CA2214395C

CA2214395C - Proteinase k variants having decreased adsorption and increased hydrolysis

Info

Publication number: CA2214395C
Application number: CA002214395A
Authority: CA
Inventors: Philip Frederick Brode, Iii; Bobby Lee Barnett; Donn Nelton Rubingh
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 1995-03-09
Filing date: 1996-03-06
Publication date: 2001-10-30
Anticipated expiration: 2016-03-06
Also published as: AU5092796A; MX9706841A; CA2214395A1; MA23820A1; HUP9801638A3; WO1996028556A3; BR9607677A; JPH11501814A; US6451574B1; IL117350A0; CN1183809A; WO1996028556A2; CZ279997A3; ZA961820B; HUP9801638A2; EP0813601A2

Abstract

The present invention relates to proteinase K variants having a modified ami no acid sequence of wild-type proteinase K amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loo p region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type proteinase K (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the proteinase K variant has decreased adsorption to, and increased hydrolys is of, an insoluble substrate as compared to the wild-type proteinase K. The present invention also relates to DNA sequences encoding such proteinase K variants. The present invention also relates to compositions comprising such proteinase K variants for cleaning a variety of surfaces.

Description

WO 96!28556 PCT/US96/03005 PROTEINASE K VARIANTS HAVING DECREASED ADSORPTION
AND INCREASED HYDROLYSIS
TECHNICAL FIELD
The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences encoding such enzyme variants.
BACKGROUND
Enzymes make up the largest class of naturally occurring proteins.
Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains.
Unfortunately, the efficacy level of these proteins in their natural.
bacterial environment, frequently does not translate into the relatively unnatural wash environment. Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
The amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces.
Objects of the Present Invention It is an object of the present invention to provide Proteinase K
enzyme variants having improved hydrolysis versus the wild-type of the enzyme.
It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants.
SUMMARY
The present invention relates to Proteinase K variants having a modified amino acid sequence of wild-type Proteinase K amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third bop region, a fourth bop region, a fifth ~ bop region and a sodh bop region; n the modified amino aad sequence comprises di~-rt amino acids than that occurring in wild-type Proteinase K (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Proteinase K variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Proteinase K. The present invention also relates to DNA sequences encoding such Proteinase K variants. The present invention also relates to compositions comprising such Proteinase K variants for cleaning a variety of surfaces.
DESCRIPTION
I. Proteinase K Variants .
This invention pertains to subtilisin enzymes, in particular Proteinase K, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (hereinafter, "Proteinase K
variants") of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to DNA sequences encoding for such Proteinase K variants.
The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site.
The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented. It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration. This has been shown to be the case. (Erode, P.F. III and D. S. Rauch, LANGMUIR, "SubtIIISIn BPN':
Activity on ari Immobilized Substrate", Vol. 8, pp. 1325-1329 (1992)). In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, "Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface," in POLYMER SOLUTIONS, BLENDs AND INTERFACES, Ed. by I. Noda and D. N. Rubingh, Elsevier, p. 464 (1992)).
Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance. In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance).
While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another. is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface.
The mutations described herein are designed to change (i.e., decrease) the adsorption' of the enzyme to surface-bound soils. In Proteinase K, certain amino acids form exterior loops on the enzyme molecule. For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions. Specifically, positions 71 form the first loop region; positions 95-107 form the second loop region;
positions 133-140 form the third loop region; positions 160-170 form the fourth loop region; positions 190-194 form the fifth loop region; and positioins 203-223 form the sixth loop region (position numbering analagous to positions in the amino acid sequence for wild-type subtilisin Proteinase K

(SEQ ID N0:1 )).
It is believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Proteinase K
molecule for at least two reasons. First, the amino acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of the loop regions to the active-site and binding pocket of the Proteinase K molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).
As used herein, "variant" means an enzyme having an amino acid sequence which differs from that of wild-type.
As used herein, "mutant Proteinase K DNA" means a DNA sequence coding for a Proteinase K variant.
As used herein, "wild-type Proteinase K" refers to an enzyme represented by SEQ ID N0:1. The amino acid sequence for Proteinase K is further described by Gunkel, F.A. and Gassen H.G., "Proteinase K from Tritirachium album Limber: Characterization of the Chromosomal Gene and Expression of the cDNA in Escherichia coli", EuR. J. BIOCHEM., Vol. 179, pp.
185-194 ( 1989).
As used herein, the term "Proteinase K wild-type amino acid sequence" encompasses SEQ ID N0:1 as well as SEQ ID N0:1 having modifications to the amino acid sequence other than at any of positions 64-71, 95-107, 133-140, 160-170, 190-194 and 203-223.
As used herein, "more hydrophilic amino acid" refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1 ) lists amino acids in descending order of increasing hydrophilicity (see Hopp, T.P., and Woods, KR., "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", PROCEEDINGS of THE
NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981).

Amino Acid Hydrophilicity Value Trp -3.4 Phe -2.5 Tyr -2.3 Leu, Ile -1.g Val -1.5 Met -1.3 Cys -1.0 Ala, His -0.5 Thr -0.4 Pro, Gly -0.0 Gln, Asn 0.2 Ser 0.3 Arg+, Lys+, Glu-, Asp- 3.0 Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9). ~ The positively charged amino acids are Arg and Lys, the negatively charged amino acids are Glu and Asp, and the remaining amino acids are neutral. In a preferred embodiment of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).
Therefore, for example, the statement "substitute Gln with an equally or more hydrophilic amino acid which is neutral or has a negative charge"
means Gln would be substituted with Asn (which is equally hydrophilic to Gln), or Ser, Glu or Asp (which are more hydrophilic than Gln); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gln. Likewise, the statement "substitute Pro with a more hydrophilic amino acid which is neutral or has a negative charge" means Pro would be substituted with Gln, Asn, Ser, Glu or Asp.
In one embodiment of the present invention, the Proteinase K variant has a modified amino acid sequence of Proteinase K wild-type amino acid sequence, wherein the wild-type amino acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region; whereby the Proteinase K variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Proteinase K.
In a preferred embodiment of the present invention, the substituting amino acid for one or more of the positions in one or more of the loop regions is, with reference to Table 1, neutral or negatively charged and equally or more hydrophylic, preferably more hydrophylic, than the amino acid at the subject position in the wild-type amino acid sequence.
A. Substitutions in the First Loop Region When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 64, 65, 66, 67, 68, 70 or 71.
When a substitution occurs at position 64, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 65, the substituting amino acid is Glu.
When a substitution occurs at position 66, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 67, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 68, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 70, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 71, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
B. Substitutions in the Second Loop Region When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 or 107.
When a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 96, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser , Thr or Val.
When a substitution occurs at position 97, the substituting amino acid is Glu. .
When a substitution occurs at position 98, the substituting amino acid is Glu.
When a substitution occurs at position 99, the substituting amino acid is Asp, Gln, Glu or Ser.

When a substitution occurs position100,the substituting at amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs position101,the substituting at amino acid is Asp or Glu.

When a substitution occurs position102,the substituting at amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs position103,the substituting at amino acid is Asn, Asp, Glu or Ser.

When a substitution occurs position104,the substituting at amino acid is Ala, Asn, Asp, Cys, ly, e, Gln, Glu, G His, Leu, Il Met, Pro, Ser, Thr, or Val.

When a substitution occurs at position 105, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 106, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 107, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.
C. Substitutions in the Third Loop Re4ion When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 133, 134, 135, 136, 137, 138, 139 or 140. wherein When a substitution occurs at position 133, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 134, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 135, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 136, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 137, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 138, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 139, the substituting amino acid is Asp or Glu. and When a substitution occurs at position thesubstitutingamino 140, acid is Asp or Glu.

D. Substitutions in the Fourth Loop Region When a substitution occurs in the fourthregion, loop the substitution occurs at one or more of positions 160, 6, 167, 161, 162, 163, 164, 165, 16 168, 169 or 170. wherein When a substitution occurs at position thesubstitutingamino 160, acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position thesubstitutingamino 161, acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position thesubstitutingamino 162, acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position thesubstitutingamino 163, acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position thesubstitutingamino 164, acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position thesubstitutingamino 165, acid is Glu.

When a substitution occurs at position thesubstitutingamino 166, acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position thesubstitutingamino 167, acid is Asp or Glu.

When a substitution occurs at position thesubstitutingamino 168, acid is Asp, Gln, Glu or Ser.

When a substitution occurs at position thesubstitutingamino 169, acid is Ala, Asn, Asp, Cys, Gln, Glu, et, Pro, Thr or Gly, His, Ile, Leu, M Ser, Val. and When a substitution occurs at position thesubstitutingamino 170, acid is Asp or Glu.

E. Substitutions in the Fifth Loop Re4ion When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 190, ' 191, 192, 193 or 194. wherein When a substitution occurs at position thesubstitutingamino 190, acid is Asp or Glu.

When a substitution occurs at position thesubstitutingamino 191, acid is Asp or Glu.

When a substitution occurs at position thesubstitutingamino 192, acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val.
When a substitution occurs at position 193, the substituting amino acid is Asp or Glu. and When a substitution occurs at position 194, the substituting amino acid is Asp, Gln, Glu or Ser. and F. Substitutions in the Sixth Loop Region When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223. wherein When a substitution occurs at position 203, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 204, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser.
When a substitution occurs at position 205, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 206, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 207, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 209, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 210, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 211, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 212, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val.
When a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 215, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.

When a substitution occurs at position 216; the substituting amino acid is Asp or Glu.
When a substitution occurs at position 217, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 218, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 219, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 220, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 221, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 222, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser. and When a substitution occurs at position 223, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
G. Preparation ofi enzyme variants Example 1 Mutant Proteinase K DNA
A phagemid ("PKP") containing the wild type Proteinase K gene is constructed. The 2.8 Kbp Pvu II restriction enzyme fragment of plasmid pUC119, (Vieira, J. and Messing, J.. "Production of Single-Stranded Plasmid DNA", 153 METhioDS w ENZYMOLOGY 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus, OH 1 E9). The pUC119-pUB110 hybrid plasmid is named pJMA601. Into pJMA601 is cloned the Bacillus amyloliquefaciens subtilisin gene. The subtilisin gene is modified to contain two BamHl sites. One of the BamHl sites is between DNA encoding Gly and Lys, the second and third amino acid residues of the pro region. The other BamHl site follows the TAA stop sequence. A Proteinase K cDNA is amplified from RNA using reverse transcriptase and the polymerise chain reaction with oligonucleotides, each containing a BamHl site in addition to sequences identical to the Proteinase K cDNA. The amplified region consists of the DNA extending from the Ala at the beginning of the pro region of Proteinase K to the carboxy terminal Ala of the mature protease. The amplified segment is used to replace the DNA between two BamHl sites within the Bacillus amyloliquefaciens subtilisin gene. Phagemid PKP is transformed into Escherichia coli Ung-strain CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel, T.A., J.D. Roberts and R.A. Zakour. "Rapid and efficient site-specific mutagenesis without phenotypic selection", METHODS IN ENZYMOIOGY. Vol. 154, pp. 367-382, (1987); as modified by Yuckenberg, P.D., F. Witney, J. Geisselsoder and J.
McClary, "Site-directed in vitro mutagenesis using uracil-containing DNA
and phagemid vectors", DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed. M.J. McPherson, pp. 27-48, (1991 )). A single primer site-directed mutagenesis modification of the method of Zoller and Smith (Zoller, M.J., and M. Smith, "Oligonucieotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA", Nuc~Elc ACIDS RESEARCH, Vol. 10, pp. 6487-6500, (1982)), is used to produce all mutants (basically as presented by Yuckenberg, et al., 1991, above).
Oligonucleotides are made using an Applied Biosystem Inc. 3808 DNA
synthesizer. Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli.
33625). All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M. Y., E. Ferrari and D. J. Henner, (1984). "Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an In Vitro-derived Deletion Mutation", JOURNAL OF BACTERIOLOGY, Vol. 160, pp. 15-21 ). For some of the loop mutants a modified PKP with a frameshift-stop codon mutation in the corresponding loop is used to produce the uracil template. Oligonucleotides are designed to restore the proper reading frame and to encode for random substitutions at positions 64, 65, 66, 67, 68, 70, 71, 95, 96, 97, 93, 99, 100, 101, 102, 103, 104, 105, 106, 107, 133, 134, 135, 136, 137, 138, 139, 140, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 190, 191, 192, 193, 194, 203, 204, 205, 206, 207 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223 (equimolar andlor variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing.

1.
Example 2 Fermentation The Bacillus subtilis cells (BG2036) containing a subtilisin mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B. Braun Biotech, Inc., Allentown, Pennsylvania) in a total volume of 10 liters. The fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see FERMENTATION: A PRACTICAL APPROACH, Ed. B. McNeil and L. M.
Harvey, 1990). The broth is kept at a constant pH of 7.0 during the fermentation run. Chloramphenical is added for antibiotic selection of mutagenized plasmid. The cells are grown overnight at 37°C to an A600 of about 60 and harvested.
Example 3 Purification The fermentation broth is taken through the following steps to obtain pure enzyme. The broth is cleared of Bacillus subtilis cells by centrifugation, and clarified by removing fine particulates with a 100K cutoff membrane. This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5.5 using 0.025M MES buffer (2-(N-morpholino)ethanesulfonic acid). The enzyme is further purified by loading it onto either a ration exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCI or a propylene glycol gradient (see Scopes, R. K..
PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Springer-Verlag, New York (1984)).
The pNA assay (DeIMar, E.G., C. Largman, J.W. Brodrick and M.C. Geokas, ANAL. BIOCHEM., Vol. 99, pp. 316-320, (1979)) is used to determine the active enzyme concentration for fractions collected during gradient elution. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The active enzymeltotal-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution.

t3 To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type II-T: turkey egg white purchased from Sigma Chemical Company (St. Louis, Missouri). The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA.
In preparation for use, the enzyme stock solution is eluted through a TM
Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01 M CaCt2 and pH
adjusted to 8.6 with HCI. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C.
H. Characterization of enzyme variants Example 4 Model Surface Preparation Aminopropyl controlled pore glass (CPG) purchased from CPG Inc.
(Fairfield. New Jersey) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, California).
The_ reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3-(dimethylamino)propylj carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPGaAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water. This is followed by oven drying with a N2 purge at about 70°C. . The reaction scheme and preparation of the immobilized substrate are conducted as described by Erode, P.F. III, and D.S. Rauch, "Subtilisin BPN': Activity on an immobilized Substrate," LANGMUIR, Vol. 8, p. 1325-1329, (1992).
The CPG surface will have 62,000 ~ 7,000 pNA moleculeslwm2. The surface area will remain unchanged from the value of 50.Om2/g reported by CPG Inc. for the CPG as received.. This suggests that the procedure used WO 96!28556 PCT/US96/03005 to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).
Example 5 Surface Hydrolysis Assay Using CPG:sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment. A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been degassed. The flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis -e.g., the first 20 minutes - and every 10 minutes towards the end of the experiment). The CPG:sAAPF-pNA is allowed to settle and the solution is sampled. Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode et al., 1992, above.
All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment.
Therefore, enzyme adsorption can be determined by measuring solution depletion. The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed. The amount of pNA hydrolyzed from the surtace is measured by taking an absorbance reading on an aliquot of the sample at 410 nm. The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask. This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme.
Example 6 Soluble Substrate Kinetic Analysis The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer. The enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 ~.M sAAPF-pNA for each kinetic determination. An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LoTUSTM
spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts).
Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give vo. The vo and so data are plotted in the standard inverse fashion to give KM and kcat~
I. Example Proteinase K variants Proteinase K variants of the present invention which have decreased adsorption to and increased hydrolysis of surtace bound substrates are exemplified in Tables 2-36, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third.

