td><td> 2</td><td>N57</td><td>G2</td><td></td><td></td><td></td><td></td><td>N</td>

<td> 44</td><td> 3</td><td>N40</td><td>R14</td><td>K5</td><td></td><td></td><td></td><td>N</td>

<td> 45</td><td> 2</td><td>F58</td><td>Y</td><td></td><td></td><td></td><td></td><td>F</td>

<td> 46</td><td> 11</td><td>K39</td><td>Y5</td><td>E4 S2 V2</td><td>D2</td><td>RH</td><td>T</td><td>AL K</td>

<td> 47</td><td> 2</td><td>S36</td><td>T23</td><td></td><td></td><td></td><td></td><td>S</td>

<td> 48</td><td> 11</td><td>A23</td><td> 111</td><td>E6 Q6 L4</td><td>K2</td><td>T2</td><td>W2</td><td>ARDS</td>

<td> 49</td><td> 8</td><td>E37</td><td>K8</td><td>D6 Q3 A2</td><td>PH</td><td>T</td><td></td><td>AND</td>

<td> 50</td><td> 7</td><td>E27</td><td>D25</td><td>K2 L2 M</td><td>QY</td><td></td><td></td><td>D</td>

<td> 51</td><td> 2</td><td>C58</td><td>TO</td><td></td><td></td><td></td><td></td><td>C</td>

<td> 52</td><td> 9</td><td>M17</td><td>R15</td><td>E8 L7 K6</td><td>Q2</td><td>T2</td><td>H</td><td>VM</td>

<td> 53</td><td> 11</td><td>R37</td><td>E6 &lt;</td><td>Q5 K2 C2</td><td>H2.</td><td>AN</td><td>G</td><td>DWR</td>

<td> 54</td><td> 8</td><td>T41</td><td>Y5.</td><td>A4 V3 12</td><td>E2:</td><td>Μ K</td><td></td><td>T</td>

<td> 55</td><td> 1</td><td>C59</td><td></td><td></td><td></td><td></td><td></td><td>C</td>

<td> 56</td><td> 10</td><td>G33</td><td>V9:</td><td>R5 14 E3</td><td>THE</td><td>ST</td><td>K</td><td>G</td>

<td> 57</td><td> 12</td><td>G34</td><td>V6</td><td>-5 A3 R2</td><td> 12</td><td>P2 D</td><td>K</td><td>SLNG</td>

<td> 58</td><td> 10</td><td>A25</td><td> -15</td><td>P7 K3 S2</td><td>Y2</td><td>G2</td><td>F</td><td>DRA</td>

Legend to Table 15

BPTI

Synthetic BPTI, Tan &amp; Kaiser, Biochem. 16 (8) 1531-41

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Semi synthetic BPTI, TSCH87

Recombinant BPTI, AUER87

BPTI Auerswald et al. GB 2 208 511A

BPTI Auerswald et al. GB 2 208 511A

Recombinant BPTI from MARK87

Recombinant BPTI from MARK87

BPTI (KR15, ME52): Auerswald '88, Biol Chem Hoppe-Seyler, 369 Suppl, pp 27-35.

BPTI CA30 / CA51 Eigenbrot et al, Protein Engineering 3 (7) 591-598 ('90)

Isoaprotinin 2 Siekmann et al. '88, Biol Chem Hoppe-Seyler, 369: 157-163.

Isoaprotinin G-2: Siekmann et al. '88, Biol Chem Hoppe-Seyler, 369: 157-163.

IS 2 124 203 T3

Recombinant BPTI, Auerswald et al. GB 2 208 511A

Recombinant BPTI, Auerswald et al. GB 2 208 511A

Recombinant BPTI, Auerswald et al. GB 2 208 511A

Isoaprotinin G-1 Siekmann et al. '88, Biol Chem Hoppe-Seyler, 369: 157-163.

Recombinant BPTI, Auerswald et al. GB 2 208 511A

Recombinant BPTI, Auerswald et al. GB 2 208 511A

Bovine serum (in Dufton '85)

Bovine spleen TI II (FIORBS)

Snail mucus (Helix pomatia0) (WAGN78)

HHV II of Hemachatus hemachates (Ringhal's cobra) (in Dufton '85)

Red Sea Turtle Egg White (in Dufton '85)

Bovine Colostrum (in Dufton '85)

NNV II of Naja nivea (Cape cobra) (in Duft'85)

Bungarus fasciatus toxin VIII B (in Dufton '85)

TI toxin from Vipera ammodytes (in Dufton '85)

Domain 1 of the porcine ITI, (in CREI87)

Human Alzheimer's beta APP protease inhibitor, (SHIN9O)

Equine ITI Domain 1, at Creighton and Charles

Bos taurus BI-8e (inactive) (ITI domain 1)

5 Anemonia sulcata II (Sea Anemone) (in Dufton '85)

Dendroaspis polylepis polylepes (black mamba) toxin E (in Dufton '85)

Vipera russelli's RVV II (Russel's Viper) (TAKA74)

Tachypleus tridentatus (horseshoe crab) hemocyte inhibitor (NAKA87)

LACI 2 (Factor Xa) (WUNT88)

CTI toxin from Vipera ammodytes (in Dufton '85)

Identification codes for Tables 14 and 15

Dendroaspis polylepis polylepis (black mamba) poison K (in Dufton '85)

HI-8e "inactive" domain of Homo sapiens (in Dufton '85)

Green Mamba toxin K, (in CREI87)

Dendroaspis angusticeps (eastern green mamba) toxin C13 S1 C3 (in Dufton '85)

LACI 3

Equine ITI Domain 2, (CREI87)

LACI 1 (VIIa)

Dendroaspis polylepis polylepes (black mamba) toxin B (in Dufton '85)

IS 2 124 203 T3

Swine ITI Domain 2, Creighton and Charles

Homo sapiens HI-8t "active" domain (at Dufton '85)

Bos taurus BI-8t (active)

Tripstatin Kito et al. ('68) JBiol Chem 263 (34) 18104-07

Dendroaspis angusticeps (eastern green mamba) toxin C13 S2 C3 (in Dufton '85)

Green Mamba Venom I Creighton and Charles '87 CSHSQB 52: 511-519.

Bungarotoxin beta B2 (in Dufton '85)

Dendroaspis polylepis polylepis (black mamba) poison I (in Dufton '85)

Bungarotoxin beta B1 (in Dufton '85)

SCI-III of Bombyx mori (silkworm) (SASA84) (Table goes to next page)

IS 2 124 203 T3

TABLE 61

Variability of natural Kunitz domains

<td colspan="4">Id amino acids</td><td rowspan="2">BPTI</td>

<td>of</td><td>beef</td><td>different</td><td>Contents</td>

<td> 1</td><td></td><td> 12</td><td>R16 K6 T4 A3 H2 G2 Μ NPIL -</td><td>R</td>

<td> 2</td><td></td><td> 9</td><td>P18 R5 A4 V4 E3 NF Η I</td><td>P</td>

<td> 3</td><td></td><td> 10</td><td>D14 K8 S4 A3 T3 G2 E2 LRY</td><td>D</td>

<td> 4</td><td></td><td> 9</td><td>F17 A6 L4 S4 Y3 D2 VWI</td><td>F</td>

<td> 5</td><td></td><td> 1</td><td>C39</td><td>C</td>

<td> 6</td><td></td><td> 12</td><td>L7 N7 E6 K4 Q4 13 D2 S2 Y2 RFTA</td><td>L</td>

<td> 7</td><td></td><td> 5</td><td>L29 E7 FST</td><td>AND</td>

<td> 8</td><td></td><td> 9</td><td>P27 H3 D2 G2 IKLEQ</td><td>P</td>

<td> 9</td><td></td><td> 11</td><td>A10 PIO 14 V4 R3 Y3 HQ Ε KL</td><td>P</td>

<td> 10</td><td></td><td> 9</td><td>E8 V6 Y6 D5 A4 S3 N3 R3 I</td><td>Y</td>

<td> 11</td><td></td><td> 11</td><td>T31 Q8 P7 R3 A3 Y2 KSDVI</td><td>T</td>

<td> 12</td><td></td><td> 1</td><td>G39</td><td>G</td>

<td> 13</td><td></td><td> 4</td><td>P27 R7 L4 I</td><td>P</td>

<td> 14</td><td></td><td> 1</td><td>C39</td><td>C</td>

<td> 15</td><td></td><td> 6</td><td>K18 Rll L4 Y3 M2 N</td><td>K</td>

<td> 16</td><td></td><td> 7</td><td>A25 G7 D2 12 FQR</td><td>TO</td>

<td> 17</td><td></td><td> 12</td><td>K7 R7 F5 M4 H3 Y3 A2 G2 S2 L2 NI</td><td>R</td>

<td> 18</td><td></td><td> 8</td><td>P23 M6 F4 L2 KA Ε T</td><td>I</td>

<td> 19</td><td></td><td> 10</td><td>P13 16 R6 K4 S4 Q2 LN Ε T</td><td>I</td>

<td> 20</td><td></td><td> 5</td><td>R21 A7 L5 S5 Q</td><td>R</td>

<td> 21</td><td></td><td> 5</td><td>F16 Y16 W5 IL</td><td>Y</td>

<td> 22</td><td></td><td> 5</td><td>Y17 F14 A5 H2 N</td><td>F</td>

<td> 23</td><td></td><td> 2</td><td>Y32 P7</td><td>Y</td>

<td> 24</td><td></td><td> 4</td><td>N29 D7 K2 S</td><td>N</td>

<td> 25</td><td></td><td> 13</td><td>All S6 G4 W4 Q3 K2 L2 P2 RITVN</td><td>TO</td>

<td> 26</td><td></td><td> 12</td><td>K13 A9 T5 V3 S2 HDQREFG</td><td>K</td>

<td> 27</td><td></td><td> 8</td><td>A13 S12 K5 Q3 T3 IEL</td><td>TO</td>

<td> 28</td><td></td><td> 7</td><td>G14 K10 N5 M4 H2 Q2 R2</td><td>G</td>

<td> 29</td><td></td><td> 8</td><td>K13 Qll A5 L4 F2 R2 GM</td><td>L</td>

<td> 30</td><td></td><td> 1</td><td>C39</td><td>C</td>

<td> 31</td><td></td><td> 10</td><td>E16 Q8 L5 V4 ANIRKY</td><td>Q</td>

<td> 32</td><td></td><td> 10</td><td>Pll T7 K5 L4 R3 Q3 E2 G2 SV</td><td>T</td>

<td> 33</td><td></td><td> 1</td><td>F39</td><td>F</td>

<td> 34</td><td></td><td> 12</td><td>110 V6 T5 N3 D3 K3 Q2 H2 F2 SPL</td><td>V</td>

<td> 35</td><td></td><td> 2</td><td>Y36 W3</td><td>Y</td>

<td> 36</td><td></td><td> 2</td><td>G31 S8</td><td>G</td>

<td> 37</td><td></td><td> 1</td><td>G39</td><td>G</td>

<td> 38</td><td></td><td> 1</td><td>C39</td><td>C</td>

<td> 39</td><td></td><td> 8</td><td>G14 R9 K6 Q3 M3 L2 Ε P</td><td>R</td>

IS 2 124 203 T3

BPTI

TABLE 61 (continued)

Different beef amino acids ID Content

<td> 40</td><td> 2</td><td></td><td>G33</td><td>A6</td><td></td><td></td><td></td><td></td><td></td>

<td> 41</td><td> 2</td><td></td><td>N33</td><td>K6</td><td></td><td></td><td></td><td></td><td></td>

<td> 42</td><td> 10</td><td></td><td>A13</td><td>G8</td><td>S6</td><td>R4</td><td>N2</td><td>Q2</td><td>EKL</td>

<td> 43</td><td> 1</td><td></td><td>N39</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 44</td><td> 3</td><td></td><td>N20</td><td>R14</td><td colspan="2">l K5</td><td></td><td></td><td></td>

<td> 45</td><td></td><td> 2</td><td>F38</td><td>Y</td><td></td><td></td><td></td><td></td><td></td>

<td> 46</td><td> 11</td><td></td><td>K19</td><td>Y5</td><td>E4</td><td>R2</td><td>V2</td><td>D2</td><td>HST</td>

<td> 47</td><td> 2</td><td></td><td>T22</td><td colspan="2">S17</td><td></td><td></td><td></td><td></td>

<td> 48</td><td> 10</td><td></td><td> 112</td><td>Q6</td><td>TO 5</td><td>E5</td><td>L3</td><td>K2</td><td>T2 W2</td>

<td> 49</td><td> 6</td><td></td><td>E19</td><td>K8</td><td>D7</td><td>Q3</td><td colspan="2">PA</td><td></td>

<td> 50</td><td> 6</td><td></td><td>E27</td><td>D7</td><td>K2</td><td>Μ ζ</td><td>) Y</td><td></td><td></td>

<td> 51</td><td> 1</td><td></td><td>C39</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 52</td><td> 9</td><td></td><td>R13</td><td>M7</td><td>L7</td><td>K6</td><td>Q2</td><td>NB</td><td>[EV</td>

<td> 53</td><td> 10</td><td></td><td>R20</td><td>E6</td><td>Q4</td><td>H2</td><td>K2</td><td>AN</td><td>[GDW</td>

<td> 54</td><td> 6</td><td></td><td>T24</td><td>Y5</td><td>A4</td><td>V3</td><td> 12</td><td>M</td><td></td>

<td> 55</td><td> 1</td><td></td><td>C39</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 56</td><td> 9</td><td></td><td>G15</td><td>HE SAW</td><td>R5</td><td> » 13</td><td>E2</td><td>TO</td><td>STK</td>

<td> 57</td><td> 10</td><td></td><td>G17</td><td>V5</td><td> -5</td><td>A3</td><td>R2</td><td> 12</td><td>P2 SD</td>

<td> 58</td><td> 9</td><td></td><td> -15</td><td>Q7</td><td>A7</td><td>K3</td><td>S2</td><td>G2</td><td>RFD</td>

M

K

TO

K

R

N

N

AL K

TABLE 62

Kunitz sequences used to compile Table 61

BPTI

Isoaprotinin 2 (SIEK88)

Isoaprotinin G-2 (SIEK88)

Isoaprotinin G-1 (SIEK88)

Bovine serum (in DUFT85)

Bovine spleen TI II (FIOR85)

Snail mucus (Helix pomatia) (WAGN78)

HHV II from Hemachatus hemachates (Ringhal's cobra) (in DUFI85)

Red Sea Turtle Egg White (in DUFT85)

Bovine Colostrum (in DUFT885)

NNV II of Naja nivea (Cape cobra) (in DUFT85)

Bungarus fasciatus toxin VIII B (in DUFT85)

Vipera ammodytes IT toxin (in Dufton '85)

Domain 1 of the porcine ITI (in CREI87)

Inhibitor of human Alzheimer's β APP protease (SINH90)

Equine ITI Domain 1 (CREI87)