Loop 1 - Single Mutation Variants Arg64Asp Arg64Glu Asp65Glu G1y66Asn Gly66Asp Gly66Gln G1v66G1u G1y66Pro G1y66Ser Asn67Asp Asn67Gln Asn67Glu Asn67Ser G1v68Asn Gly68Asp G1y68G1n Gly68Glu Gly68Pro G1y68Ser Gly70Asn G1 v-1 OAsp Gly70Gln G1y70G1u G1y70Pro Gly'70Ser Thr7lAsn Thr7lAsp Thr7lGln Thr7lGlu Thr7lGly Thr7lPro Thr7lSer Loop 1 - Double Mutation Variants Asn~7Ser + Thr7lAsp Gly66Ser + Thr7lSer Arg64Asp + Thr7lAsn Arg64Asp + G1y68Ser G1y66G1u + Gly70Ser Arg64Glu + Gly68Ser Arg64Glu + Gly66Gln Asn67Glu + G1y70Asn Asn67Gln + Thr7lAsp G1y66Ser + G1y70Asn Asp65Glu + G1y70Ser Arg64Glu + G1y70Asn G1y66Pro + Thr7lAsn G1y70G1n + Thr71G1n G1y68G1u + G1y70Asn Asp65Glu + Thr7lSer Gly68Asp + Gly70Gln Asp65Glu + G1y68G1n G1y70G1n + Thr7lAsp G1y68G1n + Gly'70Pro Asn67Asp + G1y70G1n G1y66Ser + Asn67Gln Asp65Glu + G1y68Asn G1y68Asp + G1y70Pro G1y66G1n + Thr7lPro Asp65Glu + Thr7lPro Arg64Asp + G1y68Asn G1y68Asn + Thr7lGlu G1y66Asn + Thr7lGlu G1y68Ser + Thr7lAsp G1y68Ser + Thr7lGly G1y66Ser + G1y68G1n G1y66G1n + G1y70Ser Gly66Glu '+ G1y70Pro Arg64Glu + G1y70Pro Asp65Glu + Thr7lGly Arg64Glu + Thr7lGly Asp65Glu + Gly70Pro Arg64Asp + G1y68G1n Arg64Glu + Gly68Pro G1y66Asn + Gly70Ser TABLE 4 ' Loop 1 - Triple Mutation Variants Asn67Ser + G1y68Asp + Giy70Ser Asn67Asp + G1y70Asn + Thr7lGly Arg64Glu + G1y66Asn + G1y70Pro WO 96/28556 PC"T/US96/03005 Gly66Asp + G1y68Ser + Gly70Ser Asp65Glu + G1y70G1n + Thr7lGln Gly66Glu + Gly68Pro + G1y70Asn Arg64Asp + G1y66Ser + G1y70G1n Gly66Gln + Asn67Gln + G1y68G1u Gly66Glu + Asn67Ser ~ Thr7lPro Gly66Asn + G1y68Pro + Thr7lGln Arg64Glu + G1y66G1n + G1y68G1n G1y66Asn + Gly68Ser + Gly70Pro Arg64Asp + G1y70G1n + Thr7lGly Asp65Glu + Gly68Gln + G1y70Ser Arg64Glu + G1y66Pro + Gly68Ser Gly66Gln + Asn67Gln + Thr7lGly Arg64Glu + G1y68Pro + Thr7lSer Asn67Asp + Gly70Ser + Thr7lGly Arg64Glu + Gly~)OPro + Thr7lGln Asp65Glu + Gly68Gln + G1y70G1n Gly&6Pro + G1y68G1n + Thr7lPro Gly66Glu + Asn67Gln + Gly70Pro G1y66G1n + G1y70Ser + Thr7lGln Asp65Glu + Gly68Gln + G1y70Asn Arg64Glu + G1y66Ser + Thr7lPro Asp65Glu + G1y68Ser + G1y70Asn Asp65Glu + Asn67Ser + Gly70Ser Arg64Glu + G1y66Ser + Gly70Pro Asp65Glu + G1y70G1n + Thr7lGly Asp65Glu + Gly68Asn + Gly70Asn Gly68Asn + G1y70Pro + Thr7lAsp Arg64Asp + G1y68Ser + Gly70Pro G1y66Asn + G1y68G1u + G1y70G1n Asn67Glu + G1y68G1n + G1y70Asn G1y66G1u + Gly68Pro + Gly70Gln Asn67Asp + G1y68G1u + G1y70Ser Asn67Asp + Gly68Asp + Gly70Asn Arg64Asp + Asp65Glu + Asn67Ser Arg64Asp + Asp65Glu + G1y70Pro Arg64Glu + Asp65Glu + Asn67Gln Loop 1 - Quadruple Mutation Variants Asp65Glu + Gly68Pro + Gly70Asn + Thr7lAsn Asp65Glu + Asn67Gln + G1y68G1n + Gly70Ser Gly66Glu + Asn67Gln + G1y68Asn + G1y70G1n Asp65Glu + G1y66Asn + Gly70Ser + Thr7lGln Asn67Gln + G1y68G1u + Gly70Gln + Thr7lGly Arg64Glu + G1y66G1n + G1y70Asn + Thr7lGln G1y66Pro + G1y68Ser + Gly70Asn + Thr7lGln Arg64Glu + G1y68Pro + G1y70G1n + Thr7lPro Gly66Glu + Gly68Pro + G1y70Asn + Thr7lPro Asp65Glu + Gly66Glu + Asn67Ser + G1y70Pro Asp65Glu + G1y66Asp + G1y68Asn + Thr7lSer Asp65Glu + G1y66G1u + Gly68Pro-+ G1y70Ser Asp65Glu + G1y66G1u + G1y70Asn + Thr7lGln Arg64Asp + Asp65Glu + Asn67Gln + G1y70Asn Arg64Asp + Asp65Glu + G1y70Pro + Thr7lGln Giy66Glu + Asn67Asp + G1y68Ser + G1y70Ser Gly66Glu + Asn67Glu + G1y70Asn + Thr7lSer Asp65Glu + G1y66Asp + Asn67Glu + G1y68Ser Arg64Glu + Asp65Glu + G1y66Asp + Gly68Pro G1y66G1u + Asn67Asp + G1y68Asp + G1y70Asn Asp65Glu + Asn67Glu + G1y68Ser +-G1y70G1n Asp65Glu + Asn67Asp +-G1y68G1n + G1y70Ser Asp65Glu + Asn67Glu + G1y68G1n + G1y70Asn Asp65Glu + G1y66Asn + Asn67Glu + G1y70Pro -Asp65Glu + G1y66Asp + Gly68Asp + Thr7lAsn Asp65Glu + G1y56G1u+ Asn67Gln + Gly68Asp Loop 2 - Single Mutation Variants Val 95A1a Val 95Asn Val 95Asp Val 95Cys Val 95G1n Val 95G1u Val 95G1y Val 95His Val 95Met Val 95Pro Val -95Ser Val 95Thr Leu 96Aia Leu 96Asn Leu 96Asp Leu 96Cys Leu 96G1n Leu 96G1u Leu 96G1y Leu 96His Leu 96I1e Leu 96Met Leu 96Pro Leu 96Ser Leu 96Thr _ Leu 96Va1 ' Asp 97G1u Asp 98G1u Asn 99Asp Asn 99G1n Asn 99G1u Asn 99Ser Glyl 00Asn G1y100Asp G1yl00Gln G1yl00Glu Glyl00Pro G1yl00Ser Ser101Asp Ser101G1u G1y102Asn G1y102Asp G1y102G1n G1y102G1u G1y102Pro G1y102Ser G1n103Asn G1n103Asp G1n103G1u G1n103Ser Tyr104A1a Tyr104Asn Tyr104Asp Tyr104Cys Tyr104G1n Tyr104G1u Tyr104G1y Tyr104His Tyr104I1e Tyr104Leu Tyr104Met Tyr104Pro Tvr104Ser Tyr104Thr Tyr104Va1 Ser105Asp Ser105G1u Thr106Asn Thr106Asp Thr106G1n Thr106G1u Thr106G1y Thr106Pro Thr106Ser I1e107A1a I1e107Asn I1e107Asp I1e107Cys I1e107G1n I1e107G1u I1e107G1y I1e107His I1e107Leu I1e107Met I1e107Pro Iie107Ser I1e107Thr I1e107Va1 Loop 2 - Double Mutation Variants Val 95G1n + Ser101G1u Tyr104His + I1e107Asp Val 95G1u + I1e107Asn Asn 99G1n + G1n103Asp Leu 96G1y + Ser105G1u Val 95Met + G1n103Asp Ser101Asp + Iie107Thr Val 95His + Asp 97G1u Asp 98G1u + Tyr104Leu Leu 96Cys + Asp 97G1u G1n103G1u + Tyr104G1n G1y102Ser + Tyr104G1y Leu 96A1a + Thr106Pro Thr106G1n + I1e107Asn Asn 99G1n + I1e107Asp Asn 99G1u + Thr106G1y G1y102Pro + G1n103Asp Asn 99Asp + Tyr104Thr Leu 96I1e-+ Ser101G1u Val 95G1y + G1n103Ser Tyr104Leu + Ser105G1u G1y102Asn + Tyr104Pro Leu 96Asp + I1e107Leu Asp 98G1u + G1y102Asn Leu 96Pro + Glyl00Glu SerlOiAsp + Thr106Ser G1yl00Ser + G1n103Asn G1y102Pro + Tyr104Thr Leu 96G1u + I1e107Leu Leu 95I1e + Glyl00Asp G1yl00Pro + I1e107Ser Asp 97G1u + Asn 99G1n Asp 97G1u + I1e107Cys G1y102Ser + G1n103G1u G1yl00Asn + Tyr104Asp G1yl00Pro + Thr106Asp Val 95Pro + Asp 97G1u Val 95Thr + G1yl00Glu Thr106Pro + I1e107G1u Ser101Asp + G1y102Asn Ser105G1u + Iie107Thr Asn 99G1n + Ser105A5p G1n103Ser + I1e107Met Leu 96Asp + Thr106G1y Val 95Thr + G1y102Asp Val 95A1a + Ser105Asp G1yl00Asn + G1n103Ser.
GlylC2Asp + Thr106G1n Leu 9nAsn + Ser105Asp G1yl00Glu + G1v102Pro G1n103Asp + I1e107His Tyr104A1a + I1e107Pro Asp 97G1u + I1e107Pro Asn 99G1u + Tyr104Asn Val 95Thr + Asp 98G1u Glyl00Gln + Thr106G1y Asn 99G1u + G1n103Ser Val 95His + Ser105Asp G1y102Pro + I1e107A1a Asp 97G1u + Glyl00Gln Loop 2 - Triple Mutation Variants Val 95G1n + Leu 96Thr + Ser101G1u Ser101Asp + Thr106Ser + I1e107Leu Val 95Asn + Leu 96Asn + Asp 98G1u G1n103G1u + Tyr104Ser + I1e107Ser Val 95Cys + Leu 96G1u + G1y102Ser Val 95His + Tyr104Thr + Ser105G1u Leu 96Pro + Asn 99Asp + G1n103Ser Val 95Ser + Leu 96Asp + G1n103Ser Leu 96Cys + G1y102G1n + I1e107Leu VaJ_ 95Ser + Leu 96His + Thr106Pro Leu 96G1n + G1y102Pro + Ser105Asp Leu 96Met + Asn 99Ser + Serl05Asp Leu 96His + Ser101Asp + Tyr104Va1 Asn 99Asp + G1yl00Asn + G1y102Ser Va1 95G1n + Glyl00Asp -~- Thr106Pro Leu 96Asn + Tyr104G1u + I1e107G1n Asn 99Asp + G1y102Ser + Tyr104His Val 95Met + Leu 96G1y + G1yl00Pro Val 95A1a + Asp 98G1u + Asn 99Ser Asp 97G1u + Tyr104Thr + I1e107His Leu 96Thr + G1y102G1n + G1n103Asp Tyr104Met + Thr106G1u + I1e107Leu Glyl00Ser + G1n103Asn + Tyr104I1e G1n103Asn + Tyr104Pro + Thr106G1u Val 95Met + Asp 98G1u + Asn 99G1n Leu 96His + Asp 97G1u + I1e107Met G1yl00Asn + G1n103Ser + Ser105Asp G1y102Asn + G1n103G1u + Thr106Pro Val 95Met + G1yl00Ser+ G1y102Asp Val 95Asp + Leu 96Met+ Tyr104Asn G1n103G1u + Tyr104Cys + I1e107Asn Val 95Met + Leu 96I1e + Tyr104Met Val 95Ser + SerlOlGlu + Thr106G1y Val 95Pro + Asn 99Asp + G1yl00Ser WO 96/28556 PCT/US96l03005 Glyl00Ser + G1n103G1u + I1e107Va1 G1n103Asp + Tyr104Va1 + I1e107His G1yl00Gln.-+ Tyr104Met + Thr106Asp Asn 99Asp + Glyl00Ser + Tvr104His SerlOlAsp + G1y102Asp + G1n103Ser ' Asp 98G1u + Asn 99Asp + Tyr104A1a Asp 98G1u + Asn 99G1u + G1yl00Gln G1y102Asp + G1n103Asp + Thr106G1n Tyr104G1u + Ser105Asp + Thr106Pro Asp 97G1u + Asp 98G1u + Tyr104Thr Asp 97G1u + Asp 98G1u + Asn 99Asp Asn 99Asp + G1yl00Asp + Ser101G1u G1n103G1u +-Tyr104Asp + I1e107G1u Leu 96G1u + Asp 97G1u + G1y102Asp Leu 96G1u + SerlOlAsp + G1n103Asn Asp 97G1u + G1yl00Asp + I1e107His Asp 97G1u + Asn 99Ser + G1yl00Asp G1n103Asp + Ser105G1u + Thr106Asn Leu 96Ser + Ser105Asp + I1e107Asp Asp 97G1u + SerlOlGlu + G1n103Ser Asp 97G1u + Serl~OlGlu + Tyr104Met Asn 99Asp + G1yl00Ser + Ser101Asp G1n103Asp + Tyr104His + I1e107G1u Asp 97G1u + G1y102G1u + Tyr104Pro Val 95Asp + G1yI02Glu + Tyr104G1y Val 95Asp + Asp 97G1u + I1e107Asn Loop 2 - Quadruple Mutation Variants Vai 95G1n + Asp 97G1u + Glyl00Ser + I1e107Gin Val gSSer + Leu-96Pro-+-As-p 97G1u + G1n103Asn Leu 96G1n + Glyl00Ser + Ser101Asp + Thr106Asn -Val 95Cys + Asp 97G1u + Glyl00Gln + G1y102Ser Leu 96Pro + G1y102Asn + Ser105Asp + I1e107G1n Val 95Ser + Glyl00Gln + Ser101G1u + I1e107Asn Leu 96Met + Ser101G1u + G1n103Asn + Thr106Pro Asn 99Asp + G1yl00Pro + G1y102Ser + Tyr104A1a Glyl00Pro + G1y102Pro + Thr106G1y + I1e107G1u Val 95Ser + G1yl00Gln + Ser101Asp + G1y102Pro Val 95G1y + Leu 96Va1 + Thr106Pro + I1e107Asp Val 95G1n + Leu 96Va1 + Asn g9p,,sp + Z1e107Thr Leu 96Ser + Asp 97G1u + Thr106G1n + I1e107Met Leu 96Thr + Asn 99G1n + Tyr104Va1 + I1e107Cys Asn 99G1u + G1y100G1n + G1y102Asn + G1n103Ser Val 95G1y + Ser101G1u + G1n103Ser + Thr106G1y Val 95Asn + Leu 96G1ri-+ Asp 98G1u + G1yl00Asn Val 95Cys + G1y102Pro + Tyr104Va1 + I1e107Asp Val 95Met + heu 96Met + G1y102G1u + Tyr104Thr Val 95Asp + Leu 96Cys + Thr106Ser + I1e107G1y Leu 96Met + Gl.yl00Glu + SerlOlGlu + Tyr104Cys Asp 98G1u + Asn 99G1u + G1y102Asn + I1e107Ser Val 95Ser + Asp 97G1u + Asp 98G1u + Asn 99Ser Val 95G1y + Asp 97G1u + Asp 98G1u + Tyr104I1e Leu 96G1y + Asp 97G1u + Asp 98G1u + Asn 99Asp Asp 98G1u + Asn 99G1u + G1yl00Glu + Thr106G1y Leu 96G1u + Asp 97G1u + Ser101Asp + Tyr104I1e Asp 97G1u + G.1yl00Asp + SerlOlGlu + Tyr104Leu Asp 97G1u + Asp 98G1u + Glyl00Glu + Tyr104Met G1yl00Glu + Ser101Asp + G1y102Asp + I1e107G1y Val 95Met + Leu 9nGlu + Asp 97G1u + G1y102Asp Asp 97G1u + G1yl00Glu + Tyr104A1a + Thr106G1n .
Asp 97G1u + Asn 99G1u + Tyr104His + Thr106G1y G1y102Ser + G1n1C3Asp + Ser105Asp + Thr106Ser Asp 97G1u + Ser101Asp + G1y102Pro + Tyr104I1e Asn 99Asp + Glyl00Gln + SerlOlAsp + Thr106Pro Asn 99Asp + G1yl00Ser + SerlOlAsp + I1e107Cys G1y102Ser + G1n103G1u + Ser105Asp + I1e107Asp Asp 98G1u + G1yl00Glu + Ser101G1u + G1y102G1n Va'_ 95G1y + Asp 98G1u + Asn 99G1u + Ser101Asp Asp 97G1u -+- G1y102Asp + G1n103Ser + I1e107Met Leu 96Ser + Asp 97G1u + G1yl00Glu + G1y102G1u Val 95Thr + Leu 96G1u + Asp 98G1u + Ser101Asp G1y102G1u + Tyr104A1a +-Ser105Asp + Thr106Asp Leu 96Met + Ser101Asp + G1n103G1u + I1e107G1n SerlOlAsp + G1n103Asp z Tyr104Ser + I1e107Thr Asp 98G1u + G1yl00Asn + SerlOlGlu + Tyr104Va1 Val 95G1u + G1y102G1u + G1n103Asn + TyrlQ4Glu Val 95Thr + SerlOlGlu + G1n103Asp + I1e107G1u Ser101G1u + G1n103Asp + Tyr104Met + I1e107G1u Val 95G1u + Ser101Asp + Tyr104His + I1e10-7Glu Val 95G1y + Asp 97G1u + G1y102G1u + Thr106G1u Val 95Ser + Leu 96Asp + Asn 99G1n + G1n103G1u Leu 96G1u + G1y102Asn + Tyr104G1n + Thr106G1u Val 95Asp + Asp 98G1u + Glyl00Asp + Thr106G1y Leu 96G1u + G1y102Asp + G1n103Ser + Ser105G1u G1yl00Ser + Ser101G1u + G1n103Asp + Ser105Asp Val 95Met + Glyl00Glu + G1y102Asp + I1e107Asp Leu 96A1a + Asp 97G1u + Ser101Asp + I1e107Asp Leu 96G1n + Asp 97G1u + G1n103Asp + I1e107G1u Loop 3 - Single Mutation Variants Leu133A1a Leu133Asn Leu133Asp Leu133Cys Leu133G1n Leu133G1u Leu133G1y Leu133His Leu133I1e Leu133Met Leu133Pro Leu133Ser Leu133Thr Leu133Va1 G1y134Asn G1y134Asp G1y134G1n G1y134G1u G1y134Pro G1y134Ser G1y135Asn G1y135Asp G1y135G1n G1y135G1u G1y135Pro G1y135Ser G1y136Asn G1y136Asp G1y136G1n G1y136G1u G1y136Pro G1y136Ser Tyr137A1a Tyr137Asn Tyr137Asp Tyr137Cys Tyr137G1n Tyr137G1u Tyr137G1y Tvr137His Tyr137I1e Tyr137Leu Tyr137Met Tyr137Pro Tyr137Ser Tyr137Thr Tyr137Va1 Ser138Asp Ser138G1u Ser139Asp Ser139G1u Ser140Asp Ser140G1u Loop 3 - Double Mutation Variants G1y134Asn + Ser140Asp Leu133A1a + G1y135G1u Leu133Thr + Ser139Asp Leu133G1n + Ser140Asp G1y136G1n + Ser138G1u G1;a134Ser+Ser138Asp G1y136Pro +Ser139Asp G1y135Asn +Ser138Asp Glyl3SGlu +G1y136Pro Leu133I1e +Ser140Asp Leu133Cys +Ser140Asp G1y134Asn +G1y135Pro Leu133Cys +G1y136Asp Tyr137Asn +Ser139Asp G1y136Asn +Ser140G1u G1y134G1u +GlyI35Pro Tyr137Met +Ser140Asp G1y135G1n +Ser139G1u Tyr137Thr +Ser138G1u Leu133Asn +Ser139G1u G1y135Ser +G1v136G1n Leu133G1y +G1y136Ser Leu133A1a +Tyr137His Leu133Va1 +G1y136G1u Tyr137I1e +Ser139Asp Leu133I1e +Tyr137G1n G1y136Ser +Ser140Asp G1y134Asn +Ser138G1u G1y134Ser +Ser138G1u G1y135Ser +Ser138Asp G1y136Ser -+Ser140G1u G1y136Ser +Ser138G1u Leu133G1u +G1y134Asn Leu133G1u +G1y135G1n G1y135Asn +Tyr137G1u Tyr137Thr +Ser139G1u Tyr137A1a +Ser139G1u G1y134G1n +Tyr137G1y G1y135Pro +Tvr137His Leu133Pro +Ser138G1u Leu133Thr +G1y135Ser G1y136Pro +Ser138G1u G1y134Ser +Ser140Asp Leu133Met +Tyr137A1a Tyr137Va1 +Ser138G1u G1y134Pro +-Ser140G1u Leu133Thr +Tyr137Ser G1y135G1u +G1y136Asn Leu133A1a +Ser140G1u G1y134G1n +Ser139G1u Leu133Pro +Tyr137G1y Leu133Va1 +G1y136Ser G1yi36Asp +Tyr137Pro G1y136Asn +Tyr137Va1 Leu133A1a +Ser138Asp G1y135G1n +G1y136Pro Tyr137Met +Ser138G1u Leu133G1n +Tyr137Asp Tyr137Ser + Ser138G1u G1y134Asp + G1y135Pro Loop 3 - Triple Mutation Variants Leu133Cys + G1y135G1n + T'yr137G1u Leu133I1e + G1y134Ser + G1y136G1u Leu133G1y + G1y135G1u + Tyr137Va1 Leu133G1n + G1y135G1n + Ser139Asp Leu133Asp + G1y135Pro + Tyr137Asn Leu133Va1 + G1y135G1n + G1y136Ser G1y135Asn + Tyr137I1e + Ser139G1u Leu133Met + G1y136Pro + Ser140Asp G1y13-4Pro + G1y136Pro + Ser138G1u G1y135Asn + Tyr137Met + Ser138Asp Leu133Ser + G1y136Asn + Tyr137Va1 G1y134G1u + G1y135Ser + G1y136Pro G1y136Asn + Tyr137Met + Ser140G1u LeuI33Ile + G1y136Pro + Tyrl-37G1u Leu133Met + G1y136G1n + Tyr137G1n Leu133Va1 + G1y135Pro + Ser139Asp Leu133Asp + G1y135G1n + Tyr137Cys G1y135Pro + Tyr137Met + Serl3BGlu G1y136G1n + Tyr137G1y + Ser140Asp Leu133Ser + G1y134Pro + Ser140Asp Leu133Thr + G1y134Asp + G1y135Pro Leu133I1e + G1y135Asn + Ser139G1u G1y135G1u + G1y136Pro + Tyr137His G1y135Asn + G1y136Pro + Ser140G1u Leu133Met + G1y134Ser + G1y136G1n G1y134Pro + G1y135Asn + Ser140G1u G1y135G1n + G1y136G1n + Ser138Asp G1y136Ser + Tyr137Met + Ser138G1u G1y134Pro + G1y136Ser + Ser139Asp G1y134Ser + Tyr137Thr + Ser138Asp Leu133Thr + G1y134Pro + Ser140Asp G1y134G1n + G1y135Pro + Ser140G1u G1y134Pro + Tyr137His + Ser138Asp Leu133Met + G1y134G1n + Ser139Asp Leu133Ser + G1y134Asn + Ser138G1u G1y136G1n + Tyr137A1a + Ser139G1u Leul3-3Ile + G1y13~Asn + Ser139Asp Leu133A1a + G1y135Asp + G1y136Pro Leu133Asn + Ser138Asp + Ser139G1u G1y136Asn + Serl3-8Asp + Ser139Asp G1y135Asn + Ser138Asp + Ser139G1u Leu133Asp + G1y134Asp + Tyr137His Leu133Asp + G1y134G1u + G1y135Pro Leu133G1y + G1y136G1u + Tyr137Asp Tyr137Ser + Ser139G1u + Ser140Asp G1y134G1n + Ser139Asp + Ser140Asp G1y136Asn + Ser139Asp + Ser140G1u Tyr137Pro + Ser139Asp + Ser140Asp G1y135Ser + Tyr137G1u + Ser138G1u Seri38Asp + Ser139Asp + Ser140Asp Tyr137G1u + Ser138Asp + Ser139GIu Tyr137A1a + Ser138Asp + Ser140Asp Tyr137Thr + Ser138Asp + Ser140G1u Tyr137G1n + Ser138Asp + Ser140Asp Leu133Thr + Ser138Asp + Ser140G1u G1y135Ser + Ser138Asp + Ser140G1u _ G1y136G1n + Ser138Asp + Ser140Asp G1y135Ser + Ser138G1u + Ser140Asp G1y136G1n + Ser1.38G1u + Ser140Asp Leu133Va1 + Ser138G1u + Ser140G1u Loop 3 - Quadruple Mutation Variants Leu133A1a + GIy134Asn + Tyr137Ser + Ser138Asp G1y134G1u + G1y135Asn + G1y136Asn + Tyr137Cys Leu133Cys + G1y135Asn + G1y136G1n + Ser138Asp Leu133Pro + G1y134Asn + G1y135Ser + Ser139Asp Leu133Va1 + G1y134G1n + G1y136G1n + Ser140G1u Leu133Asp + G1y134G1n + G1y136G1n + Tyr137Thr Leu133His + G1y135G1n + G1y136Pro + Tyr137Ser Leu133G1y + G1y134Ser + Tyr137A1a + Ser138Asp Leu133G1n + G1y134G1r_ + G1y135Asn + Ser140G1u Leu133Ser + G1y135Ser + Tyr137pro ~ Ser139G1u Leu133Asn + G1y134Asn + Glyi 36G1u -~ T yr 137Cys Leu133Met + G1y134Asn + G1y135G1n ~ Ser138G1u Leu133Asn + G1y134Ser + T_,:ri37Val + Ser138GIu G1y135Asn + G1y136Pro. + Tyr137Cys + Ser139Asp Leu133I1e + G1y135Asp + G1y136Asn + Tyr137I1e G1y134Asp + G1y135G1u + G1y136G1n + Tyr137Thr Leu133G1n + G1y134G1u + G1y135Asp + Tyr137Thr G1y134Ser + Tyr137Va1 + Ser138G1u + Ser139G1u G1y134Ser + Tyr137Va1 + Ser138Asp + Ser139G1u Leu133His + G1y135G1n + Ser138Asp + Ser139Asp Leu133G1y + G1y135Pro + Ser138G1u + Ser139Asp Leu133Va1 + G1y135Pro + Ser138Asp + Ser139G1u Leu133Ser + G1y134G1n + G1y135Asp + G1y136G1u Leu133I1e + G1y134G1n + Ser139Asp + Ser140Asp Leu133Cys + Tyr137Asn + Ser139Asp + Ser140G1u Leu133Ser + Tyr137His + Ser139G1u + Ser140G1u G1y134Pro + G1y136G1n + Ser139Asp + Ser140G1u G1y134G1n + G1y135Ser + Ser139Asp + Ser140Asp Leu133Cys + G1y134Pro + Ser139G1u + Ser140Asp Leu133A1a + G1y136Asn + Tyr137G1u + Ser138Asp G1y136Pro + Ser138Asp + Ser139G1u + Ser140Asp Tyr137Leu + Ser138Asp + Ser139G1u + Ser140G1u G1y135Asn + Ser138Asp + Ser139Asp + Ser140G1u Leu133G1y + Ser138Asp + Ser139G1u + Ser140G1u WO 96!28556 PCT/US96/03005 Leu133Ser + Tyr137Asp + Ser138Asp + Ser139G1u G1y136G1n + Tyr137Asp + Ser138Asp + Ser139Asp G1y13oAsn + Tyr137Asp + Ser138Asp + Ser139G1u G1y134G1u + G1y135Asp + G1y136Asp + Tyr137I1e Leu133G1n -. Tyr137Va1 + Ser138G1u + Ser140Asp G1y134Pro + Tyr137G1y + Se-r138Asp + Ser140Asp Leu133His + Tyr137Leu + Ser138G1u + Ser140G1u G1y134G1n + G1y135Ser + Ser138G1u + Ser140G1u ' Leu133Cys + G1y136Ser + Serl38Glu + Ser140G1u G1y135G1n + G1y136G1n_+ Serl38Asp + Ser140Asp G1y134Pro + Tyr137His + Ser138Asp + Ser140G1u _ Leu133G1n + G1y134Pro-+ G1y136G1u + Ser138Asp Leu133His + G1y134Pro + G1y136Asp + Ser138G1u Leu133I1e + G1y135G1u + Tyr137G1u + Ser138G1u G1y134Asn + G1y135Asp + Tyr137Asp + Ser138G1u Giy135G1n ~ Tyrl37G1u + Ser138Asp + Ser140G1u G1y136Asn + Tyr137G1u + Seri38-Asp + Ser140G1u Leu133Asn + Tyr137Asp + Ser138G1u + Ser140Asp Leu133Cys - G1y136Pro + Tyr137G1u + Ser139Asp G1y134G1u + G1y135Pro + G1y136Asp + Tyr137G1u G1y136Pro + 'Tyr137G1u + Ser139Asp + Ser140Asp G1y135Ser + Tyr137G1u + Ser139G1u + Ser140G1u Leu133Pro + G1y134G1u + G1y1.36Asp + Tyr137Cys G1y134G1n + G1y136Asp + Ser138G1u + Ser139Asp G1y135Ser + G1y136G1u + Ser138G1u + Ser139Asp G1y134Asp + G1y135G1u + G1y136Ser + Ser138Asp Loop 4 - Single Mutation Variants G1y160Asn G1y160Asp G1y160G1n G1y160G1u G1y160Pro G1y160Ser Asn161Asp Asn161G1n Asn161G1u Asn161Ser Asn162Asp Asn162G1n Asn162G1u Asn162Ser Asn163Asp Asn163G1n Asn163G1u Asn163Ser A1a164Asn A1a164Asp A1a164G1n A1a164G1u A1a164Giy A1a164His A1a164Pro A1a164Ser A1a164Thr Asp165G1u A1a166Asn A1a166Asp A1a166G1n A1a166G1u A1a166G1y A1a166His A1a166Pro A1a166Ser A1a166Thr Arg167Asp Arg167G1u Asn168Asp Asn168G1n Asn168G1u Asn168Ser Tyr169A1a Tyr169Asn TyrlE9Asp Tyr169Cys Tyr159G1n Tyr169G1u Tyr169G1y Tyr169His Tvr169I1e Tyr169Leu Tyr169Met Tyr169Pro Tyr169Ser Tyr169Thr Tyr169Va1 Ser170Asp Ser170G1u Loop 4 - Double Mutation Variants G1y160G1n + Asp165G1u Asn161Ser + Asn162Ser A1a164Thr + Asn168Asp G1y160Asn + Ser170G1u Asn161G1n + Asn163G1u Asn162Ser + Ser170Asp G1y160G1n + Asn168G1u A1a166G1y + Ser170Asp Asn161G1u + Asn162Ser Asn162G1n + Ser170Asp A1a166Thr +Ser170G1u Asn162Ser +A1a164Pro G1y160Asn +Asn161G1n G1y160G1u +A1a166Ser A1a166G1y +Ser170G1u A1a164His +A1a166G1y A1a164Asn +Asp165G1u Asn162Asp +Asn168Ser A1a164Pro +Asn168Asp Asn162Asp +A1a164Asn Tyr169Thr +Ser170Asp A1a164G1u +Tyr169His AspI65Glu +Tvr169His Asn162G1u +Asn168Ser G1y160Asp +Tyr169Met Asn162G1u +A1a166Asn Asn161G1u +A1a166Thr Asp165G1u +A1a166Asn Asn161G1n +Tyr169Pro Asn162Asp +A1a164Thr Asn162Ser +Arg167G1u A1a166Pro +Arg167Asp A1a164Thr +Ser170Asp Asn163Asp +A1a164Asn Asp165G1u +Asn168G1n Asn163Asp +Tyr169Thr Asn168Asp +Tyr169G1y Arg167Asp +Asn168Ser Asn161Ser +Arg167G1u Asn161Ser +Ser170G1u G1y160Asp +A1a166G1y A1a164Asn +Aia166G1n G1y16CAsn +Asn161G1u Asn162G1u +A1a166Ser-A1a166His +Asn168Asp G1y160Pro +A1a164G1y G1y160Asn +A1a164G1y A1a164G1n +Ser170Asp Asn161G1u +A1a164Thr G1y160G1u +A1.a166G1y A1a166Pro +Ser170Asp A1a166Pro +Tyr169A1a G1y160Ser ~Argl67Glu G1y160Asp +A1a164Ser Asn161G1n +A1a164Pro Asn163G1u +A1a166G1n A1a164Ser +Asn168G1n G1y160G1u +A1a164Ser G1y160Asp +A1a166G1n A1a164Thr +Asp165G1u WO 96/28556 PCT/US96/0300>