IS 2 124 203 T3

Bos taurus BI-8e (inactive) (ITI domain 1) (in CREI87)

5 Anemonia sulcata II (Sea Anemone) (in DUFT85)

Dendroaspis polylepis polylepes (black mamba) toxin B (in DUFT85)

Vipera russelli's RVV II (Russel's Viper) (TAKA74)

Tachypleus tridentatus (horseshoe crab) hemocyte inhibitor (NAKA87)

LACI 2 (FactorXa) (WUNT88)

Vipera ammodytes CTI toxin (in DUFT85)

Naja naja naja poison (SHAF9O)

Dendroaspis polylepis polylepis (black mamba) venom K (in DUFT85)

HI-8e Homo sapiens "inactive" domain (in DUFT85)

Green Mamba toxin K, (in CREI87)

Dendroaspis angusticeps (eastern green mamba) toxin C13 S1 C3 (in DUFT85)

LACI 3 (WUNT88)

Equine ITI Domain 2 (CREI87)

LACI 1 (VIIa) (GIRA9O)

Dendroaspis polylepis polylepes (black mamba) toxin B (in DUFT85)

Domain 2 of the porcine ITI (CREI87)

Homo sapiens HI-8t "active" domain (in DUFT85)

Bos taurus BI-8t (active) (at CREI87)

Tripstatin (KITO88)

Dendroaspis angusticeps (eastern green mamba) toxin C13 S2 C3 (in DUFT85)

Green Mamba Poison I (in CREI87)

Dendroaspis polylepis polylepis (black mamba) poison I (in DUFT85)

TABLE 63

Histogram of (number of residues with given variability) as a function of variability

<td>N different 58</td><td>locations 51</td><td>central sites</td>

<td> 1</td><td> 10</td><td> 10</td>

<td> 2</td><td> 7</td><td> 7</td>

<td> 3</td><td> 1</td><td> 1</td>

<td> 4</td><td> 2</td><td> 2</td>

<td> 5</td><td> 4</td><td> 4</td>

<td> 6</td><td> 4</td><td> 4</td>

<td> 7</td><td> 2</td><td> 2</td>

<td> 8</td><td> 4</td><td> 4</td>

<td> 9</td><td> 7</td><td> 5</td>

<td> 10</td><td> 8</td><td> 7</td>

<td> 11</td><td> 3</td><td> 3</td>

<td> 12</td><td> 5</td><td> 4</td>

<td> 13</td><td> 1</td><td> 1</td>

IS 2 124 203 T3

TABLE 64

Natural Kunitz Domains Table References (Table 62) CREI87 Creighton &amp; Charles (1987) Cold Spring Harbor Symp QuantBiol 52: 511-519. DUFT85 Dufton (1985) Eur J Biochem 153: 647-654.

FIOR85 Fioretti et al. (1985) J Biol Chem 260: 11451-11455.

GIRA90 Girard et al. (1990) Science 248: 1421-24.

KITO88 Kito et al. (1988) J Biol Chem 263 (34) 18104-07

NAKA87 Nakamura et al. (1987) J Biochem 101: 1297-1306.

SHAF90 Shafqat et al. (1990) Eur J Biochem 194: 337-341.

SIEK 88 Siekmann et al. (1988) Biol Chem Hoppe-Seyler, 369: 157-163.

TAKA74 Takahashi et al. (1974) JBiochem 76: 721-733.

WAGN78 Wagner et al. (1978) Eur J Biochem 89: 367-377.

WUNT88 Wun et al. (1988) J Biol Chem 263: 6001-4.

(Table goes to next page)

IS 2 124 203 T3

TABLE 65

Effect of mutations in Kunitz domains on binding to serine proteases

Lessons:

A No major effects are expected if the molecular charge remains in the range of -1 to + 1.

B No major effects are expected, but they are more likely than in “A”

C Residual at junction interface, all changes must be tried X Substitutions are not allowed.

Id.

EpiNEl Substitutions Class res

<td> 1</td><td>R</td><td>any</td><td>TO</td>

<td> 2</td><td>P</td><td>any</td><td>TO</td>

<td> 3</td><td>D</td><td>any</td><td>TO</td>

<td> 4</td><td>F</td><td>Y, W, L</td><td>B</td>

<td> 5</td><td>C</td><td>C</td><td>X</td>

<td> 6</td><td>L</td><td>different from Pro</td><td>TO</td>

<td> 7</td><td>AND</td><td>L, S, T, D, N, K, R</td><td>TO</td>

<td> 8</td><td>P</td><td>any</td><td>TO</td>

<td> 9</td><td>P</td><td>any</td><td>TO</td>

<td> 10</td><td>Y</td><td>pref. different from Pro</td><td>B</td>

<td> 11</td><td>T</td><td>any</td><td>C</td>

<td> 12</td><td>G</td><td>it has to be G</td><td>X</td>

<td> 13</td><td>P</td><td>any</td><td>C</td>

<td> 14</td><td>C</td><td>C highly preferred, anyone but Pro</td><td>C</td>

<td> 15</td><td>I</td><td>GOES</td><td>c</td>

<td> 16</td><td>TO</td><td></td><td>c</td>

<td> 17</td><td>F</td><td>L, I, N, Y, W, Η, V</td><td>c</td>

<td> 18</td><td>F</td><td>Y, W, H</td><td>c</td>

<td> 19</td><td>P</td><td>any</td><td>c</td>

<td> 20</td><td>R</td><td>pref. different from Pro</td><td>c</td>

<td> 21</td><td>Y</td><td>F, And more pref .; W, I, L pref .; Μ, V allowed</td><td>c</td>

<td> 22</td><td>F</td><td>Y, F more pref .; pref. different from Pro</td><td>Y, FB</td>

<td> 23</td><td>Y</td><td>Y, F very pref.</td><td>F, YB</td>

<td> 24</td><td>N</td><td>pref. different from Pro</td><td>TO</td>

<td> 25</td><td>TO</td><td>any</td><td>TO</td>

<td> 26</td><td>K</td><td>any</td><td>TO</td>

<td> 27</td><td>TO</td><td>any</td><td>TO</td>

<td> 28</td><td>G</td><td>pref. different from Pro</td><td>TO</td>

<td> 29</td><td>L</td><td>pref. different from Pro</td><td>TO</td>

<td> 30</td><td>C</td><td>it has to be C</td><td>X</td>

<td> 31</td><td>Q</td><td>pref. different from Pro</td><td>B</td>

<td> 32</td><td>T</td><td>pref. different from Pro</td><td>B</td>

<td> 33</td><td>F</td><td>F very strongly pref .; And possible</td><td>X</td>

<td> 34</td><td>V</td><td>any</td><td>C</td>

<td> 35</td><td>Y</td><td>And more pref .; W pref .; F allowed</td><td>B</td>

IS 2 124 203 T3

TABLE 65 (continued)