Loop 4 - Triple Mutation Variants G1y160G1r_ + Asn161Ser + Asp165G1u Alai66Gly + Arg167G1u + Tyr169Cys Asn162Ser +- A1a1.66Asn + Ser170Asp G1y160Gin + Asnl62Asp + Asn168Ser A1a164Asn + Asn168G1n + Ser170G1u A1a164Asp + A1a166Thr + Asn168Ser Asn163G1n + A1a166G1y + Arg167G1u Asn162Ser + A1a166His + Asn168G1n Asp165G1u + A1a166Thr + Asn168G1n Asn163Ser + A1a164Pro + Asn168Asp Glyl~OGlu + A1a164His + Tyrl69Gln Asr_'_6lGln + Asn163Ser + Ser170G1u A1a166Thr + Tyr169Leu + Ser170Asp Gly_oOSer + Aia164Asn + Asn168G1u Asn161G1n + Asn162G1u + Asnl63Ser Asnlo'3Ser + Alal665er + Arg167G1u A1a164Thr + A1a166G1y + Arg167G1u Alalei4Ser + A1a166G1y + Ser170G1u Asn162G1n + A1a164G1n + A1a166Pro Asn162G1u + Asn163G1n + A1a164G1y Asn161Asp + A1a164Asn + Tyr169Va1 G1y160Asn + A1a164G1u + A1a166Pro Asn162G1n + Alal64Pro + Arg167Asp Asn163Ser + Ala_64Ser + Tyr169G1y A1a166Asn + Arg_~7Glu + Asn168Ser Asnlo3Gln + AspI~SG'_~ ~ Alai~oSer Asn162Ser + Aia164His r Asn168Asp A1a164Ser + Arg167Asp + Asn168Asp Asn162Ser + Arg167Asp + Asn168Asp Asn161Asp + Asn162G1u + Asn163G1n Asn161Asp + Asn162G1u + Asn168G1n G1y160G1u + Asn161G1u + A1a166Asn G1y160G1u + Asn161G1u + A1a164Ser G1y160Pro + Asn162Asp + Asn163Asp Asn162Asp + Asni63Glu + A1a166Asn Asn161G1u + Asn162Asp + Asn163G1u Asn162G1u + Asn163Ser + A1a164Asp Asp165G1u + Arg167Asp + Tyr169Pro Asp165G1u + Arg167Asp + Asn168Asp A1a164G1n + Arg167Asp + Tyr169Asp Asn161Asp + Asn162Asp + A1a164Asp Asn161Asp + Asn163Asp + Tyr169Thr Asn161Asp + Asn163Asp + A1a164Asn G1y160G1u + Asn162G1u + Asn163G1n A1a164Asp + Asp165G1u + Arg167G1u A1a164Pro + Asp165G1u + Asn168Asp G1y160Asp + Asn162G1u + A1a164G1u G1y160Pro + Asn168G1u + Ser170G1u A1a164G1u + Arg167G1u + Asn168G1u Asr_161G1u + Asn163G1u + Asp165G1u G1y160Asp + Asn162G1u + Aspl-65G1u Asp165G1u + A1a166G1y + Tyr169G1u G1,;150G1u +Asn161Asp + Asp165G1u ' ArglG7Asp + Asn168G1n + Ser170Asp Asr.l o2Gln + Arg167Asp + Ser170-Gl~a AsnI63Gln + Argl6-7Glu + Ser170G1u Asr~162Asp + Asp165G1u + Asn168G1u Asn162Asp + Asp165G1u + Arg167G1u G1y160Asp + Asn168G1n + Ser170G1u G1y160Asp + Asp165G1u + Tyr169G1y Loop 4 - Quadruple Mutation Variants G1y160G1r~ + Asn161Ser + Asn162Ser + Asp165G1u G1y160G1u - Asn161Ser + Asnl6-2Gln + Asn168Ser Asn161Ser + Asn163Asp + A1a164Asn + Tyr169Thr G1y160Ser + Asp165G1u + A1a166Asn + Tyr169Leu -G1y160G1n + A1a166Ser + Arg167Asp + Tyr169His G1y1605er + Asri163Ser + A1a166G1y + Ser170Asp Asn161G1n + Asn163G1n + A1a164G1u + A1a166G1y Asn161G1u + Asn163G1n + A1.a166His + Tyr169Asn Asn163G1n + A1a166His + Arg167Asp + Asn168G1n G1y160G1n + Asn162Ser + Asn163Asp + Tyr169G1n AsnlE2Glu + A1a166G1n + Asn168Ser + Tyr169G1y Asn163G1u + A1a166His + Asn168G1n + Tyr169G1n G1y160Asn + Asnl615er + A1a1~4Thr + A1a166Ser Asn161G1n + Asn162Ser + Asn168G1u + Tyr169Ser Asn161G1n + A1a166His + Arg167G1u + Tyr169A1a Asn161G1n + A1a164G1y + A1a166G1n + Tyr169His A1a164G1n + Asp165G1u + Asn168Ser + Tyr169Met A1a164G1n + A1a166His + Arg167G1u + Asn168G1u G1y160Asn + Asn162Ser + p~ a164G1u + Asp165G1u Asn161Asp + Asn162Asp + A1a164Asn + A1a166Ser Asn161Asp + Asn162Asp + A1a164Asn + A1a166Thr-A1a164G1n + Alal6&Asn + Asn168Asp + Tyr169Asp G1y160Pro + Asn163Asp + A1a164Asp + A1a166Thr Asn161Ser + Asn163Asp + A1a164Asp + Asn168G1n Asn162Asp + Asn163Asp + A1a166Pro + Tyr169Met Asn161Ser + Asn163G1n + Tyr169Asp + Ser170Asp Asn161Ser + A1a166His + Tyr169Asp + Ser170G1u Asn161G1u + Asn162Asp + Asn163G1u + Tyr169Cys , Asn161G1u + Asn162G1u + Asn163Asp + Tyr169A1a Asp165G1u + A1a166G1y + Arg167Asp + Asn168G1n Asp165G1u + Arg167Asp + Asn168G1n + Tyr169Va1 .-Asn161Asp + Asn163G1u + A1a164Asn + A1a166G1y Asn163G1n + A1a164Asp + Asp165G1u + Arg167Asp Asp165G1u + A1a166Asn + Asn168Asp + Tyr169G1u Asn163G1n + Asp165G1u + Arg167G1u + Tyr169Asp Asp165G1u + Arg167Asp + Asn168Ser + Tyr169Asp Asn163Ser + Asp165G1u + Arg167Asp + Tyr169G1u Asni63Gln + p1a166Thr + Asn168G1u + Ser170G1u Asn161G1n + A1a166G1y + Asn168Asp + Ser170G1u Asr_163G1u + Asp165G1u + A1a166G1y + Tyr169Leu G1y160Pro + Asn162G1n + Asn163Asp + Asp165G1u G1y160G1u + Asn163G1n + Asn168Asp + Tyr169G1u G1y160Asp + A1a164G1u + Asp165G1u + Tyr169Ser Asn161G1u + A1a164Asp + Asp165G1u + Tyr169G1n G1y160Asp + Asn162Asp + A1a166G1y + Tyr169Asp Asn161G1u + Asn162G1u + Asp165G1u + A1a166Asn Asn161Asp + Asn162Asp + Asp165G1u + A1a166G1n Asn161Asp + Asn163Asp + Asp165G1u + Asn168G1n G1y160Asp + Asn161Ser + Asn163G1u + Tyr169G1y G1y160Asp + Asn162Asp + Asp165G1u + Asn168G1n Asn161Ser + Arg167G1u + T~.Tr169Thr + Ser170Asp Asn161G1n + Arg167G1u + Asn168G1n + Ser170Asp G1y160Asp + Asn163Asp + Asp165G1u + Tyr169A1a G1y160Ser + Asn162G1u + A1a164Pro + Asp165G1u G1y160G1u + Arg167G1u + Asn168G1u + Tyr169G1n Asni6lSer + A1a164Asp + Arg167G1u + Tyr169Asn Asn162Ser + Asn163Asp + A1a164Asp + Asn168Asp Asn163G1u + Asp165G1u + A1a166Asn + Arg167G1u Asn163Asp + Asp165G1u + Arg167Asp + Asn168Ser G1y160G1u + A1a164G1y + Asn168Asp + Ser170Asn Loop 5 - Single Mutation Variants Ser190Asp Ser190G1u Ser.191Asp Ser191G1u Phe192A1a Phe192Asn Phe192Asp Phe192Cys Phe192G1n Phe192G1u Phe192G1y Phe192His Phe192I1e Phe192Leu Phe192Met Phe192Pro Phe192Ser Phe192Thr Phe192Tyr Phe192Va1 Ser193Asp Ser193G1u Asn194Asp Asn194G1n WO 96/28556 PCT/US96l03005 Asn194G1u Asn194Ser Loop 5 - Double Mutation Variants Ser190Asp + Phe192Leu Ser190G1u + Phe192G1n Phe192G1u + Asn194G1n Phe192Tyr + Asn194G1u Ser190Asp + Phe192I1e Ser191G1u + Phe192Ser Ser191Asp + Phe192G1y Ser191Asp + Asn194Ser Ser191G1u + Phe192Thr Ser191G1u + Asn194G1n Ser191G1u + Phe192His Ser191Asp + Phe192Asn Ser190Asp + Phe192G1y Ser191Asp + Asn194G1n Ser191Asp + Phe192A1a Ser190G1u + Phe192Cys Ser190G1u + Phe192I1e Ser191Asp + Phe192Ser Ser190Asp-+ Asr.194G1n Ser191G1u + Phe192Cys Phe192Ser + Asn194G1u Ser1.91G1u + Phe192G1y Phe192G1y y Asn194G1n Phei 92Cys -t- Asn194G1n Phe192Asn + Asn194Asp Ser190G1u t Phe192Tyr Ser191G1u + Phe192A1a Ser190G1u + Asn194Ser -Phe192I1e + Asn194Ser Ser191G1u + Phe192Met Ser190Asp + Phe192Met Ser190Asp + Asn194Ser Phe192G1y + Asn194Asp Phe192His + Asn194Ser Ser190G1u + Asn194G1n Phe192A1a + Asn194G1u Phe192Thr + Asn194G1u Ser190G1u + Phe192Ser Ser191G1u + Phe192I1e Phe192Va1 + Asn194G1u Phe192Thr + Asn194Asp Ser191G1u + Phe192Pro ' Ser191Asp + Phe192Leu Phe192I1e + Asn194G1u Ser190Asp + Phe192Thr Ser191Asp + Phe192I1e Phe192Thr + Asn194G1n Phe192A1a + Asn194G1n Ser191Asp + phe192Tyr Ser190G1u + Phe192Thr Phe192Leu + Asn194G1u Phe192His + Asn194G1n Ser190Asp + Phe192Tyr Phe192Va1 + Asn194Ser Phe192Asp + Asr.194G1n Phe192G1y + Asn194Ser Ser191Asp + Phe192G1n Phe192Asp + Asn194Ser Phe192Thr + Asn194Ser Ser190G1u + Phe192A1a Loop 5 - Triple Mutation Variants Ser191Asp + Phe192Met + Asn194G1n Ser191G1u + Phe192A1a + Asn194G1n Ser191Asp + Phe192Leu + Asn194Ser Ser191G1u + Phe192Asn + Asn194G1n Ser190Asp + Phe192G1n + Asn194G1n Ser191Asp + Phe192I1e + Asn194G1n Ser190Asp + Phe192Asn + Asn194Ser Ser191G1u + Phe192I1e + Asn194Ser Ser190Asp + Phe192Leu + Asn194G1n Ser190Asp + Phe192Va1 + Asn194Ser Ser190G1u + Phe192G1n + Asn194Ser -Ser191Asp + Phe192Vai + Asn194Ser Ser191G1u + Phe192A1a + Asn194Ser Ser191G1u + Phe192Cys + Asn194G1n Ser190G1u + Phe192Va1 + Asn194Ser Ser190G1u + Phe192I1e + Asn194G1n Ser190G1u + Phe192Ser + Asn194Ser Ser190G1u + Phe192Tyr + Asn194G1n Ser191Asp + Phe192I1e + Asn194Ser Ser190Asp + Ser191G1u + Asn194Ser Ser190Asp + Ser191Asp + Phe192Met Ser190G1u + Ser191G1u + Phe192Va1 Ser190Asp + Ser191G1u + Phe192Ser Ser190Asp + Ser191Asp + Phe192Thr Ser190G1u + Ser191Asp + Asn194Ser Ser190G1u + Ser191Asp + Phe192Asn Ser190Asp + Ser191Asp + Phe192Asn Ser190Asp + Ser191Asp + Phe192Va1 Ser190G1u + Serl9lGlu + Phe192Cys Ser190Asp + Ser191G1u + Phe192Asn Ser190G1u + Ser191G1u + Phe192Leu Ser190G1u + Ser191Asp + Phe192G1y Ser190G1u + Ser191G1u + Phe192Ser Ser190G1u + Ser191G1u + Asn194Ser WO 96!28556 PCT/US96l03005 Ser190Asp + Ser191Asp + Asn194G1n Ser190Asp + Ser191Asp + Phe192Tyr Ser190Asp + Ser191G1u + Phe192Met Serl-90Asp + SeY191Asp + Phe192G1y Ser190G1u + Ser191Asp + Asn194G1n ' Ser190Asp + Ser191G1u+ Phe192Leu Ser190Asp + Ser191G1u + Phe192G1y Ser190G1u + Ser191Asp + Phe192Tvr Serl90Asp + Ser191G1u + Phe192Thr Ser190G1u + Ser191Asp + Phe192His Ser190Asp + Ser191Asp + Phe192G1n Ser190G1u + Ser191Asp + Phe192I1e Ser190Asp + Ser191G1u + Asn194G1n Ser190Asp + Ser191~1sp + Phe192Pro Ser190G1u + Ser191Asp + Phe192Cys Ser190Asp + Ser191Asp + Asn194Ser-Seri90Asp + Ser191G1u + Phe192G1n Ser190G1u + Ser191Asp + Phe192Pro Ser191Asp + Phe192Asp + Asn194G1n Ser191Asp + Phe192G1u + Asn194Ser Ser191Asp + Phe192G1u + Asn194G1n Ser190Asp + Ser191G1u + Phe192Asp Ser190Asp + Ser191Asp + Phe192G1u Ser190G1u + Ser191G1u + Phe192Asp Ser190G1u + Ser191Asp + Phe192Asp Ser190G1u + Ser191Asp + Phe192G1u Loop 5 - Quadruple Mutation Variants Ser190G1u + Ser191Asp + Phe192I1e + Asn194Ser Ser190Asp + Ser191Asp + Phe192G1y + Asn194G1n Ser190G1u + Ser191Asp + Phe192His + Asn194Ser Ser190G1u + Ser191Asp + Phe192G1n + Asn194G1n Ser190Asp + Ser191Asp + Phe192Met + Asn194Ser Ser190Asp + Ser191G1u + Phe192Leu + Asn194G1n Ser190G1u + Ser191G1u + Phe192A1a + Asn194Ser Ser190Asp + Ser191Asp + Phe192Pro + Asn194G1n Ser190G1u + Ser191G1u + Phe192Leu + Asn194Ser Ser190G1u + Ser191Asp + Phe192His + Asn194G1n Ser190G1u + Ser191Asp + Phe192Cys + Asn194G1n Ser190G1u + Ser191Asp + Phe192Thr + Asn194Ser Ser190G1u + Ser191Asp + Phe192Ser + Asn194G1n Ser190G1u + Ser191Asp + Phe192Thr + Asn194G1n ' Ser190Asp + Ser191G1u + Phe192I1e + Asn194Ser Ser190G1u + Ser191Asp + Phe192G1n + Asn194Ser Ser190Asp + Ser191Asp + Phe192Cys + Asn194G1n Ser190Asp + Ser191Asp + Phe192Pro + Asn194Ser Ser190G1u + Ser191G1u + Phe192A1a + Asn194G1n Ser190Asp + Ser191G1u + Phe192Thr + Asn194Ser Ser190G1u + Ser191Asp + Phe192Leu + Asn194G1n Ser190G1u + Ser191Asp + Phe192G1y + Asn194G1n Ser190Asp + Ser191Asp + Phe192Cys + Asn194Ser Ser190G1u + Ser191G1u + Phe192His + Asn194G1n Ser190G1u + Ser191Asp + phe192Asn + Asn194Ser Ser190Asp + Ser191Asp + Phe192His + Asn194G1n Ser190G1u + Ser191G1u + Phe192I1e + Asn194G1n Ser190Asp + Ser191Asp + Phe192Tyr + Asn194Ser Ser190G1u + Ser191Asp + Phe192Tyr + Asn194G1n Ser190G1u + Ser191Asp-+ Phe192A1a + Asn194Ser Ser190Asp + Ser191G1u + Phe192A1a + Asn194Ser Ser190Asp + Ser191G1u + Phe192Asn + Asn194Ser Ser190G1u + Ser191Asp + Phe192Va1 + Asn194Ser Ser190Asp + Ser191Asp + Phe192G1n + Asn194Ser Ser190Asp + Ser191Asp + Phe192G1n + Asn194G1n Ser190Asp + Ser191Asp + Phe192Leu + Asn194G1n Ser190Asp + Ser191Asp + Phe192Ser + Asn194G1n Ser190G1u + Ser191Asp + Phe192G1u + Asn194Ser -Ser190Asp + Ser191Asp + Phe192Asp + Asn194Ser Ser190G1u + Ser191Asp + Phe192G1u + Asn194G1n Ser190G1u + Ser191G1u + Phe192G1u + Asn194Ser -Ser190Asp + Ser191G1u + Phe192Giu + Asn194G1n Ser190Asp + Ser191Asp + Phe192G1u + Asn194Ser Ser190G1u + Ser191G1u + Phe192Asp + Asn194Ser -Ser190G1u + Ser191Asp + Phe192Asp + Asn194Ser Ser190G1u + Ser191G1u + Phe192Asp + Asnl94Gln Ser190Asp + Ser191Asp + Phe192Asp + Asn194G1n Ser190Asp + Ser191G1u + Phe192G1u + Asn194Ser Ser190G1u + Ser191G1u + Phe192Tyr + Asn194G1u Ser190G1u + Ser191G1u + Phe192Met-+ Asn194G1u Ser190Asp + Ser191G1u + Phe192G1n + Asn194Asp Ser190Asp + Ser191Asp + Phe192A1a + Asn194Asp Ser190G1u + Seri9lGlu + Phe192Leu + Asn194Asp Ser190G1u + Ser191G1u + Phe192Ser + Asn194Asp Ser190G1u + Ser191G1u + Phe192Met + Asn194Asp Ser190Asp + Ser191G1u + Phe192Tyr + Asn194G1u Ser190Asp + Ser191Asp + Phe192His + Asn194Asp Ser190G1u + Ser191Asp + Phe192G1n + Asn194Asp Ser190Asp + Ser191Asp + Phe192Asn + Asn194G1u Ser190Asp + Serl-9lGlu + Phe192I1e + Asn194Asp Loop 6 - Single Mutation Variants G1y203Asn G1y203Asp G1y203G1n G1y203G1u G1y203Pro G1y203Ser Pro204Asn Pro204Asp Pro204G1n Pro204G1u WO 96/28556 PCT/US96l03005 Pro204G1y Pro204Ser G1y205Asn G1y205Asp G1y205G1n G1y205G1u G1y205Pro G1y205Ser Thr206Asn Thr206Asp Thr206G1n Thr206G1u Thr206G1y Thr206Pro Thr206Ser Ser207Asp Ser207G1u I1e208A1a I1e208Asn I1e208As~
I1e208Cys I1e208G1n I1e208G1u I1e208G1y I1e208His I1e208I~eu I1e208Met I1e208Pro I1e208Ser Iie~~eThr I1e208Va1 Leu209A1a Leu209Asn Leu209Asp Leu209Cys Leu209G1n Leu209G1u Leu209G1y Leu209His Leu209I1e Leu209Met Leu209Pro Leu209Ser Leu209Thr Leu209Va1 Ser210Asp Ser210G1u Thr211Asn Thr211Asp Thr211G1n Thr211G1u Thr211G1y Thr211Pro Thr211Ser Trp212A1a Trp212Asn Trp212Asp Trp212Cys Trp212G1n Trp212G1u Trp212G1y Trp212His Trp212I1e Trp212Leu Trp212Met Trp212Phe Trp212Pro Trn212Ser Trt~212Thr Trp212Tyr Trp212Va1 I1e213A1a I1e213Asn I1e213Asp I1e213Cys I1e213G1n I1e213G1u I1e213G1y I1e213His I1e213Leu I1e213Met I1e213Pro I1e213Ser I1e213Thr I1e213Va1 G1v214Asn G1y214Asp G1y214G1n G1y214G1u G1y214Pro G1y214Ser G1y215Asn G1y215Asp G1y215G1n G1y215G1u G1y215Pro G1y215Ser Ser216Asp Ser216G1u Thr217Asn Thr217Asp Thr217G1n Thr217G1u Thr217G1y Thr217Pro Thr217Ser Arg218Asp Arg218G1u Ser219Asp Ser219G1u I1e220A1a I1e220Asn I1e220Asp I1e220Cys I1e220G1n I1e220G1u I1e220G1y I1e220His I1e220Leu I1e220Met I1e220Pro I1e220Ser~
I1e220Thr I1e220Va1 Ser221Asp Ser221G1u G1y222Asn G1y222Asp G1y222G1n G1y222G1u G1y222Pro G1y222Ser Thr223Asn Thr223Asp Thr223G1n Thr223G1u Thr223G1y Thr223Pro Thr223Ser Loop 6 - Double Mutation Variants G1y203G1n + Ser219G1u Thr206G1y + Leu209Ser Trp212Phe + Ser221G1u Arg218G1u + G1y222Pro G1y214Asp + I1e220Ser G1y215Asp + Thr217G1y G1y203Asn-+-Ser219Asp Trp212I1e + Ser221G1u Trp212G1n + Ser219Asp Trp212Cys + Ser219Asp Thr217G1u + Thr223Pro G1y214Pro + I1e220Va1 I1e208Pro + Thr223Ser Leu209G1n + I1e220G1u Thr206Asn + I1e220His WO 96128556 PCT/US96/0300~

I1e208A1a + Thr217Ser Pro204G1n + Ser221Asp Trp212Thr + Thr223Asn G1y203Asn + Ser219G1u G1y203G1n + G1y214Pro G1y214Asp + G1y215G1n G1y203Ser + Arg218G1u Thr206G1n + Trp212Asn Pro204Ser + I1e213G1u I1e208he-a +-G1y222G1u G1y215G1n + Thr217G1n I1e208Thr + Ser219Asp G1y205Ser + G1y214Asp I1e208Ser + Arg218G1u I1e208Ser + I1e220Va1 Thr211G1y + Arg218Asp I1e213Asp + I1e220Ser Pro2_04G1n + Ser219G1u Thr211G1y + I1e220Met __ I1e208His + Leu209A1a G1y205Asn + Ser207G1u Thr206Ser + G1V215Asn G1y205Asn + G1y222Ser G1y203Pro + I1e213Cvs Thr206G1n + G1y215Ser I1e208Ser + G1y214G1u Thr217G1n + Ser219G1u Pro204Ser + Ser216Asp G1y214Asn ~ Ser2lo'Asp I1e208Leu - T.hr~llAsr.
Pro204G1y - ~er~07Glu Ser219Asp + G~y~~2Asn Trp212Tyr + I1e213Thr Thr206Ser + Trp212Va1 I1e220Asp-+ Thr223Pro I1e208Pro + Trp212Pro I1e208Leu + Thr217Asp Pro204Ser + G1y214Ser G1y203Ser + G1y222Pro G1y205Ser + Arg218G1u G1y203Pro + Ser207Asp I1e208Va1+ Ser219G1u Ser216Asp +-G1y222G1n Ser207Asp + Trp212A1a I1e220Cys + G1y222G1u Thr206Asn + I1e213Cys Thr206Asp + G1y215G1n Ser207Asp + Thr211Ser I1e208Leu + Ser216G1u Leu209Va1 + G1y214Asp Thr206G1u + I1e213Asn Pro204Ser + Thr211Ser I1e213Asp + G1y215Pro G1y214Asp + Thr223Ser G1y205Asn + Leu209Gys G1y205G1n + I1e208A1a I1e208Leu + Ser219As.p Leu209G1u + Thr211Pro I1e208Cys + Thr217G1n Pro204Asn + Leu209Asn G1y214Pro + I1e220Pro G1y203G1n + Thr211G1y Pro204G1y + Thr223Asp G1y215Ser + Thr217Pro Thr206G1n + Thr211Asn G1y205Pro + G1y215Pro ~1y203Pro + Ser207G1u Ser207G1u + I1e208His Pro204Ser + Thr223G1y G1y205Asn + G1y215Asp Leu209G1n + Ser219G1u G1y215Pro + Thr223Ser I1e208G1n + Ser221Asp G1y203G1n + Arg218Asp G1y203Asn + I1e22OAsp G1y205Pro + Arg218Asp Thr211Asn + Arg218G1u G1y205Asn + Thr223G1u Thr206G1u + Leu209His Thr206Pro + G1y215Asp Trp212G1n + Ser221Asp Ser216G1u + I1e220Met G1y215Asn + G1y222Asp I1e213Cys + Ser219Asp Pro204Ser + Thr211Asn Pro204Ser + Thr223Pro Pro204G1n + I1e213G1u G1y2145er + Ser221Asp I1e208Leu + Thr223Pro Ser207Asp + I1e208G1y I1e208Leu + Arg218G1u Leu2095er + I1e220His I1e213Leu + Arg218Asp Pro204Ser + Ser219Asp G1y203Ser + G1y222G1u G1y205Pro + Thr223Asn Trp212Thr + G1y214Ser Thr206Ser + Ser216Asp G1y203Pro + Thr206G1y Thr211Pro + G1y222Ser I1e213Thr + Thr223G1u Thr206Pro + I1e213G1u Ser207Asp + G1y222Asn I1e213Va1 + G1y214Asn Thr211Asn + Thr223Asn I1e213Cys + Thr223Pro WO 96!28556 PCT/US96/03005 Pro204Ser + G1y222G1n Gly2-05G1n+ G1y215G1u Leu209Pro + G1y222G1n G1y205Ser + G1y222Asp Pro204Ser T I1e208Leu Pro204G1n + Thr211G1y Leu209Ser + Ser219Asp Thr217G1u + Thr223Asn G1y203G1n + Thr217Ser G1y205Pro + -I1e213G1n Leu209G1u + Thr217Ser Ser216Asp + Thr217Ser Trp212G1n + I1e213A1a Arg218G1u + I1e220G1n Leu209I1e + .Thr223Asn I1e208Met + G1y215G1n Arg218Asp + I1e220A1a G1y203Asn + Pro204Asn I1e208His + Ser221Asp I1e213Asn + Ser219Asp Trp212Met + Ser219G1u Thr206Pro + Ser216G1u Pro204G1n + Thr206Asp Thr206G1y + G1y215Pro Pro204Asn + G1y222G1n Ser219G1u + G1y222Ser I1e220Asn + G1y222Asn G1y205Asn + I1e213Asn G1y203Ser + I1e213Leu Ser207Asp + T'hr211Asn Leu209Pro + I1e213G1u Iie208Pro + Thr223Asp Thr211Ser + G1y222Asp Thr206Ser + Thr217Asp G1y203G1n + G1y214G1u G1y205Ser + G1y215Ser G1y203G1n + Leu209A1a G1y203G1n + G1y222Asn Ser207Asp + Thr211G1y G1y205G1n + Ser219G1u Trp212Cys + Ser216Asp G1y203G1n + Thr217G1u Arg218G1u + I1e220Cys Thr211G1y + Ser221G1u Pro204Asn + Ser216Asp Arg218Asp + G1y222Pro G1y205Pro + Ser221G1u Arg218G1u + Thr223Ser Leu209Asn + Ser216G1u G1y205Pro + I1e220Met Thr206Asn + Thr217Asp G1y203Ser + I1e208Thr Thr217Ser + Arg218G1u WO 96/28556 PCTlUS96/03005 Pro204Asn + Thr211Asn G1y205Asn + Thr217G1u Ser207Asp + Thr223G1y Trp212Tyr + I1e213Pro G1y214G1n + Ser216Asp Thr206Asp + I1e208Thr Loop 6 - Triple Mutation Variants G1y203G1n + Thr206G1y + Ser219G1u I1e213A1a + G1y215Asp + Thr217G1y G1y203Asn + Ser219Asp + Thr223Ser G1y215Pro + Ser216G1u + Thr223Ser G1y203G1n + Trp212Pro + G1y222Asp Pro204Ser + G1y205Ser + Ser207G1u G1y205Pro + I1e208Met + Ser219Asp Thr211G1y + G1y215G1n + Thr217Asn Thr211Ser + Thr217G1n + Thr223Asp G1y205G1n + Thr211Asn + G1y222Asn Thr211Pro + G1y214Asn + Thr217Asn G1y205Pro + Leu209Asn + Thr223G1n G1y203G1n + Trp212Leu + I1e220A1a Trp212Thr + G1y215G1u + I1e220Thr G1y205G1n + Ser207Asp + Trp212A1a G1y205Asn + Trp212Phe + Ser216G1u Ser207G1u + Thr211G1y + G1y222Asn Pro204G1n + G1y205Ser + Ser221G1u Thr206Pro + Leu209G1y + Thr223Asn I1e208Met + Thr217G1y + Thr223G1n Thr211Ser + G1y215Pro + G1y222G1u I1e208Pro + Trp212Pro + Ser216G1u G1y203Ser + Thr211G1n + G1y222Pro G1y203Pro + I1e208Va1 + Ser219G1u Thr206Pro + G1y215Ser + Ser216G1u Thr206Pro + I1e220Asn + Thr223G1y Pro204G1n + G1y205Pro + Arg218G1u Trp212His + I1e213Met + G1y215Asp I1e208Ser + G1y222G1n + Thr223G1n G1y205G1n + Trp212Leu + Arg218Asp G1y214G1n + Ser216G1u + G1y222Ser G1y203Asn + Ser216G1u + G1y222G1n Leu209Asn + G1y222G1u + Thr223Ser Thr206Asp + Thr211Asn + G1y214Ser Pro204Asn + Thr211G1y + Trp212Thr G1y203Ser + I1e208A1a + Ser216G1u Thr211Ser + G1y214Pro + G1y222G1u G1y215G1n + Arg218Asp + I1e220Va1 G1y215Ser + Ser216Asp + Thr223Ser Thr206Asn + Thr211Asn + Arg218G1u Pro204Asn + Thr217G1u + Thr223Ser Thr206Ser + I1e213Met + G1y215Ser Pro204G1y + G1y205Ser + Trp212G1n Thr211Pro + Trp212A1a + Thr223Asp G1y215G1u + I1e220A1a + Thr223G1n Thr206G1n + I1e208Ser + Leu209Asp Thr211Ser + Trp212Va1 + Ser219G1u G1y205Pro + Thr211Pro + Ser2lnAsp G1y205Pro + Trp212His + Ser219G1u G1y205Pro + Trp212Asn + Ser219Asp Leu2Q9Ser + Thr211G1n + Arg218Asp Pro204G1y + Thr211Asn + G1y215G1u G1y203Pro + Pro204Ser + Ser216Asp G1y203G1n + I1e213Va1 + Arg218G1u I1e208G1y + Thr217Asn + G1y222Ser Pro204Ser + G1y205G1n + Trp212I1e Pro204G1n + G1y214G1u + G1y215G1n Trp212Cys + G1y21-SGln + Ser221G1u G1y203Asn + Leu209Va1 + Thr217G1u G1y205Asn + Ser207Asp + Thr217G1y Pro204G1n + Thr206Asp + G1y215Ser Ile20eGln + 'T'hr211Pro + Ser219Asp G1y205Ser + Ile~I3His + Ser219G1u G1y203Ser + Ile2-08Ser + Trp212Met Thr211Pro + Trp212Tyr + Ser219Asp G1y205G1n + I1e208His + G1y222Asn G1y203Ser + Thr211Ser + I1e220G1u Pro204G1y + Leu209I1e + Arg218G1u Trp212Tyr + I1e213Ser + Thr217Pro I1e208Asn + I1e213Pro + G1y214Ser G1y203G1n + I1e213His + Ser219Asp G1y205Asn + Thr211G1n + Ser221G1u G1y205Asn + Ser-207G1u + G1y214G1n I1e208Va1 + G1y214G1u + G1y222G1n G1y203Ser + Thr205G1y + I1e213Leu Pro204G1y + I1e208Pro + G1y215Asp Thr206G1u + Thr217G1y + I1e220Pro I1e213Ser + Thr217Asp + G1y222G1n I1e208Va1 + G1y215Asn + Thr223Ser G1y205Pro + Ser207G1u + Thr217Ser G1y203Pro + Thr206Giu + I1e208His G1y205Ser + Trp212Cys + Ser216Asp Pro204Ser + Thr'-_'06Asp + Leu209I1e Thr206G1u + I1e4~8Thr-+ I1e220G1y I1e213G1n + I1e220G1u + G1y222G1n I1e208Va1 + G1y214G1n + G1y215Pro Leu209Pro + Ser216G1u + Thr217Asn Pro204G1n + Trp212Met + G1y222G1u I1e208Met + Thr211Asn + Thr223G1n Pro204G1n + Leu209I1e + A~g218Asp I1e208Leu + Ser216G1u + I1e220Pro Pro204G1y + G1y205G1n + Thr206G1u I1e208Ser + Leu20gHis + G1y21-4Prc G1y203Pro + I1e208G1n + Trp212Ser G1y2~5Asn + G1~215G1u + G1y222Pro G1y203Ser + Thr217G1n + I1e220Leu Leu209Ser T -G1y214Ser+ G1y222G1n I1e208G1y + Leu209Pro + Ser221Asp G1y205Asn ~+Thr206Ser + I1e220G1y Ser=_'07G1u+ Leu209Met + G1y222Asn G1y203Ser + Trp212G1n + Ser216Asp G1y205Asn + Iie208Cys + T'hr211G1n Thr211G1y + Trp212G1y + Thr223G1u G1y205Asn + G1y214Pro + Ser221G1u G1y205Ser + Trp212A1a + Ser216Asp G1y203G1n + Arg218G1u + I1e220Leu Ser207Asp + Thr211Asn + Thr223G1y Trp212Cys + G1y215Asn + Thr223Pro G1y205Asn + G1y214Ser + Arg218G1u G1y214Pro -+Thr217G1u + G1y222Asn Thr206G1n + I1e208Asn + Thr211Ser G1y203G1n + G1y214Pro + Ser219G1u Trp212His + I1e213G1n + Arg218G1u I1e208A1a + I1e21.3A1a+ Ser221G1u Pro204Asn + I1e208Leu + Trp212Pro G1y205Asn + Ser219Asp + I1e220His G1y205G1n + I1e208Leu + Thr211G1n Trp212Tyr + I1e213G1y + G1y215G1n Trp212Met + G1y215G1u + G1y222Asn Thr206Ser + Thr211Ser -+Thr217Asn I1e208Leu + I1e213Ser + I1e220Pro I1e208Ser + G1y214Pro + G1y222Asn Thr211Ser + Ser216G1u + Thr223Pro G1y203Ser + Thr217Pro + Thr223G1y G1y203Ser--+Trp212Aia + G1y214Asp G1y205Asn + I1e208Va1 + 5er219Asp G1y203Ser + Pro204G1n + Ser221Asp G1y203Pro + Trp212G1y + G1y222Pro I1e208G1n + Thr217G1y + Arg218G1u Thr206G1n + Ser207G1u + Thr223G1y Ser207Asp + Leu209His + Thr211Pro I1e208Thr + Trp212Va1 + I1e220Met Leu209A1a + Ser221Asp + Thr223G1n G1y203Asn + Thr211G1y + G1y214G1u G1y214Pro + G1y215G1u + I1e220Pro G1y205Ser + G1y214Asn + Ser216G1u Thr206G1n + I1e213Thr + Ser219G1u Pro2Q4Gly + Thr211G1y + Ser221Asp G1y203Pro + G1y205Asn + G1y215Asp G1y203Pro + Thr211Asn +~Thr223Asn Pro204G1y + Leu209Pro + G1y215G1u G1y203Pro + I1e213G1u + I1e220G1y Pro204G1n + I1e213Met + I1e220Pro G1y214Pro + Thr217G1n + G1y222G1u I1e208His + Ser221G1u + Thr223Asn G1y203Pro + G1y21.4Asn+ Arg218Asp I1e213A1a + Ser216Asp + G1y222Ser G1y203Asn + Thr211G1y + Thr217Asn Leu209Me~._ +. Thr211G1n + Ser219Asp Thr206Ser + Leu209l1e + Thr211G1y I1e213Met + G1y215G1n + Ser221Asp Pro204Asn + Ser207Asp + I1e220His G1y203G1n + I1e208Va1 + Ser221Asp Thr217G1n + I1e220Va1 + Thr223G1u G1y205Asn + I1e208Va1 + I1e213His Thr206Asn + Thr211G1y + Thr217Asp Ser207G1u + I1e208Cys + G1y215Pro Pro204Asn + G1y205Pro-+ Trp212I1e Pro204G1y + I1e208Va1 + Thr223Asp Pro204G1y + G1y215Asn + I1e220His G1y205Asn + Thr211Asn + Trp212Pro Pro204Ser + Thr206G1u + I1e213Va1 G1y203Pro + Ser207G1u + G1y214Asn Pro204G1n + G1y214G1u + G1y215G1u G1y205Ser + Thr217Asp + Arg218Asp Leu209Met + Thr217Asp + Arg218Asp Thr217Asp + Arg218Asp + I1e220Cys Thr211G1n + Ser221G1u + G1y222Asp G1y205Pro + Thr206Asp + Ser207G1u Pro204G1y + Thr206Asp + Ser207G1u Thr211G1n + I1.e220G1u + Ser221Asp Trp212Tyr + Arg218Asp + Ser219G1u I1e208Va1 + Arg218G1u + Ser219G1u G1y205G1n + G1y215Asp + Ser216Asp Leu209G1u + Trp212Phe + Ser219Asp Thr206G1u + I1e208A1a + Ser221Asp G1y205Asn + Ser207G1u + Ser221G1u Ser207G1u + Thr211Asn + Ile22OAsp G1y203Pro + Ser207G1u + I1e220G1u Ser207Asp + Trp212His + Ser219Asb Loop 6 - Quadruple Mutation Variants G1y203G1n + Thr206G1y + Leu209Ser + Ser219G1u I1e208Va1 + Thr217Ser + Ser219Asp + G1y222Pro G1y203Asn + I1e213Thr + G1y214Pro + Ser219G1u G1y203G1n + Trp212Phe + G1y214Asn + G1y215Asp Ser207Asp + Leu209Thr + Thr211Pro + Trp212G1y G1y205Pro + Trp212Asn + I1e213His + I1e220Asp G1y203G1n + Pro204Ser + Trp212I1e + I1e220Ser Pro204G1n + Thr211G1y + Ser219G1u + I1e220Met G1y203G1n + Trp212G1y + G1y215G1u + I1e220A1a Thr206G1n + Thr211Pro + G1y215Asn + I1e220Thr Leu209Va1 + Thr211Ser + Arg218Asp + I1e220Ser G1y203G1n + I1e208Cys + Leu209Ser + G1y214G1u G1y205G1n + Ser207G1u + G1y215G1n + I1e220A1a I1e208A1a + Leu209Va1 + Ser216G1u + Thr223Pro Pro204G1y + I1e208His + Ser219G1u + I1e220Ser _ Thr206Asp + I1e208G1y + Thr217Asn + Thr223Ser G1y203Asn + Leu209Ser + G1y214Ser + Ser221Asp I1e213Asp + G1y214Ser + I1e220G1n + G1y222Asn Trp212Thr + I1e213G1y + G1y214Ser + I1e220A1a G1y203Asn + Leu209Asp + Trp212Phe + G1y222G1n Thr206Asn + Thr211Ser + I1e213G1y + Ser219Asp Pro204Asn + G1y205G1n + Thr206Asn + Ser207G1u G1y203G1n + G1y205Asn + Ser207Asp + Leu209Thr G1y205G1n + I1e213Va1 + G1y214Pro + Arg218G1u ' Thr206Pro + I1e213Pro + G1y222Pro + Thr223Asp G1y203Asn + G1y214Asn + Ser221Asp + Thr223G1y I1e213Asn + Ser216Asp + I1e220Asn + G1y222Asn Pro204Ser + Leu209Pro + I1e213G1u + I1e220Pro G1y203G1n + G1y205Ser + Leu209A1a + G1y215Ser G1y203G1n + Ser207Asp + Thr211G1y + G1y222Asn Pro204G1n + Leu209His + Thr217G1y + Ser219G1u Thr206Asp + I1e208Thr + Leu209A1a + Thr217Asn I1e208Thr + Leu209G1y t Arg218Asp + I1e220Leu Pro204Asn + I1e208Va1 + Leu209His + Arg218G1u I1e208Asn + Thr211G1n + Ser219G1u + G1y222G1n Thr206G1y + Ser207G1u + I1e208Thr + I1e213G1y Leu209G1u + G1y214G1n + I1e220Va1 + G1y222G1n G1y205G1n + G1y214Asn + 'Thr217G1n + Thr223G1u G1y203G1n + Leu209Va1 + G1y215Pro + I1e220G1u Thr211G1n + Trp212G1n + G1y215Asn + Ser219G1u Pro204Ser + I1e208Ser + G1y222G1u + Thr223G1n Pro204G1y + I1e208Ser.-+- G1y215G1n + Ser221Asp G1y203G1n + Thr211G1y + I1e213Leu + G1y214Asn Pro204Asn + Thr211Ser + G1y214Asp + Thr217Asn Pro204G1y + Trp212Met + G1y215G1u + G1y222Asn Thr206G1u + I1e208Leu +- I1e213Ser + I1e220Pro -Thr206G1y + Trp212Leu + G1y214Asp + G1y222Ser -G1y203Ser + G1y214Asp + Thr217Pro + Thr223G1v G1y203Ser + Pro204Asn + I1e208A1a + G1y215G1u G1y203Ser + Thr211Ser + Ser219G1u + G1y222Asn Ser207G1u + I1e208G1y + Thr211Pro + I1e220Pro G1y203Asn + G1y205Pro + Leu209A1a + G1y222G1u Leu209Asn + Thr211G1y + I1e213Thr + Thr223G1n G1y203Pro + Pro204Asn + I1e213His + Thr217G1n I1e208Asn + Thr217Pro +-I1e220L.eu + Ser221G1u Thr206Ser + Thr211G1n + I1e213G1y + Ser221Asp G1y205Pro + G1y215Asp + I1e220His + Thr223G1v Pro204G1n + I1e208Thr + Ser219Asp + G1y222Asn G1y203Pro + Leu209Met +- Thr211Pro + I1e220Cys Thr206G1y ~- Leu209Cys + I1e213His + Ser216Asp I1e208G1y + Leu209Met + Thr217G1y + I1e220Pro .
G1y203Asn + Thr206Ser + G1y214G1n + Ser221G1u I1e208His + Thr211Ser + Trp212Pro + Arg218Asp Thr206Ser + I1e208Pro + Leu209I1e + G1y214Asp G1y205Pro + Thr206G1u + I1e213His + G1y222Asn Thr211Asn + I1e213G1n + Thr217Asp + Thr223Ser Thr206Asn + Trp212Va1 + Arg218G1u + I1e220Pro Pro204Asn + Ser207Asp + Thr211G1n + G1y214Ser Pro204Ser + I1e213Met + G1y214Asp + Thr217G1y G1y203Pro + I1e208A1a + Ser216G1u + Thr217Pro Ser207Asp + Trp212Cys + G1y215Pro + Thr217G1y Pro204Asn + G1y214G1n + Ser216G1u + I1e220His Pro204Ser + Trp212A1a + G1y214Asp + G1y215Pro Pro204Asn + Leu209G1y + Thr211G1y + I1e213Thr Pro204Ser + G1y205G1n + I1e208His + Ser219Asp G1y203Asn + G1y205Ser + Leu209Ser + Arg218G1u -Thr206Pro + Thr211G1n + I1e213G1u + G1y214Asn Ser207Asp + G1y214Asn + I1e220Va1 ~ G1y222Asn Trp212Tyr + I1e213Va1 + G1y214Asn + G1y222Ser G1y205Pro + I1e208G1y + Ser216Asp + I1e220His G1y203Asn + Leu209G1y + G1y214Pro + Arg218Asp Pro204G1n + I1e208Cys + I1e213G1y + G1y215G1u Pro204Asn + G1y205Pro + Leu209A1a + Thr211Pro G1y203Pro + I1e213A1a + -Thr217G1-y + Ar.~218G1u G1y203G1n + Pro204G1y + I1e220G1y + Ser221G1u G1y205Asn + I1e213G1y + G1y214Pro t G1y222Ser G1y203Asn + Thr211G1n + G1y214G1u.+ G1y222G1n I1e213Thr + G1y215G1n + Ser219Asp + I1e220Pro G1y203Asn + Pro204Ser + Arg218Asp + Thr223Pro -G1y203Ser + Thr211G1n + Arg218G1u + I1e220Vai G1y203Ser + Pro204Ser + I1e208Ser + Thr211G1n Pro204G1y + G1y214Asn + G1y215G1u + Thr223Asn I1e208Cys + Thr211Pro + Trp212Ser + G1y215Pro Pro204Asn + I1e213Met + Thr217G1u + Ile22QGln Pro204G1y + G1y205Pro + Iie208G1y + Thr221Pro G1y203Ser + Thr206G1y + I1e208Asn + Ser219Asp Pro204Asn + I1e213G1u + G1y215Pro T Thr217Asn G1y203Asn + Thr211G1n + Arg218G1u + I1e220G1y G1y203G1n + I1e208Cys + G1y~15Ser + Arg218Asp Pro204G1n + G1y205G1n + G1y214Ser + Thr223G1y Pro204G1n + Leu209Met + Trp212Phe + Ser219G1u G1y205Asn + I1e208Cys + Leu209I1e + I1e220Asn G1y203Pro + Leu209Cys + Thr217G1y + Ile2ZOGlv G1y205Asn + Thr206G1y + I1e208Asn + Ser216G1u Pro204G1n + Thr206Pro + Ser207G1u + G1y214Asn G1y203G1n + I1e208Met + Trp212His + Thr223Pro Pro204Asn + Leu209Va1 + Thr211Asn + G1y215Ser G1y203Asn + I1e208His + Ser221Asp + G1y222Ser Pro204Asn + Leu209Pro + Ser219G1u + I1e220G1u Thr211Pro + I1e213Asn + G1_y214G1u + G1y215Asp Thr211G1r_ + G1y215Ser + Thr217Asp + Arg218Asp G1y205Asn + Thr217Asp + Arg218Asp + I1e220G1n I1e208Thr + Leu209Thr + Ser221Asp + G1y222G1u G1y203Asn + G1y205Asn + Ser221Asp + G1y222G1u Thr206Ser + Trp212Pro + Ser221Asp + G1y222Asp I1e208Pro + Thr211Ser + G1y222Asp + Thr223Asp Pro204Ser + Trp212Met + G1y222G1u + Thr223Asp G1y203G1n + Thr206G1u + Ser207G1u + Thr211G1n G1y203Asn + Thr206G1u + Ser207Asp + Thr211Asn Thr206Asp + Ser207Asp + I1e220Thr + Thr223Pro G1y203G1n + Thr217Ser + I1e220G1u + Ser221Asp G1y205Ser + Thr211G1n + Arg218G1u + Ser219Asp Leu209G1y + G1y214Ser + Arg218G1u + Ser219G1u 1e208G1n + G1y214Pro + Ser216Asp + Thr217G1u I1e208G1y + I1e213Met + G1y215G1u + Ser2-l6Glu I1e208Ser-~ Leu20-9Asp + Thr211G1y + Ser219Asp Pro204G1n + I1e208Thr + Leu209Asp + Ser219Asp Thr206Asp + Trp212G1n + Ser221Asp + Thr223G1y G1y203Asn + Thr206Asp + Trp212Phe + Ser221G1u Pro204G1n + Thr206GIu + Leu209I1e + Ser221G1u Pro204G1n + G1y205G1n + Ser207G1u + Ser221G1u Ser207Asp + I1e208Leu + Trp212I1e + Ser221G1u Ser207G1u + Leu209Met + I1e220G1u + Thr223Ser Thr217Asn + Arg218Asp + Ser219Asp + I1e220Asp Lro204ASn + Ser207Asp + Ser219G1u + G1y222G1n Pro204G1y + Gly2Q5Pro + Ser207G1u + Ser219G1u G1y205G1n + Ser207Asp + Trp.212Va1 + Ser219G1u Ser207G1u ~ I1e208Ser + Ser219G1u + Thr223G1y Thr206Asp + Thr217Ser + I1e220Asp + Ser221G1u Trp212G1n + G1y214Asp + Ser216G1u + I1e220Va1 G1y214Asp + G1y215G1n + Ser216G1u + Thr223Asn G1y203G1n + Leu209Asp + Thr211Asn + Arg218G1u Pro204Ser + Leu209G1u + Arg218Asp + I1e220Cys Ser207Asp + I1e208G1y + Ser221G1u + G1y222Asp Pro204Asn + Ser207G1u-+ Ser221Asp + G1y222Asp Pro204Ser + G1y205Ser + I1e213Asp + Ser216Asp Thr206Asn + Ser219G1u + Ser221G1u + Thr223Asn Thr206Pro + Trp212Pro + Ser216G1u + Arg218G1u G1y203Ser + G1y215Asn + Ser216Asp + Arg218G1u Thr206G1n + Trp212Asn + Ser216Asp + Arg218G1u Leu209Va1 + Trp212I1e + Ser216Asp + Arg218G1u Pro204G1y + I1e208Pro + Ser216Asp + Arg218Asp I1e208G1y + Thr211Asn + Ser216G1u + Arg218G1u Trp212His + I1e213Asn + Thr217G1u + Ser219G1u I1e208Leu + Thr211Pro + Thr217Asp + Ser219G1u G1y203Asn + Thr217Asp + Ser219G1u + I1e220A1a Thr206Pro + Thr217G1u + Ser219G1u + I1e220A1a G1y203G1n + G1y215G1u + Thr217G1u + I1e220Cys G1y203Asn + Arg218G1u + Ser219Asp + Ser221G1u Leu209Met + Ser219Asp + Ser221G1u + G1y222Asp Thr211Ser + I1e213G1u + Ser216Asp + Arg218Asp Thr211Asn + Ser216Asp + Arg218Asp + Ser219Asp Ser216G1u + Arg218Asp + Ser219G1u + Thr223Ser G1y203Pro + Thr206G1u + Leu209Asp + I1e220Asp Ser207G1u + Leu209G1n + G1y222Asp + Thr223Ser G1y205G1n + Ser207G1u + I1e213G1n + G1y222Asp G1y205Asn + Ser207G1u + I1e213Leu + G1y222Asp ' G1y205G1n + Ser207G1u + G1y222Asp + Thr223G1u Ser207Asp + Trp212G1y + Ser219G1u + G1y222G1u Trp212Thr + G1y214G1u + Ser216Asp + Arg218Asp ' Ser207Asp + Arg218Asp + Ser221Asp + G1y222Asn Ser207Asp + Trp212His + Arg218G1u + Ser221G1u Pro204Asn + Thr206Asp + Thr211Ser + Ser219Asp Thr206Asr. + I1e213Pro + G1y214G1u + Thr217Asp Thr211G1n + G1y214G1u + G1y215Ser + Thr217G1u WO 96!28556 PCT/US96/03005 Leu209Asp + G1y214Pro + Ser221G1u + G1y222Asp Ser207G1u + Leu209A1a + I1e213A1a + Arg218G1u Pro204G1n + Ser207G1u + Trp212Leu + Arg218Asp Pro204G1y + I1e213G1u + Arg218G1u + Ser219G1u Pro204G1y + Thr206G1u + Leu209Asp + Thr223G1y Loop 6 - Quintuple Substitution Variants Pro204G1n t Gly20SGln + I1e208A1a + Ser216Asp +
G1y222Pro Pro204G1n + Ser207G1u + I1e208Ser + Thr211G1y +
I1e220A1a G1y205G1n + Trp212Phe + G1y214Pro + G1y215Asp +
Thr217G1y G1y203Asn - Ile2Q8Gln + Leu209G1n + Thr211Pro +
Thr223Asp G1y203Ser ~ I1e208Cys + Giy214G1n + 5er2I9Glu +
I1e220Thr Trp212G1n + I1e213Ser + G1y214Asn + Thr217G1u +
I1e220Ser G1y205Ser + I1e208Pro + Trp212Tyr + G1y214G1n +
Ser221Asp G1y205G1n + Thr206G1u + I1e208Thr + Leu209Thr +
Trp2i2Cys G1y203Asn + G1y205Pro + I1e208Va1 + G1y214Asn +
Ser219G1u G1y203G1n + G1y205G1n t I1e208G1y + Thr217Asn +
G1y222Ser I1e208Met + Thr211G1y + I1e213Cys + G1y214Pro +
I1e220Leu Thr211Pro + I1e213Ser + Thr217Asp + I1e220Pro +
G1y222G1n Pro204Asn + G1y205Ser + Thr211G1y + I1e220Thr +
G1y222Asn G1y203Asn + G1y205Asn + Leu209His + I1e220His +
Thr223G1n Pro204G1y + I1e208Asn + Thr217G1n + G1y222G1u +
Thr223G1n G1y205G1n + Ser207Asp + I1e208Met + I1e213C_vs +
G1y214Pro G1y205Asn + Thr211G1n + Trp212Met + Thr217G1u +
G1y222Asn G1y205G1n + Thr206Pro + Leu209G1u + I1e213Thr +
I1e220G1y Thr211Asn + G1y214G1n + G1y215Ser + Thr217Ser +
G1y222Ser Pro204G1y + G1y205G1n + Thr211G1y + Ser221G1u +
Thr223G1n G1y203Ser + I1e208Thr + I1e220Va1 + G1y222Pro +
Thr223G1u G1y205Asn + Thr206Pro + Ser216G1u + Thr217G1n +