Go

<td>of beef</td><td>EpiNEl</td><td>Substitutions</td><td>Class</td>

<td> 36</td><td>G</td><td>G very pref .; S, A pref.</td><td>C</td>

<td> 37</td><td>G</td><td>has to be G as long as 38 is C</td><td>X</td>

<td> 38</td><td>C</td><td>C very pref.</td><td>X</td>

<td> 39</td><td>M</td><td>any</td><td>C</td>

<td> 40</td><td>G</td><td>A, S, N, D, T, P</td><td>C</td>

<td> 41</td><td>N</td><td>K, Q, S, D, R, T, A, E</td><td>c</td>

<td> 42</td><td>G</td><td>any</td><td>c</td>

<td> 43</td><td>N</td><td>has to be N</td><td>X</td>

<td> 44</td><td>N</td><td>S, K, R, T, Q, D, E</td><td>B</td>

<td> 45</td><td>F</td><td>Y</td><td>B</td>

<td> 46</td><td>K</td><td>any different from Pro</td><td>B</td>

<td> 47</td><td>ST</td><td>N, A, G</td><td>B</td>

<td> 48</td><td>TO</td><td>any</td><td>B</td>

<td> 49</td><td>AND</td><td>any</td><td>TO</td>

<td> 50</td><td>D</td><td>any</td><td>TO</td>

<td> 51</td><td>C</td><td>it has to be C</td><td>X</td>

<td> 52</td><td>M</td><td>any</td><td>TO</td>

<td> 53</td><td>R</td><td>any</td><td>TO</td>

<td> 54</td><td>T</td><td>any</td><td>TO</td>

<td> 55</td><td>C</td><td>it has to be C</td><td>X</td>

<td> 56</td><td>G</td><td>any</td><td>TO</td>

<td> 57</td><td>G</td><td>any</td><td>TO</td>

<td> 58</td><td>TO</td><td>any</td><td>TO</td>

pref. means preferred (Table goes to next page)

IS 2 124 203 T3

TABLE 203

Effect of pH on the dissociation of the phages BPTI-IIIMK and BPTI (K15L) -IIIMA bound to immobilized HNE

<td rowspan="2">pH</td><td colspan="2">BPTI-III MK</td><td colspan="2">BPTI (K15L) -III MA</td>

<td>Total plaque-forming units in fraction</td><td>% of the added phage</td><td>Total plaque-forming units in fraction</td><td>% of the added phage</td>

<td> 7,0</td><td> 5,0-10⁴</td><td> 2 · 10³</td><td> 1,7-10⁵</td><td> 3,2-10^</td>

<td> 6,0</td><td> 3,8-10⁴</td><td> 2-10³</td><td> 4,5-10⁵</td><td> 8,5-10^-2</td>

<td> 5,0</td><td> 3,5-10⁴</td><td> 1-10³</td><td> 2,1-10⁶</td><td> 4,0-10^-1</td>

<td> 4,0</td><td> 3,0-10⁴</td><td> 1-10³</td><td> 4,3-10⁶</td><td> 8,2-10^-1</td>

<td> 3, 0</td><td> 1,4-10⁴</td><td> 1-10³</td><td> 1,1-10⁶</td><td> 2,1-10^-1</td>

<td> 2,2</td><td> 2,9-10⁴</td><td> 1-10³</td><td> 5,9-10⁴</td><td> 1,1-10^-2</td>

Percentage of Percentage of phage added = 8.0-10^-3 phage added = 1.56 recovered recovered

The total of phage BPTI-III MK added was 0.030 ml x (8.6 10¹⁰ pfu / ml) = 2.6 · 10⁹.

The total phage BPTI (K15L) -III MA added was 0.030 ml x (1.7 10¹⁰ pfu / ml) = 5.2 10⁸.

Since the infectivity of the BPTI (K15L) -III MA phage is 5 times less than that of the BPTI-III MK phage, the aggregate amount of phage used ensures that there is an equivalent number of phage particles added to the immobilized HNE.

(Table goes to next page)

IS 2 124 203 T3

TABLES 207-208 (combined)

<td colspan="3">Sequences of the EpiNE clones in the P1 region</td>

<td>IDENTIFIER</td><td>SEQUENCE</td><td></td>

<td>OF THE CLONE</td><td> 111111122 345678901</td><td></td>

<td>BPTI (only comp.</td><td>) PCKARIIRY</td><td>(BPTI)</td>

<td></td><td>PCVAMFQRY CCT.TGC.GTG.GCT.ATa.TTC.CAA.CGC.TAT</td><td>Epinea</td>

<td> 3, 9, 16,</td><td>PCVGFFSRY</td><td>EpiNE3</td>

<td> 17, 18, 19</td><td>CCT.TGC.GTC.GGT.TTC.TTC.TCA.CGC.TAT</td><td></td>

<td> 6</td><td>PCVGFFQRY CCT.TGC.GTC.GGT.TTC.TTC.CAA.CGC.TAT</td><td>EpiNE6</td>

<td> 7, 13, 14</td><td>PCVAMFPRY</td><td>EpiNE7</td>

<td> 15, 20</td><td>CCT.TGC.GTC.GCT.ATG.TTC.CCA.CGC.TAT</td><td></td>

<td> 4</td><td>PCVAI FPRY CCT.TGC.GTC.GCT.ATC.TTC.CCA.CGC.TAT</td><td>EpiNE4</td>

<td> 8</td><td>PCVAI FKRS CCT.TGC.GTC.GCT.ATC.TTC.AAA.CGC.TCT</td><td>EpiNE8</td>

<td> 1, 10</td><td>PCIAFFPRY</td><td>EpiNEl</td>

<td> 11, 12</td><td>CCT.TGC.ATC.GCT.TTC.TTC.CCA.CGC.TAT</td><td></td>

<td> 5</td><td>PCIAFFQRY CCT.TGC.ATC.GCT.TTC.TTC.CAA.CGC.TAT</td><td>EpiNE5</td>

<td> 2</td><td>PCIALFKRY CCT.TGC.ATC.GCT.TTG.TTC.AAA.CGC.TAT</td><td>EpiNE2</td>

IS 2 124 203 T3

TABLE 209

DNA sequences and predicted amino acid sequences around the P1 region of BPTI analogs selected to bind cathepsin G

Clone Ρ1

<td></td><td colspan="2"> 10</td><td> 15</td><td> 16</td><td> 17</td><td> 18</td><td> 19</td><td> 39</td><td> 40</td><td> 41</td><td> 42</td><td> 52</td><td>F</td>

<td>BPTI</td><td></td><td>TYR</td><td>LYS</td><td>TO</td><td>ARG</td><td>ILE</td><td>ILE</td><td>ARG</td><td>TO</td><td>LYS</td><td>ARG</td><td>MET</td><td> -</td>

<td colspan="2">BRINK</td><td>TYR</td><td>PHE</td><td>TO</td><td>PHE</td><td>ILE</td><td>ILE</td><td>ARG</td><td>TO</td><td>LYS</td><td>ARG</td><td>GLU</td><td> -</td>

<td>EpiC</td><td> 1</td><td>TYR</td><td>MET</td><td>GLY</td><td>PHE</td><td>TO BE</td><td>LYS</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 3/7</td>

<td>EpiC</td><td> 7</td><td>TYR</td><td>MET</td><td>TO</td><td>LEU</td><td>PHE</td><td>LYS</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 1/7</td>

<td>EpiC</td><td> 8</td><td>ASN</td><td>PHE</td><td>TO</td><td>ILE</td><td>THR</td><td>PRO</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 1/7</td>

<td>EpiC</td><td> 10</td><td>TYR</td><td>MET</td><td>TO</td><td>LEU</td><td>PHE</td><td>GLN</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 1/7</td>

<td>EpiC</td><td> 20</td><td>TYR</td><td>MET</td><td>TO</td><td>ILE</td><td>TO BE</td><td>PRO</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 1/7</td>

<td>EpiC</td><td> 31</td><td>TYR</td><td>MET</td><td>TO</td><td>ILE</td><td>TO BE</td><td>PRO</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 2/15</td>

<td>EpiC</td><td> 32</td><td>TYR</td><td>MET</td><td>TO</td><td>ILE</td><td>TO BE</td><td>PRO</td><td>GLU</td><td>TO</td><td>LYS</td><td>ARG</td><td>MET</td><td> 7/15</td>