WO 96!28556 PCT/US96/0300~

G1y222Ser G1y203Asn + Pro204G1n + I1e208Met + G1y215G1n +

Ser219Asp G1y205G1n + Thr206Pro + Trp212Ser + I1e213G1n +

Ser221G1u ' _.e42 09G1r_- G1y214G1n + Thr217Ser + Ser221Asp +

Thr223Asn Ser207G1u + Trp212Tyr + I1e213G1y + G1y214Pro +

I1e220Pro G1y203Pro + Trp212Pro + G1y214G1u + G1y215Pro +

G1y222Pro Pro204Ser T Thr206Asn + Thr211G1y + Trp212G1n + -Thr223G1n Pro204G1y + Thr206Pro + Trp212Va1 + G1y214Pro +

Arg218G1u G1y203G1n + Thr211G1n + G1y215Asn + Thr217G1u +

I1e220Va1 G1;r203G1n+ G1y205Pro t Leu209Va1 + -Thr211G1n+

G1y214Asp Pro204Asn + I1e213Thr + G1y215G1n + Ser219Asp +

I1e220Pra Pro204G1n + Thr206Asn + I1e208Met + G1y215G1u +

I1e220A1a G1y203Ser + G1y205Pro + I1e208Asn + Leu209G1u +

Thr211G1n Prc204G1y + G1y205G1n + Thr211Ser + Trp212Met +

Thr217Pro G1y205Ser + Thr206G1y ~ Ile2G8Va1 + Gl=a214Asp+

Thr~~3Pro Ser207G1u - I1e208A1a + ;;i,~=lSPro+ Thr217G1n + -Thr223Pro G1y203Ser + G1y205G1n + Trp212Asn + I1e213Ser +

Thr223Pro Pro204G1n + I1e208A1a + Leu209G1n + Thr211Ser + -Giy214Ser Pro204G1n + I1e208His + Thr211G1n + G1y215Pro + _ Ser216Asp Pro204G1n + Leu209I1e + Thr211Ser + G1y214Asp +

I1e220Cys Leu209I1e + Thr211G1n + Trp212Asn + I1e213Va1 +

Thr217Asp Pro204Asn + I1e208His + Leu209Va1 + Thr211Asn +

G1y222Ser G1y203G1n + Pro204Asn + G1y205Asn + I1e213A1a +

G1y214Asp .