<td>EpiC</td><td> 33</td><td>TYR</td><td>MET</td><td>ASP</td><td>ILE</td><td>TO BE</td><td>PRO</td><td>MET</td><td>GLY</td><td>ASN</td><td>GLY</td><td>MET</td><td> 1/15</td>

<td>EpiC</td><td> 34</td><td>TYR</td><td>MET</td><td>ASP</td><td>ILE</td><td>TO BE</td><td>PRO</td><td>GLU</td><td>TO</td><td>LYS</td><td>ARG</td><td>MET</td><td> 4/15</td>

<td>EpiC</td><td> 35</td><td>TYR</td><td>MET</td><td>ASP</td><td>ILE</td><td>TO BE</td><td>PRO</td><td>GLU</td><td>TO</td><td>LYS</td><td>ARG</td><td>MET</td><td> 1/15</td>

TABLE 211

Effect of antisera on phage infectivity

<td>Phage (dilution)</td><td>Incubation conditions</td><td>pfu / ml</td><td>Title relative</td>

<td>MA-ITI</td><td>PBS</td><td></td><td></td>

<td> 1,2-10¹¹</td><td> 1,00</td><td></td><td></td>

<td> (10^_1)</td><td>NRS</td><td> 6, 8-10¹⁰</td><td> 0,57</td>

<td></td><td>anti-ITI</td><td> 1,1-10¹⁰</td><td> 0,09</td>

<td>MA-ITI</td><td>PBS</td><td> 7,7-10®</td><td> 1,00</td>

<td> (10'³)</td><td>NRS</td><td> 6,7-10¹⁰</td><td> 0, 87</td>

<td></td><td>anti-ITI</td><td> 8,0-10⁶</td><td> 0,01</td>

<td>MA</td><td>PBS</td><td> 1, 3-10¹²</td><td> 1, 00</td>

<td> (10’¹)</td><td>NRS</td><td> 1,4-10¹²</td><td> 1,10</td>

<td></td><td>anti-ITI</td><td> 1, 6-10¹²</td><td> 1,20</td>

<td>MA</td><td>PBS</td><td> 1,3-10¹⁰</td><td> 1,00</td>

<td>(ΙΟ '³)</td><td>NRS</td><td> 1,2-10¹⁰</td><td> 0, 92</td>

<td></td><td>anti-ITI</td><td> 1,5-10¹⁰</td><td> 1,20</td>

IS 2 124 203 T3

TABLE 212

Fractionation of EpiNE-7 and MA-ITI phages on hNE spheres

<td></td><td colspan="2">EpiNE-7</td><td colspan="3">MA-ITI</td>

<td>Show</td><td>total pfu in the</td><td>Fraction of</td><td colspan="2">total pfu in the</td><td>Fraction of</td>

<td></td><td>show</td><td>aggregate</td><td colspan="2">show</td><td>aggregate</td>

<td>AGGREGATE Final wash</td><td> 3,3-10⁹</td><td> 1,00</td><td> 3,4</td><td> 10¹¹</td><td> 1, 00</td>

<td colspan="2">with TBS-TWEEN 3,8-10⁵</td><td> 1,2-10^-4</td><td> 1,8</td><td> 10^-6</td><td> 5,3-10^-6</td>

<td>pH 7.0</td><td> 6,2-10⁵</td><td> 1,8-10^-4</td><td> 1,6</td><td> 10⁶</td><td> 4,7-10^-6</td>

<td>pH 6.0</td><td> 1,4-10⁶</td><td> 4,1-10^-4</td><td> 1,0</td><td> 10⁶</td><td> 2,9-10^-6</td>

<td>pH 5.5</td><td> 9,4-10⁵</td><td> 2,8-10^-4</td><td> 1,6</td><td> 10⁶</td><td> 4,7-10^-6</td>

<td>pH 5.0</td><td> 9,5-10⁵</td><td> 2,9-10^-4</td><td> 3,1</td><td> 10⁵</td><td> 9,1-10^-7</td>

<td>pH 4.5</td><td> 1,2-10⁶</td><td> 3,5-10^-4</td><td> 1,2</td><td> 10⁵</td><td> 3,5-10^-7</td>

<td>pH 4.0</td><td> 1,6-10⁶</td><td> 4,8-10^-4</td><td> 7,2</td><td> 10⁴</td><td> 2,1-10^-7</td>

<td>pH 3.5</td><td> 9,5-10⁵</td><td> 2,9-10^-4</td><td> 4,9</td><td> 10⁴</td><td> 1,4 · 10^-7</td>

<td>pH 3.0</td><td> 6,6-10⁵</td><td> 2,0-10^-4</td><td> 2,9</td><td> 10⁴</td><td> 8,5-10^-8</td>

<td>pH 2.5</td><td> 1,6-10⁵</td><td> 4,8-10^-5</td><td> 1,4</td><td> 10⁴</td><td> 4,1-10^-8</td>

<td>pH 2.0</td><td> 3,0-10⁵</td><td> 9,1-10^-5</td><td> 1,7-</td><td> 10⁴</td><td> 5,0-10^-8</td>

<td>Sum*</td><td> 6,4-10⁶</td><td> 3-10^-5</td><td> 5,7-</td><td>Tó *</td><td> 2-10^-5</td>

* Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 213

Fractionation of EpiC-10 and MA-ITI phages on Cat-G spheres

<td></td><td>EpiC-</td><td> 10</td><td></td><td>MA-ITI</td><td></td>

<td>Show</td><td colspan="2">total pfu in the show</td><td>Fraction of aggregate</td><td>total pfu in the show</td><td>Fraction of aggregate</td>

<td>AGGREGATE</td><td> 5, 0</td><td> 10¹¹</td><td> 1,00</td><td> 4,6-10¹¹</td><td> 1,00</td>

<td>Washed with TBS-</td><td>final TWEEN 1.8</td><td> 10⁷</td><td> 3,6-10^-5</td><td> 7,1-10⁶</td><td> 1,5-10^-5</td>

<td>pH 7.0</td><td> 1,5</td><td> 10⁷</td><td> 3,0-10^-5</td><td> 6,1-10⁶</td><td> 1,3-10^-5</td>

<td>pH 6.0</td><td> 2,3</td><td> 10⁷</td><td> 4,6-10^-5</td><td> 2,3-10⁶</td><td> 5,0-10^-6</td>

<td>pH 5.5</td><td> 2,5</td><td> 10⁷</td><td> 5,0-10^-5</td><td> 1,2-10⁶</td><td> 2,6-10^-6</td>

<td>pH 5.0</td><td> 2,1</td><td> 10⁷</td><td> 4,2-10^-5</td><td> 1,1-10⁶</td><td> 2,4 · 10^-6</td>

<td>pH 4.5</td><td> 1,1</td><td> 10⁷</td><td> 2,2-10^-5</td><td> 6,7-10⁵</td><td> 1,5-10^-6</td>

<td>pH 4.0</td><td> 1,9</td><td> 10⁶</td><td> 3,8-10^-6</td><td> 4,4-10⁵</td><td> 9, 6-10^-7</td>

<td>pH 3.5</td><td> 1,1</td><td> 10⁶</td><td> 2,2-10^-6</td><td> 4,4 · 10⁵</td><td> 9, 6-10^-7</td>

<td>pH 3.0</td><td> 4,8</td><td> 10⁵</td><td> 9, 6-10^-7</td><td> 3,6-10⁵</td><td> 7,8-10^-7</td>

<td>pH 2.5</td><td> 2,0</td><td> 10⁵</td><td> 4,0-10^-7</td><td> 2,7-10⁵</td><td> 5, 9-10^-7</td>

<td>pH 2.0</td><td> 2,4</td><td> 10⁵</td><td> 4,8-10^-7</td><td> 3,2-10⁵</td><td> 7,0-10^-7</td>

<td>Sum*</td><td> 9,9</td><td> 10^z</td><td> 2-10^-4</td><td> 1,4-10⁷</td><td> 3-10^-5</td>

★ Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 214

Abbreviated fractionation of phage on hNE spheres

<td rowspan="2"></td><td colspan="4">FAGO</td>

<td>EpiNE-7</td><td>MA-ITI 2</td><td>MA-ITI-E7</td><td>1 MA-ITI-E7 2</td>

<td>AGGREGATE</td><td> 1, 00</td><td> 1,00</td><td> 1,00</td><td> 1,00</td>

<td>(pfu)</td><td> (1,8-10⁹)</td><td> (1,2-10⁹)</td><td> (3,3-10⁹)</td><td> (1,1-10⁹)</td>

<td>WASHED</td><td> 6-10^-5</td><td> 1-10^-5</td><td> 2-10^-5</td><td> 2-10^-5</td>

<td>pH 7.0</td><td> 3-10^-4</td><td> 1-10^-5</td><td> 2-10⁵</td><td> 4-10^-5</td>

<td>pH 3.5</td><td> 3-10^-3</td><td> 3-10'⁶</td><td> 8-10”⁵</td><td> 8-10^-5</td>

<td>pH 2.0</td><td> 1-10^-3</td><td> 1-10^-6</td><td> 6-10^-6</td><td> 2-10^-5</td>

<td>Sum*</td><td> 4,3-10^-3</td><td> 1,4-10^-5</td><td> 1,1-10^-4</td><td>₁^_4-1θ 4</td>

* Sum is the total fraction of the aggregate obtained from all fractions eluted by pH

IS 2 124 203 T3

TABLE 215

Fractionation of EpiNE-7 and MA-ITI-E7 phages on hNE spheres

<td></td><td colspan="2">EpiNE-7</td><td></td><td></td><td></td><td colspan="2">MA-ITI-E7</td><td></td><td></td><td></td>

<td>Show</td><td>pfu</td><td colspan="2">totals</td><td colspan="2">Fraction</td><td>pfu</td><td>totals</td><td colspan="2">Fraction</td><td></td>

<td></td><td>on</td><td></td><td>the</td><td>of</td><td></td><td>on</td><td>the</td><td>of</td><td></td><td></td>

<td></td><td colspan="2">show</td><td></td><td colspan="2">aggregate</td><td colspan="2">show</td><td colspan="2">aggregate</td><td></td>

<td>AGGREGATE</td><td> 1,8</td><td> 10^to</td><td></td><td colspan="2"> 1,00</td><td> 3,0</td><td> 10^to</td><td colspan="2"> 1,00</td><td></td>

<td>pH 7.0</td><td> 5,2</td><td> 10⁵</td><td></td><td> 2,9</td><td> 10^-4</td><td> 6,4</td><td> 10⁴</td><td> 2,1</td><td> 10'⁵</td><td></td>

<td>pH 6.0</td><td> 6, 4</td><td> 10⁵</td><td></td><td> 3, 6</td><td> 10'⁴</td><td> 4,5</td><td> 10⁴</td><td> 1,5</td><td> 10⁵</td><td></td>

<td>pH 5.5</td><td> 7,8</td><td> 10⁵</td><td></td><td> 4,3</td><td> 10’⁴</td><td> 5,0</td><td> 10⁴</td><td> 1,7</td><td>1O⁵</td><td></td>

<td>pH 5.0</td><td> 8,4</td><td> 10⁵</td><td></td><td> 4,7</td><td> 10’⁴</td><td> 5,2</td><td> 10⁴</td><td> 1,7</td><td> 10'⁵</td><td></td>

<td>pH 4.5</td><td> 1,1</td><td> 10⁶</td><td></td><td> 6,1</td><td> 10'⁴</td><td> 4,4</td><td> 10⁴</td><td> 1,5</td><td> 10'⁵</td><td></td>

<td>pH 4.0</td><td> 1,7</td><td> 10⁶</td><td></td><td> 9,4</td><td> 10’⁴</td><td> 2, 6</td><td> 10⁴</td><td> 8,7</td><td> 10'⁶</td><td></td>

<td>pH 3.5</td><td> 1,1</td><td> 10⁶</td><td></td><td> 6,1</td><td> 10’⁴</td><td> 1,3</td><td> 10⁴</td><td> 4,3</td><td>1Q-6</td><td></td>

<td>pH 3.0</td><td> 3,8</td><td> 10⁵</td><td></td><td> 2,1</td><td> 10’⁴</td><td> 5, 6</td><td> 10³</td><td> 1,9</td><td> 10⁶</td><td></td>

<td>pH 2.5</td><td> 2,8</td><td> 10⁵</td><td></td><td> 1, 6</td><td> 10~⁴</td><td> 4,9</td><td> 10³</td><td> 1,6</td><td> 10⁶</td><td></td>

<td>pH 2.0</td><td> 2,9</td><td> 10⁵</td><td></td><td> 1, 6</td><td> 10^-4</td><td> 2,2</td><td> 10³</td><td> 7,3-</td><td> 10⁷</td><td></td>

<td>Sum*</td><td> 7,6</td><td></td><td></td><td> 4,1</td><td> 10³</td><td> 3,1</td><td> 10⁵</td><td> 1,1'</td><td> 10~⁴</td><td></td>

★ Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 216

Fractionation of MA-EpiNE-7, MA-BITI and MA-BITI-E7 on hNE spheres

<td></td><td colspan="3">MA-BITI</td><td colspan="2">MA-BITI-E7</td>

<td>Show</td><td colspan="2">total pfu in the show</td><td>Fraction of aggregate</td><td>total pfu in the sample</td><td>Fraction of aggregate</td>

<td>AGGREGATE</td><td> 2,0</td><td>l₀io</td><td> 1,00</td><td> 6,0-10^s</td><td> 1,00</td>

<td>pH 7.0</td><td> 2,4</td><td> 10⁵</td><td> 1,2-10^-5</td><td> 2,8-10⁵</td><td> 4,7-10^-5</td>

<td>pH 6.0</td><td> 2,5</td><td> 10⁵</td><td> 1,2-10^-5</td><td> 2,8-10⁵</td><td> 4,7-10^-5</td>

<td>pH 5.0</td><td> 9,6</td><td> 10⁴</td><td> 4,8-10^-6</td><td> 3,7-10⁵</td><td> 6,2-10^-5</td>

<td>pH 4.5</td><td> 4,4</td><td> 10⁴</td><td> 2,2-10^-6</td><td> 3,8-10⁵</td><td> 6,3-10^-5</td>

<td>pH 4.0</td><td> 3,1</td><td> 10⁴</td><td> 1, 6-10^-6</td><td> 2,4-10⁵</td><td> 4,0-10^-5</td>

<td>pH 3.5</td><td> 8,6</td><td> 10⁴</td><td> 4,3-10^-6</td><td> 9,0-10⁴</td><td> 1,5-10^-5</td>

<td>pH 3.0</td><td> 2,2</td><td> 10⁴</td><td> 1,1-10^-6</td><td> 8,9-10⁴</td><td> 1,5-10^-5</td>

<td>pH 2.5</td><td> 2,2</td><td> 10⁴</td><td> 1,1-10^-6</td><td> 2,3-10⁴</td><td> 3,8 · 10^-6</td>

<td>pH 2.0</td><td> 7,7</td><td> 10³</td><td> 3,8-10^-7</td><td> 8,7-10³</td><td> 1,4-10^-6</td>