Ser207Asp + Thr211G1n + Trp212G1y + G1y222G1n +

Thr223G1y G1y205G1n + I1e208G1y + Thr211G1n + I1e220Ser +

G1y222Asp G1y205Pro + I1e208Pro + Leu209Va1 + I1e220Leu + -G1y222G1u Pro204G1y + Thr206G1u + Leu209Cys + I1e213His +

I1e220Met Leu209G1y + Trp212Va1 + Ser216Asp + I1e220Cys +

Thr223Ser G1y205G1n + Leu209Va1 + Thr211Asn + G1y214Pro +

G1y222Asp I1e208A1a + Leu209His + G1y215Asn + I1e220Va1 +

G1y222G1u Ser207G1u + I1e208Pro + Thr211Asn + I1e213His +

G1y222Ser I1e208His + Leu209Cys + Thr217Pro + I1e220G1u +

G1y222G1n Pro204G1y + G1y205Ser + I1e208A1a + Trp212Leu +

Thr223Asn G1y203Pro + G1y205Pro + Trp212A1a + G1y215Ser +

Arg218G1u Pro204Ser + Trp212A1a + I1e2,13Thr+ G1y215Asn +

Ser216G1u Thr211Asn + Trp212Thr + Ser216G1u -+G1y222G1n +

Thr223Pro Thr206G1n + I1e208Pro +-Thr211G1y + I1e213Leu +-G1y215Asp G1y203Asn + I1e220Met + Ser221G1u + G1y222G1n +

Thr223Asn Pro204G1n + Thr206G1y + Trp212G1y + G1y215Asp +

I1e220Cys G1y203Asn + Pro204G1n + Trp212Pro + I1e220G1y +

Thr223Asp Pro204Ser + Thr206Ser + I1e208Leu + Thr211Ser +

G1y222Pro G1y203Ser + I1e208Thr + Leu209His + Thr211G1n +

I1e220G1u Pro204Asn + Thr211Pro + G1y214G1n + G1y215G1u +

Thr217Ser Pro204Asn + Thr211Pro + Thr217Asp_+ I1e220Va1 +

Thr223G1n G1y203Ser + I1e208G1n + Leu209Asn + Thr211Ser +

Trp212Asn Pro204Asn + I1e208His + Thr211G1n + I1e220G1y +

G1y222Asp G1y205Pro + Trp212Va1 +-G1y215Pro + Ser216Asp +

Thr217Pro G1y203G1n + I1e208A1a + Thr211Pro + G1y214Ser +

Arg218G1u G1y203Asn + Pro204G1n + Leu209Met + Trp212Ser +

Thr217G1u G1y205Pro - Ser207G1u + I1e208Met + Leu209Met +

I1e220Asn Thr211G1y + Trp212Asn + Ser219G1u + I1e220Asp +

Thr223Pro I1e208Met + I1e213Thr + Thr217Asp + Arg218G1u +

Thr223G1y G1y203Ser + I1e208G1n + Thr217G1u + Arg218Asp +

G1y222Pro G1y205G1n + Thr217G1u + Arg218G1u + I1e220His +

Thr223Pro ~~=~203Ser- I1e213G1n + Tizr217Asp +Arg218Asp +

G1y222G1n G1y203Pro + Pro204G1y + I1e208G1y +Ser221Asp +

G1y222Asp G1y203Asn + Thr211G1n + I1e213Asp +G1y214G1u +

G1y222G1n , Thr206G1u + Ser207G1u + Leu209Asn +Thr211G1n +

Thr217Asn G1y203G1n + Leu209Met + Thr211Pro +I1e220G1u +

Ser221G1u I1e208Va1 , Leu209His + Thr211G1n +Arg218G1u +

Ser219G1u G1y203Pro + Pro204Asn + Leu209His +Arg218Asp + -Ser219Asp I1e208A1a + Trp212G1n + Arg218G1u +Ser219G1u + -I1e220Thr Leu209G1y + Thr211Pro + G1y215Asn +Ser216G1u +

Thr217Asp I1e208Asn + Leu209Thr + I1e213His +Ser216G1u +

Thr217G1u Leu209Asn + G1y215G1u + Ser216Asp +I1e220Asn +

Thr223Asn G1y203Ser + Pro204G1y + G1y215G1u +Ser216Asp +

Thr223Ser Pro204G1n + I1e208Cys + I1e213Cys +G1y215G1u +

Ser216G1u Pro204G1n + G1y205Asn + Thr206Asp +Ser207G1u +

Ser221G1u G1y203G1n + Pro204Asn + Thr206Asp +I1e208Cys +

Ser221G1u Pro204G1y + Thr20EGlu + I1e208G1n +Trp212His +

Ser221G1u Thr206Asp + I1e208Met + Trp212Met I1e213Met +

Ser221G1u Ser207Asp + Thr211G1n + Trp212A1a +I1e213Pro +

Ser221Asp Thr206G1n + Ser207Asp + Leu209Va1 +Thr211G1y +

Ser221Asp Pro204Ser + Ser207G1u + I1e208Thr +Trp212I1e +

Ser221Asp Ser207G1u + Leu209A1a + Thr217G1y +I1e220A1a +

Ser221Asp Ser207Asp + Thr211Pro + Trp212Cys +G1y215Pro +

Ser221G1u G1y205Pro + Ser207G1u + Trp212Asn +Ser221G1u +

Thr223Ser G1y205G1n + Ser207G1u + Thr211G1y +Thr217G1y +

Ser221G1u Thr206G1n + Ser207G1u + I1e208A1a +Leu209Cys +

I1e220G1u Thr211G1n + Arg218G1u + Ser219Asp +I1e220Asp +

G1y222Ser G1-y205Asn + Thr206Pro + Ser207Asp + Ser219G1u +
G1y222G1n Pro204Ser + Ser207Asp + Ser219Asp + I1e220Met +
Thr223Pro Thr206Asn + Ser207G1u + I1e208Thr + Iie213G1y +
Ser219G1u Gl_~205G1n + Leu209Asn + Thr217Asp + Arg218G1u +
Ser219Asp Pro204'Gly + I1e208Va1 + I1e213Asp + G1y214G1u +
Ser216Asp Pro204Ser + i1e220His + Ser221G1u + G1y222G1u +
Thr223G1u Thr206Asp + Trp212Va1 + I1e220G1u + Ser221Asp +
Thr223Pro Thr206G1u + Ser207Asp + G1y214Asn + I1e220Asp +
Thr223Asn I1e208Leu + Leu209Va1 + Thr211Ser + G1y214Asp +
Ser216G1u Thr211Ser + I1e213His + G1y214Asp + Ser216Asp +
I1e220G1n G1y205Pro + Thr206Ser + Leu209G1u + Thr217Asn +
Arg218G1u G1y205Ser + Leu209Asp + G1y214Ser+ G1y215Ser +
Arg218Asp Ser207G1u + Leu209G1y + Trp212Phe + Ser219G1u +
Ser221G1u G1y203Pro + Pro204Asn + Thr211Ser + I1e213G1u +
Ser216G1u Thr206G1u + Ser207Asp + I1e208A1a + G1y215Pro +
Ser219G1u '"hr206G1u - Leu209Ser T Trp212l1e + I1e220Thr +
G1y222G1u Thr206Asp + Ser207Asp + I1e208Leu + I1e213Pro G1y222G1u G1y203Ser + Thr206Asp + Ser207G1u + I1e220G1n +
G1y222G1u Thr211G1y + I1e213A1a + G1y214G1u + Ser216G1u +
Thr217Asp Ser207Asp + Thr211G1n + Arg218G1u + Ser219G1u +
I1e220Va1 Ser207G1u + Leu209G1u + G1y214G1n + I1e220Va1 +
G1y222G1n Ser207G1u + Leu209G1u + Thr211G1n + G1y214G1n +
Thr217G1n G1y205G1n + Ser207G1u + Thr211Ser + Arg218Asp +
I1e220G1u Leu209G1u + Thr217Pro + Ser219Asp + Ser221G1u +
G1y222Ser Thr211G1y + G1y214Asn + Ser219G1u + I1e220Leu +
Ser221Asp G1y203Pro + I1e208G1n + G1y214Pro + Ser219Asp +
Ser221G1u Pro204Asn + Leu209I1e + Ser219G1u + Ser221Asp +

WO 96!28556 PCT/US96/03005 G1y222Ser Thr211Ser +Trp212G1n + G1y214G1n + Ser219Asp +

Ser221Asp Leu209A1a +G1y214Pro + Ser219G1u + I1e220Met +

Ser221G1u Pro204G1_~+G1y205G1n + I1e213Va1 -+Ser216As +
p Arg218G1u _ Prc204G1y +Thr206Pro + 'Thr211Asn + Ser216GIu +

Arg218G1u Thr206Asp +I1e208Thr + Leu209A1a + Thr217Asn + -I1e220Asp Pro204G1n +Thr206G1u + I1e208Pro + Trp212His +

I1e220G1u Thr206G1n +Th_r211Ser + Thr217Asp + Ser219Asp +

Thr223Pro G1_~203Ser+Pro204Ser + G1y205Pro + G1y215Asp +

Thr217G1u G1y20-3Pro~Pro204G1n + Gly''15G'u + Thr'l7Glu +

Gly~~2Gln Pro204Ser -Leu209Met + G1y215G1u + Thr217Asp +

Thr223Asn Pro204G1y +Trp212Leu + G1y214Ser + Ser221Asp +

Thr223Asp Leu209Ser +Thr211Asn + Arg218Asp + I1e220G1u +

Ser221G1u I1e208G1y +Leu209Asn + Ser219Asp + Ser221G1u +

G1y222Asp Pro204G1y -I1e208Leu + G1y215Asp + Ser216G1u +

Arg218Asp Pro204G1n +I1e208G1y + G1_r215Asp ~ Ser216G1u +

Arg218Giu Thr206G1y +I1e213Ser + Ser216G1u + Arg218Asp +

Ser219G1u Pro204Gin +Thr206G1u + Ser207Asp + Thr211Asn +

Thr223G1u Pro204G1y +G1y205Ser + Ser207G1u + Arg218Asp +

Ser221Asp Thr206Asp +I1e213Thr + G1y214Ser + Ser219Asp +

G1y222G1u G1y203Asn +Thr206Asn + I1e213Pro + G1y214G1u +

Thr217Asp Pro204Ser +I1e213G1y + G1y214G1u + Thr217Asp +

I1e220A1a Ser207G1u +Thr217Asp + Arg218Asp + I1e220A1a +

Thr223Ser Ser207Asp +Leu209Ser + Trp212Va1 + G1y215Ser +

Arg218G1u Pro204Asn +Ser207G1u + Leu209Va1 + Trp212Phe +

Arg218Asp G1y205Ser +Ser207G1u + G1y215G1n + Arg218G1u +

Thr223Asn G1y205Ser +Leu209Asp + I1e213Pro + G1y215Asn +

Ser221Asp G1-_;205Pro- Ser207G1u + Leu209G1u + Thr217Ser +

G1y222Asp Se=~~7Asp - Leu209Asp + Thr217G1y + G1y222Asp +

Thr223Ser G1=;=03Asn-+I1e208Ser +-Leu209Asp + Ser216G1u + -Ser219Asp Leu209A1a - Trp212Ser + G1y214G1u + G1y215G1u +

Arg218G1u G1y205G1n + Thr206G1u + I1e213G1n + Arg218G1u +

Ser221G1u Pro204G1-y+ Leu209Asn + G1y215Pro + Thr217Asp +

I1e22OAsp Thr206Pro + Trp212A1a + Arg218Asp + Ser219G1u +

G1y222Asp G1y203Asn + G1y205Pro + Trp212Asn + Arg218G1u +

Ser22lAsp Thr206G1y + I1e208A1a + Trp212Tyr + Arg218Asp +

Ser221G1u G1y205G1n + Thr211Ser + Arg218Asp + Ser221G1u +

Thr223Pro G1y205Pro + Thr211Pro + Trp212Pro + Arg218G1u +

Ser221G1u G1y203Ser + Arg218Asp + I1e220Asn + Ser221Asp +

Thr223Ser Trp2i2His + I1e213Asn + G1y214G1u + Thr217G1u +

Ser219G1u G1y205Pro + I1e213Pro + G1y215Asp + Arg218G1u +

Ser219Asp Pro204Ser t G1y215Asp + Arg218G1u + Ser219G1u +

G1y222Asn Ser207G1u + G1y214G1n + Ser216Asp + Arg218G1u + -G1v222G1n J

Ser207Asp + G1y215Pro + Thr217Asn + -+ Ser216G1u Arg218Asp G1y205Ser + I1e208Cys + Leu209A1a + G1y215Asp +

Arg218Asp G1y203Asn + Ser207Asp + Ser216G1u + Ser219Asp +

G1y222Pro Ser207G1u + Leu209I1e + Ser216G1u + Thr217G1y +

Ser219G1u Leu209Asp + Trp212Pro + G1y214G1u + Ser216G1u +

I1e220Cys G1y203G1n + Thr206G1y + Leu209Ser + Ser216G1u +

Ser219G1u I1e208Leu + G1y214Asn + Ser216G1u + Thr217Ser +

Ser219G1u Leu209Ser + Ser216G1u + Thr217Ser + Ser219G1u +

I1e220His Pro204G1n + Leu209Pro + G1y214Ser + Ser216G1u +

Ser219Asp Thr206G1y + I1e208Met + G1y215Asp + Arg218Asp '+

T~P~~n~i"

Loop 6 - Sextuple Substitution Variants G1y203Asn + Thr206Asn + I1e208Asn + Trp212Asn +
I1e213Leu + Ser221G1u Pro2-04G1n + G1y205G1n + I1e208A1a + Leu209Pro +
Ser216Asp + G1y222Pro G1y203G1n + G1y205Asn + G1y2155er + Thr217Ser +
I1e220Met + Ser221Ast~
T1e208Pro + Leu209Aia + G1y215G1n + Arg218Asp +
I1e220Va1 + Thr223Ser G1y203Ser + Leu209Cys + Trp212Tyr + Ser216Asp +
Thr217Asn + I1e220Va1 G1y203G1n + G1y205Ser + Leu209A1a + I1e213Met +
G1y215Asn + G1y222Asn G1y203Pro + G1y2055er + Leu209A1a + Trp212A1a +
G1y214G1n + Ser219Asp G1y203Pro + Leu209I1e + Thr211Pro + I1e213Va1 +
G1y215pro + I1e220Pro Thr206Ser + I1e208Asn + Thr211G1n + I1e213G1y +
I1e220Leu + Ser221Asp Pro204Ser + G1y205Pro + I1e208Thr+ G1y215Asp +
I1e220His + Thr223G1y G1y203Pro + Pro204G1n + Leu209Met + Thr211Pro +
Ser219Asp + I1e220Cys Pro204Asn + Thr206Asn + Ser207Asp + Thr211G1n +
G1y214Ser + I1e220Pro G1y203Ser + Leu209Pro + Trp212Met + I1e213Cys +
Thr217Pro + A_rg2lgGlu Pro204Asn + G1y205Ser + I1e208A1a + I1e213Pro +
G1y215Ser + I1e220G1y G1~.:2035er + G1y205Asn + Ser207G1u + Thr211Ser +
Trp212Va1 + Thr217Asn G1y203G1n + Pro204Asn + I1e208His + Leu209Pro +
I1e213Leu + G1y222Asn G1y205Asn + Thr206G1y + I1e208Pro + Thr211Pro +
Trp212I1e + Thr223Asn G1y205G1n + Thr206Ser + I1e208G1n + Ser221Asp +
G1y222G1n + Thr223Pro I1e208Leu + Trp212Asn + I1e213Thr + G1y214Ser +
Thr217G1n + Ser219G1u G1y203G1n + pro204G1n + Leu209His + Ser219G1u +
G1y222Asn + Thr223G1n Pro204G1n + G1y205Asn + I1e208Cys + Leu209I1e +
I1e220Asn + Thr223G1u G1y203Pro + G1y205G1n + Thr206G1y + Thr211G1n +
Trp212Pro + G1y215G1u Thr206G1n + I1e208Leu + Thr211G1n + G1y214Asn +
I1e220Asn + Thr223Asn I1e208Va1 + Leu209Cys + Thr211Ser + I1e213G1y +
G1y214Asp + Thr223G1y WO 96!28556 PCT/US96l03005 Prc204Ser + G1y205Asn + Leu2-09Ser + Thr217G1y +
Arg218G1u + G1y222Ser Pro204Ser + G1y205G1n + Leu209His + Thr211G1n +
G1y214Ser + Ser221G1u I1e208His + Leu209Cys + Thr211Ser + Trp212Ser +
G1y214G1u + G1y222Asn I1e208G1n + Leu209G1y + Trp212Phe + Thr217Pro +
I1e220Thr + G1v222G1u G1y205Pro + Ser207G1u + I1e2.08Met + Leu209Met +
Trp212Cys + I1e220Asn Pro204Ser + Thr206Asn + I1e208G1n + Trp212Phe +
Ser219G1u + Thr223G1y G1y205Pro + I_1e208Cys + Trp212I1e + Thr217G1n +
I1e220Leu + G1y222Pro Pro204Ser + G1y205Ser + I1e208Cys + I1e213Pro +
Ser221Asp + Thr223G1n Pra204Ser + Thr206G1n + Trp212Met + G1y214Ser +
Ser221Asp + Thr223Ser G1y205Ser + I1e208G1n + I1e213Cys + G1y215Pro +
Thr217G1n + Thr223Asp Pro204Asn + G1y205Pro + I1e208Ser + I1e213Asp +
I1e220Thr + G1y222G1n Pro204Asn + I1e208A1a + Leu209Ser + Thr211G1y +
G1y215Ser + Ser219G1u G1y203G1n + G1y205G1n + Thr206G1n + Ser207G1u +
I1e208A1a + I1e220A1a Leu209Ser + Thr211G1y + Trp212Tyr + I1e213His +
Ser216Asp + Thr223Pro Pro204G1n + Thr206Ser + I1e208Me~ + Leu209Cys +
Thr211Ser + G1y214Asp G1y203Asn + Leu209G1n + Thr211Ser ~ G1y215Pro +
I1e220Cys + G1y222Pro Leu209His + Thr211Asn + Trp212I1e + I1e213A1a +
Ser219G1u + Thr223G1y Thr206G1n + I1e208His + Trp212G1n + G1y215Ser +
Ser221Asp + Thr223G1n I1e208Cys + Leu209Met + Trp212Ser + I1e213G1n +
Ser216G1u + Thr223Pro G1y205Ser + Leu209Met + Trp212G1y + Arg218G1u +
I1e220G1y + G1y222Asn Pro204Asn + Trp212G1n + G1y215Ser + Ser216G1u +
Thr217Asn + I1e220Asn G1y203Pro + I1e208Va1 + Leu209Asp + Trp212A1a +
I1e220G1n + Thr223G1y G1y203G1n + G1y205Pro + I1e208G1y + Trp212Ser +
Thr217Asp + G1y222Asn G1y205Ser + Leu209Thr + Thr217G1n + I1e220Met +
G1y222G1n + Thr223G1u G1y203Asn + Pro204Ser + G1y205Pro + Trp212Pro +
G1y215G1n + Thr217G1u Pro204Ser + G1y205Ser + Leu209Asn + Ser216G1u +
I1e220Leu + G1v222Asn G1y203G1n + Leu209Thr + Trp212Leu + I1e213G1y +