<td>Sum*</td><td> 8,0</td><td> 10⁵</td><td> 3, 9-10^-5</td><td> 1,8-10⁶</td><td> 2,9-10^-4</td>

★ Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 216 (continued)

Fractionation of MA-EpiNE-7, MA-BITI and MA-BITI-E7 on hNE spheres

<td rowspan="2">Show</td><td colspan="3">MA-EpiNE-7</td>

<td>pfu on</td><td>sample totals</td><td>Fraction of the aggregate</td>

<td>AGGREGATE</td><td> 1,5</td><td> • 10^Y</td><td> 1,00</td>

<td>pH 7.0</td><td> 2,9</td><td> • 10⁵</td><td> 1,9-10^-4</td>

<td>pH 6.0</td><td> 3,7</td><td> • 10⁵</td><td> 2,5-10^-4</td>

<td>pH 5.0</td><td> 4,9</td><td> • 10⁵</td><td> 3,3-10^-4</td>

<td>_PH 4.5</td><td> 6, 0</td><td> • 10⁵</td><td> 4,0-10^-4</td>

<td>pH 4.0</td><td> 6,4</td><td> • 10⁵</td><td> 4,3-10^-4</td>

<td>pH 3.5</td><td> 5,0</td><td> • 10⁵</td><td> 3,3-10^-4</td>

<td>pH 3.0</td><td> 1,9</td><td> • 10⁵</td><td> 1,3-10^-4</td>

<td>pH 2.5</td><td> 7,7</td><td> • 10⁴</td><td> 5,1-10^-5</td>

<td>pH 2.0</td><td> 9,7</td><td> • 10⁴</td><td> 6,5-10^-5</td>

<td>Sum*</td><td> 3,1</td><td> • 10⁶</td><td> 2,2-10^-3</td>

★ Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 217

Fractionation of MA-BITI-E7 and MA-BITI-E7-1222 on hNE spheres

<td></td><td colspan="2">MA-BITI-E7</td><td colspan="2">MA-BITI-E7-1222</td>

<td>Show</td><td>total pfu in the show</td><td>Fraction of aggregate</td><td>total pfu in the show</td><td>Fraction of aggregate</td>

<td>AGGREGATE</td><td> 1,3-10⁹</td><td> 1,00</td><td> 1,2-10⁹</td><td> 1,00</td>

<td>pH 7.0</td><td> 4,7-10⁴</td><td> 3, 6-10^-5</td><td> 4,0-10⁴</td><td> 3,3-10^-5</td>

<td>pH 6.0</td><td> 5,3-10⁴</td><td> 4,1-10^-5</td><td> 5,5-10⁴</td><td> 4,6-10^-5</td>

<td>pH 5.5</td><td> 7,1-10⁴</td><td> 5,5-10^-5</td><td> 5,4-10⁴</td><td> 4,5-10^-5</td>

<td>pH 5.0</td><td> 9,0-10⁴</td><td> 6, 9-10^-5</td><td> 6,7-10⁴</td><td> 5,6-10^-5</td>

<td>pH 4.5</td><td> 6,2-10⁴</td><td> 4,8-10^-5</td><td> 6,7-10⁴</td><td> 5,6-10^-5</td>

<td>pH 4.0</td><td> 3,4-10⁴</td><td> 2,6-10^-5</td><td> 2,7-10⁴</td><td> 2,2-10^-5</td>

<td>pH 3.5</td><td> 1,8-10⁴</td><td> 1,4 · 10^-5</td><td> 2,3-10⁴</td><td> 1,9-10^-5</td>

<td>pH 3.0</td><td> 2,5-10³</td><td> 1,9-10^-6</td><td> 6,3-10³</td><td> 5,2-10^-6</td>

<td>pH 2.5</td><td> &lt;1,3-10³</td><td>&lt;l, 0-10^-6</td><td>&lt;l, 3-10³</td><td> &lt;1,0-10^-6</td>

<td>pH 2.0</td><td> 1,3-10³</td><td> 1,0-10^-6</td><td> 1,3-10³</td><td> 1,0-10^-6</td>

<td>Sum*</td><td> 3,8-10⁵</td><td> 2,9-10^-4</td><td> 3,4-10⁵</td><td> 2,8-10^-4</td>

★ Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 218

Fractionation of MA-EpiNE7 and MA-BITI-E7-141 on hNE spheres

<td></td><td>MA-EpiNE7</td><td>MA-BITI</td><td>-E7-141</td>

<td>Show</td><td>Total pfu Fraction</td><td>total pfu</td><td>Fraction</td>

<td></td><td>in that of</td><td>on the</td><td>of</td>

<td></td><td>sample added</td><td>show</td><td>aggregate</td>

<td>AGGREGATE</td><td> 6,1- 10^B 1, 00</td><td> 2,0-10^to</td><td> 1,00</td>

<td>pH 7.0</td><td> 5,3-10⁴ 8,7 - 10^-5</td><td> 4,5-10⁵</td><td> 2,2-10^-4</td>

<td>pH 6.0</td><td> 9,7-10⁴ 1,6-10^-4</td><td> 4,4-10⁵</td><td> 2,2-10^-4</td>

<td>pH 5.5</td><td> 1,1-10⁵ 1,8-10^-4</td><td> 4,4-10⁵</td><td> 2,2-10^-4</td>

<td>pH 5.0</td><td> 1,4-10⁵ 2,3-10^-4</td><td> 7,2-10⁵</td><td> 3, 6-10^-4</td>

<td>pH 4.5</td><td> 1,0-10⁵ 1,6-10^-4</td><td> 1,3-10⁶</td><td> 6, 5-10^-4</td>

<td>pH 4.0</td><td> 2,0-10⁵ 3, 3-10^-4</td><td> 1,1-10⁶</td><td> 5,5-10^-4</td>

<td>pH 3.5</td><td> 9,7-10⁴ 1,6-10^-4</td><td> 5,9-10⁵</td><td> 3,0-10^-4</td>

<td>pH 3.0</td><td> 3,8- 10⁴ 6, 2-10^-5</td><td> 2,3-10⁵</td><td> 1,2-10^-4</td>

<td>pH 2.5</td><td> 1,3-10⁴ 2, 1-10^-5</td><td> 1,2-10⁵</td><td> 6,0-10^-5</td>

<td>pH 2.0</td><td> 1,6-10⁴ 2, 6-10^-5</td><td> 1,2-10⁵</td><td> 5,0-10^-5</td>

<td>Sum*</td><td> 8, 6-10⁵ 1,4 · 10^-3</td><td> 5,5-10⁶</td><td> 2,8 · 10^-J</td>

★ Sum is the total pfu (or the fraction of the aggregate) obtained from all the fractions eluted by pH

IS 2 124 203 T3

TABLE 219

PH Elution Analysis of HNE Binding of BITI-E7-141 Phage Variants

AGGREGATE FRACTION

RECOVERED TO pH: _ RECOVERY

PFU PROTEIN

<td colspan="2">PRESENTED AGGREGATES⁰</td><td> 7,0^d</td><td> 3,5^d</td><td> 2,0^d</td><td colspan="2">TOTAL® RELATIVE^:</td>

<td>AMIN01^b</td><td> 0, 96</td><td> 0,24</td><td> 2,3</td><td> 0, 35</td><td> 2,9</td><td> 0, 11</td>

<td>AMIN02³</td><td> 6,1</td><td> 0, 57</td><td> 2,1</td><td> 0,45</td><td></td><td> 3 0,12</td>

<td>BITI-E7-1222^b</td><td> 1,2</td><td> 0,72</td><td> 4,0</td><td> 0,64</td><td> , 1 5,4</td><td> 0,21</td>

<td>EpiNE7^b</td><td> 0,72</td><td> 0, 44</td><td> 6,4</td><td> 2,2</td><td> 9,0</td><td> 0,35</td>

<td>MUTPl^to</td><td> 3,9</td><td> 1,8</td><td> 9,2</td><td> 1,2</td><td> 12</td><td> 0,46</td>

<td>MUT1619^b</td><td> 0,78</td><td> 0,82</td><td> 9,9</td><td> 0,84</td><td> 12</td><td> 0,46</td>

<td>MUTQE^to</td><td> 4,7</td><td> 1,2</td><td> 16</td><td> 5,3</td><td> 22</td><td> 0, 85</td>

<td>MUTT26A^b</td><td> 0, 51</td><td> 2,5</td><td> 19</td><td> 3,3</td><td> 25</td><td> 0, 96</td>

<td>BITI-E7-141³</td><td> 1,7</td><td> 2,2</td><td> 18</td><td> 5,4</td><td> 26</td><td> 1,00</td>

<td>BITI-E7-141^b</td><td> 0,75</td><td> 2,1</td><td> 21</td><td> 3,2</td><td> 26</td><td> 1,00</td>

results of the abbreviated pH elution protocol ^b results of the extended pH elution protocol ^c units are 10⁹ pfu ^d units are 10⁴^and sum of that recovered at pH 7.0, pH 3.5 and pH 2.0, the units are 10⁴^F total fraction of recovered aggregate divided by total fraction of recovered aggregate for BITI-E7-141

IS 2 124 203 T3

TABLE 220

WEAK (K_D &gt; ΙΟ⁸ M)

1. KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLQTCRGA MODERATE (ΙΟ^-8 M&gt; K_D &gt; 10'⁹ M)

two. KSDSCQLGYSAGPCVAMFPRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLQTCRGA

3. RPDFCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLQTCRGA STRONG (10⁹ M&gt; K_D &gt; 10¹¹ M)

Four. RPDFCQLGYSAGPCVAMFPRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLOTCRGA

5. RPDFCQLGYSAGPCVAMFPRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLOTCRGA

6. KEDFCQLGYSAGPCVAMFPRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLQTCRGA

7. KPDSCQLGYSAGPCVAMFPRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLQTCRGA

8. RPDFCQLGYSAGPCVAMFPRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLOTCRGA VERY STRONG (K_D &lt; 10'¹¹ M))

1111111111222222222233333333334444444444555555555 1234567890123456789012345678901234567890123456789012345678

9. RPDFCQLGYSAGPCVAMFPRYFYNGTSMACQTFVYGGCMGNGNNFVTEKDCLQTCRGA

10. RPDFCQLGYSAGPCVAMFPRYFYNGASMACQTFVYGGCMGNGNNFVTEKDCLQTCRGA

eleven. RPDFCQLGYSAGPCVAMFPRYFYNGTSMACETFyYGGCMGNGNNFVTEKDCLOTCRGA

12. RPDFCQLGYSAGPCVGMFSRYFYNGTSMACQTFVYGGCMGNGNNFVTEKDCLQTCRGA

The residues that appear underlined and in bold are those that change with respect to those present in ITI-D1. Sequence key

1. ITI-D1

two. ITI-E7

3. BITI

Four. BITI-E7

5. BITI-E7-1222

6. AMINO1

7. AMINO2

8. MUTP1

9. BITI-E7-141

10. MUTT26A

eleven. MUTQE

12. MUT1619

IS 2 124 203 T3

TABLE 221

The information is the same as in Table 220, but focuses on the sites where alterations have been made

WEAK (K_D &gt; 10⁸ M)

1. KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKDCLQTCRGA

<td> 1.</td><td>KE. S</td><td>......TO</td><td colspan="2">. . .MGMTS. . .</td><td>... T ..</td><td>. .AND.</td><td>.Q ........................</td>

<td colspan="2">Moderate</td><td> (10^-8</td><td>M</td><td>&gt; K_D &gt; 10'</td><td> ’⁹ M)</td><td></td><td></td>

<td> 2.</td><td>KS.S</td><td>......TO</td><td></td><td>.VAMFP ...</td><td>... T ..</td><td>. .AND.</td><td>.Q ........................</td>

<td> 3.</td><td>RP. F</td><td>......TO</td><td></td><td>.MGMTS ...</td><td>... T ..</td><td>. .AND.</td><td>• Q ........................</td>

<td colspan="2">STRONG</td><td>(ΙΟ^-9 M</td><td> &gt;</td><td>K_D &gt; 10'¹¹</td><td>M)</td><td></td><td></td>

<td> 4 .</td><td>RP. F</td><td>......TO</td><td></td><td>.VAMFP ...</td><td>... T ..</td><td>. .AND.</td><td>.Q ........................</td>

<td> 5.</td><td>RP. F</td><td>......TO</td><td></td><td>.VAMFP ...</td><td>... T ..</td><td>. .AND.</td><td>.Q ........................</td>

<td> 6.</td><td>KE. F</td><td>......TO</td><td></td><td>.VAMFP ...</td><td>... T ..</td><td>. .AND.</td><td>.Q ........................</td>

<td> 7.</td><td>KP.S</td><td>......TO</td><td></td><td>.VAMFP ...</td><td>... T ..</td><td>. .AND.</td><td>• Q ........................</td>

<td> 8.</td><td>RP.F</td><td>......TO</td><td></td><td>.VAMFP ...</td><td>... T ..</td><td>. .AND.</td><td> Q ........................</td>

<td>VERY</td><td colspan="2">STRONG (K_D</td><td> &lt;</td><td>ΙΟ^-11 M))</td><td></td><td></td><td></td>

<td> 9.</td><td>RP.F</td><td>......TO</td><td></td><td>.VAMFP. . .</td><td>... T ..</td><td> . .0.</td><td>. V ........................</td>

<td> 10.</td><td>RP.F</td><td>......TO</td><td></td><td>.VAMFP. ...</td><td>...TO..</td><td> . .0.</td><td>.V ........................</td>

<td> 11.</td><td>RP.F</td><td>......TO</td><td></td><td>.VAMFP ....</td><td>... T ..</td><td>. .AND.</td><td>. V ........................</td>

<td> 12.</td><td>RP.F</td><td>......TO</td><td></td><td>.VGMFS ....</td><td>.., T ..</td><td>. .Q.</td><td>. V ........................</td>

The sequence code is the same as in Table 220

TABLE 222

K CLASS_D PH FRACTION OF MAXIMUM

ESTIMATED AFFINITY UNITED AGGREGATE OF PROTEIN ELUTION

WEAK K_D &gt; 10⁴ M &lt;0.005%&gt;

MODERATE 10 '⁸ M at 0.01% at

10'⁹ M 0.03%

STRONG 10 '⁹ M at 0.03% at

10¹¹ M 0.06%

VERY K_D &lt; 10'¹¹ M&gt; O, 1%

STRONG

<td>PH</td><td> 6, 0</td><td>ITI-D1</td>

<td>PH</td><td>5.5 a</td><td>BITI</td>

<td>PH</td><td> 5,0</td><td>ITI-E7</td>

<td>PH</td><td>5.0 a</td><td>BITI-E7</td>

<td>pH</td><td> 4,5</td><td>BITI-E7-1222 AMINO1 AMINO2 MUTP1</td>

<td colspan="2">= pH 4.0</td><td>BITI-E7-141 MUTT26A MUTQE MUT1619</td>