Ser219G1u + I1e220His I1e208G1n Trp212G1y + I1e213Asn + Thr217Pro + -+

G1y222Ser + Thr223Asp-Pro204Ser I1e208His + Thr211G1y + I1e213Met +
+

Gi,~~214G1n + Ser2lnAsp ~l-_~=03Asn G1y205Pro -+ Leu209G1u+ Trp212I1e +
+

I1e213Va1 + G1y215Pro G1y203Asn Pro204G1n + Leu209Met + Trp212Ser + ' +

Thr217G1u + Arg218G1u Leu209G1n Trp212G1n + G1y214Asn + G1y215Ser +
+

G1y222G1u + Thr223G1u G1y205Pro Thr211G1y + Trp212Va1 + Arg218G1u +
+

Ser219Asp + I1e220Pro G1y203Asn Pro204G1n + G1y205G1n + G1y214Pro +
+

Arg218G1u + Ser219Asp G1y205Pro Thr211G1n + G1y215Asn + Thr217Ser +
+

Arg218G1u + Ser21-9Glu Pro204Asn G1y205Ser + I1e208G1y + Thr217Ser +
+

Arg218G1u + Ser219Asp Thr206Pro G1y214Ser + Thr217G1n + Arg218G1u +
+

Ser219G1u + Thr223Asn Thr206G1n Leu209G1u + Thr211Asn + Trp212I1e +
+

G1y215Ser + Ser219Asp Leu209Glu Thr211G1n + I1e213Va1 + G1y215G1n +
+

Ser219G1u + G1y222Pro Pro204G1n Ser207G1u + I1e208Pro + Thr211G1y +
+

I1e220Pro + Ser221Asp Pro204Asn Thr206G1n + Ser207G1u + I1e208A1a +
+

Leu209Cys + I1e220G1u Thr206G1u Thr211G1n + I1e213Pro + G1y214Asn +
+

Ser221Asp + G1y222Asp G1y203Asn Pro204G1n + Leu209Asp + G1y215Pro +
+

Ser219Asp + I1e220Asp Ser207G1u I1e208A1a + Thr211G1y + Trp212Ser +
+

Ser219G1u + I1e220G1n Ser207G1u Leu209G1y + Thr211Pro + G1y215Asn +
+

Ser219G1u + I1e220Va1 Ser207G1u I1e208Asn + Leu209Cys + G1y215Asn +
+

Ser219G1u + Thr223Ser G1y203Asn G1y205Ser + Ser207Asp + Thr217Ser +
+

Ser219Asp + I1e220Leu G1y203Asn Pro204Asn + Ser207Asp + Leu209Thr +
+

Ser219Asp + Thr223G1n G1y203Pro I1e208Pro + Thr211Ser + Ser221Asp +
+

G1y222Asp + Thr223Asp G1y205Ser + Thr217Ser +
+ Ser207G1u + Leu209G1u Ser219G1u + G1v222Asn Thr206Pro + I1e213G1u +
+ Leu209Ser + Thr2ilPro G1y215G1u + I1e220A1a G1y203Ser + Trp212G1y +
+ I1e208Ser + Leu209His G1y214Asp + Ser216G1u G1y203Ser + G1y214G1u +
+ G1y205Ser + Trp212A1a G1y215Ser + Ser216Asp G1y205G1n + Ser207G1u + I1e208Ser + Leu209G1y +
Ser219Asp + Ser221G1u Thr206Pro + Ser207G1u + Ile2G8His + Trp212A1a +
Ser219G1u + Ser221Asp G1~y205Ser + Ser207Asp + Leu209Cys + Thr217Pro +
Ser221Asp + G1y222G1u Ser207G1u + Leu209Asp + Thr211G1n + Trp212G1n +
Thr217G1n + I1e220G1u Leu209Asp + Thr211G1n + G1y214Pro + Thr217G1u +
Ser219G1u + Thr223G1n G1y203Ser + Thr206G1u + Ser207G1u + G1y215Ser +
Ser219Asp + I1e220Thr G1y205Ser + Thr206G1u + Ser207G1u + Thr211G1y +
I1e220Va1 + G1v222G1u Thr206G1u + Ser207Asp + Leu209Ser + Trp_2i2Ile +
I1e220Thr + G1y222G1u G1y203Pro + Ser207G1u + Leu209Asn + Thr217G1n +
Arg218-Asp + Ser219Asp G1y205G1n + Thr206G1n + Ser207Asp + Arg228G1u +
Ser219Asp + I1e220Pro Pro204Ser + G1y205G1n + Ser207Asp + Thr217Asn +
Arg218Asp + Ser219Asp G1y203Pro + Thr206Pro + Ser207G1u + I1e208Asn +
Leu209Asp + Thr217Ser G1y203Asn + Pro-204G1n + Thr206Asp + Trp212Phe +
Ser221G1u + Thr223Asp Pro204G1n + G1y205G1n + Ser207G1u + Leu209G1u +
Trp212MeL + Ser221G1u G1y203Asn + Trp212Phe + G1y214Ser-+ Ser219Asp +
Ser221G1u + Thr223G1n G1y205G1n + Thr206G1n + I1e208Pro + Thr217Pro +
Ser219Asp + Ser221Asp G1y203Asn + Thr211Asn + G1y215Pro + Ser219Asp +
Ser221Asp + G1y222Asn Pro204Ser + Trp212His + G1y214G1n + Ser219G1u +
Ser221G1u + G1y222G1n G1y203Pro + Thr211G1n + I1e213Met + Ser219G1u +
Ser221Asp + G1y222Ser I1e208Leu + Thr211G1y + G1y214Pro + G1y215Asn +
Ser216G1u + Arg218Asp I1e208A1a + Leu209Pro + Trp212Pro + Ser216G1u +
Arg218Asp + Thr223Pro G1y205G1n + I1e208Asn + G1y215Ser + Ser216G1u +
Arg218G1u + Thr223Asn G1y203Ser + Thr206G1y + G1y214Pro + G1y215G1n +
Ser216Asp + Arg218Asp G1y203Ser + Thr206Pro + Trp212Pro + G1y215Asn +
Ser216G1u +-Arg218G1u G1y203Asn.+ G1y205Asn + Leu209Va1 + I1e220G1u +
G1y222G1u +- Thr223Ser Pro204G1y + G1y205Pro + Leu209I1e + I1e213Met +
Ser221Asp + Thr223G1u G1y205Ser + Trp212Tyr + G1y214Pro + Arg218G1u +

Ser219Asp + Ser221Asp G1y205G1n + I1e208A1a + Leu209Cys + Arg218G1u +
Ser219Asp + Ser221Asp G1y205G1n + Leu209Va1 + Trp212Cys + I1e220Asp +
Ser221G1u + Thr223Asp ' Pro204G1n + Thr211G1n + G1y215G1u + Thr217Asp-+
Arg218G1u + Thr223Asn G1y203G1n + Leu209Asn + Ser216Asp + Arg218G1u +
Ser219Asw + Thr223Pro I1e208A1a + Leu209Pro + G1y214Ser + Ser216Asp +
Arg218G1u + Ser219Asp Pro204Asn + I1e208G1y + Thr211G1n + Ser216Asp +
Arg218G1u + Ser219G1u G1y203Ser + I1e208A1a + Trp212Ser + Ser216G1u +
Arg218Asp + Ser219G1u G1y205Pro + Thr211Ser + Ser216G1u + Thr217Asp +
Ser219Asp + G1y222Asn Pro204G1n + Leu209A1a + G1y215G1n + Ser216G1u +
Thr217Asp + Ser219Asp Pro204Asn + Ser207G1u + Thr211G1n + G1y214Ser +
G1y222G1u + Thr223Asn G1y205Asn + Ser207Asp + Trp212His + G1y214Ser +
G1y222G1u + Thr223G1u G1y203Ser + Ser207G1u + Thr211Ser + I1e213A1a +
Arg218Asp + Ser221Asp G1y203G1n + Thr206G1u + Trp212I1e + I1e213Asn +
Ser219Asp + G1y222G1u I1e208A1a + Leu209Met + Thr211Pro + I1e213Thr +
G1y214G1u + Thr217G1u G1y203Ser + Pro204Ser + G1y205Ser + G1y214Asp +
G1y215Pro + Thr217G1u G1y203Asn + I1e208Met + I1e213Cys + G1y214Asp +
Thr217G1u + Thr223Asn Pro204Asn + Leu209I1e + Trp212Va1 + I1e213Asp +
G1y215Asp + Arg218G1u Ser207Asp + I1e208Met + Thr211Pro + Trp212A1a +
G1y215Asn + Arg218G1u Pro204Asn + Thr206G1n + Ser207G1u + G1y214Asn +
G1y215G1n + Arg218G1u G1y203G1n + G1y205Pro + Ser207Asp + I1e208G1y + -Arg218G1u + G1y222G1n G1y205Pro + Thr206Asn + Ser207Asp + Trp212Tyr +
Arg218Asp + I1e220Thr Pro204Asn + G1y205Pro + Ser207G1u + I1e208G1n +
Thr217Pro + Arg218Asp Thr206G1n + Ser207G1u + Trp212Ser +Thr217Asn +
Arg218G1u + I1e220A1a G1y205Pro + Thr206G1u + Leu209Thr + G1y214G1n +
Arg218Asp + Ser219G1u G1y203Ser + Pro204Asn + Ser207Asp + G1y215G1n +
Thr217Asp + I1e220Asp I1e208G1y + Thr211G1n + I1e213G1u + Arg218G1u +
G1y222Ser + Thr223G1n WO 96/28556 PCTlUS96/03005 G1y203Pro + Thr20~Gly + G1y214G1n + Ser219G1u +
Ser221Asp + Thr223Asp Thr211G1y + Trp212Phe + Ser219Asp + Ile22OPro +
Ser221G1u + Thr223G1u Leu209Aia + I1e213A1a + Ser219Asp + I1e220G1n +
Ser221P_sp + Thr223Asp Pro204G1n + Leu209G1u + Trp212Met + G1y214Ser +
G1y21=Asp + Thr217G1u Iie208IMet + I1e213Ser + Thr217G1u + I1e220Asp +
Ser221Asp + Thr223G1n Leu209Asp + Thr211Asn + I1e213Asp + Arg218G1u +
I1e220Leu + G1y222Ser G1y203Pro + G1y205G1n + Thr217Asp + Arg218G1u +
I1e220A1a + Ser221Asp Trp212Cys + I1e213G1y + Arg218G1u + Ser219G1u +
I1e220Pro + G1y222G1u Thr206G1n + I1e208G1y + Leu209I1e + Arg218G1u +
Ser2i9Glu + G1y222Asp G1y203G1n + I1e208Met + Ser219G1u-+ I1e220G1u +
G1y222Pro + Thr223G1u G1y203G1n + G1y205Asn + Thr211G1n + Ser219Asp +
I1e220G1u + Thr223G1u Pro204Asn + G1y205Pro + Leu209Ser + G1y214Ser +
Arg218G1u + Ser221G1u I1e208Thr + Leu209Asn + Trp212Asn + Arg218Asp +
Ser221G1u + G1y222Asn G1y203Ser + Pro204G1n + G1y205Asn + Thr211G1y +
Arg218Asp + Ser221G1u Pro204Asn + Thr206Pro + G1y214Pro + Arg218G1u +
Ser??lGlu - Thr223Asn Thrl'06Asp + Ile2i3Gln + Gly~'lSAsn + Thr217Asp +
Ser219Asp + Thr223Ser I1e213Met + G1y214Asp + G1y215Ser + Thr217G1u +
Ser219Asp + I1e220Ser Pro204Asn + G1y205G1n + Ser207Asp + G1y215Pro +
Ser2i9Glu + Thr223G1u Pro204Ser + Thr206G1n + Ser207G1u + Thr217G1y +
Ser219Asp + Thr223Asp Pro204G1n + Trp212Leu + G1y215G1u + Thr217Pro +
Arg218Asp + Ser219Asp G1y203Asn + Thr211G1y + G1y214Asn + G1y215Pro +
Ser219Asp + G1y222Asp G1y203Pro + Trp212I1e + G1y214Asn + Ser219G1u +
I1e220Pro + G1y222G1u Thr206G1u + Leu209l1e + G1y215Asn + Ser219Asp +
I1e220Cys + Thr223Asp G1y203Asn + Ser207Asp + G1y214G1n + Ser216G1u +
Arg218Asp + I1e220Thr Ser207G1u + I1e208Cys + I1e213His + G1y214Ser +
Ser216Asp + Arg218G1u Thr206G1n + I1e208Ser + Leu209Asp + G1y215G1u +
Arg218Asp + I1e220A1a G1y205Ser + I1e213Cys + G1y214Asp + Arg218G1u +

Ser219Asp + G1y222G1n Pro204Asn + G1y214Asp + Arg218Asp + Ser219Asp +
G1y222Pro + Thr223G1n Prc204G1y + Thr211Asn + I1e213A1a + G1y215Asp +
Arg218G1u + I1e220G1y Ser~07Glu + Leu209I1e + Ser216G1u + Thr217G1y +
Ser219Asp + I1e220G1y G1y205Ser + Ser207Asp + Thr211G1y + Ser216G1u +
Ser219Asp + I1e220Asn Pro204G1n + G1y205Ser + Thr206Asn + Leu209G1u +
Trp212G1y + Ser216Asp G1y205Pro + Trp212G1y + I1e213His + G1y214Asp +
Ser216G1u + Ser219G1u Pro204Ser + Thr206Asn + I1e213A1a + Ser216Asp +
Ser219G1u + Thr223G1n ~1e20-8Asn + Trp2i2Asn + G1y215Asn + Ser216G1u +
Ser219G1u + I1e220Ser I1e208Leu + Thr211G1y + G1y214G1n + Ser216G1u +
Ser219G1u + G1y222Pro Pro204Ser + Thr206Ser + Ser216Asp + Thr217Pro +
Ser219G1u + Thr223Asn Leu209Va1 + Trp212Cys + G1y214Ser + Arg218G1u +
I1e220G1u + Thr223Asp G1y205G1n + Trp212Met + G1y215Asp + Arg218G1u +
I1e220G1u + G1y222Ser G1y205Pro + Thr206Ser + Ser207G1u + Leu209G1y +
G1y214Ser + Thr223G1u Thr206Asp + Leu209G1u + Thr211G1n + Thr217G1n +
I1e220Met + Thr223G1u Pro204G1y + I1e208Cys + Thr217Ser + Arg218G1u +
Ser221G1u + Thr223Asp Ser207G1u + Leu209Va1 + I1e213G1y + Ser216G1u +
Thr217Asp + G1y222G1n I1e208Thr + Ser216G1u + Ser219G1u + I1e220Leu +
Ser221G1u + Thr223Pro G1y203G1n + G1y205G1n + I1e213A1a + Ser216G1u +
Ser219Asp + Ser221Asp G1y203G1n + Pro204G1n + Thr206Asp + Leu209Asn +
Arg218G1u + G1y222G1u G1y205G1n + I1e213A1a + G1y215G1u + Ser216G1u +
I1e220G1u + G1y222Asn Thr206Asn + I1e208A1a + Leu209Asp + I1e213G1u +
G1y214G1u + G1y222Ser Gly2b3Ser + Pro204Ser + I1e208Cys + I1e213G1u +
G1y215G1u + Ser219G1u ' -Thr206Pro + Leu209Cys + I1e213G1u + G1y215G1u +
Thr217Ser + Ser219G1u Loop 6 - Heptuple Substitution Mutation Variants Pro204Asn + Leu209Thr + Thr211G1n + Trp212His +

G1y2~5Asp -+ I1e220Va1 + Thr223Ser Pro204G=y + G1y205Asn + I1e208Leu + Thr211Asn +
Trp2--Gly -+ G1y214Pro + Arg2~8Asp ..ly-03Pro t Thr2p6G1y + I1e208Pro + Thr2ilGln +
Thr217G1u + Ile22OMet + T'hr223Asn Pro2~4Gin + I1e208A1a + Leu209G1n + Thr2llSer +
G1y214Ser + Ser219G1u + Thr223Pro Pro204G1y + Giy205Pro + Thr206G1u + I1e208Pro +
Leu209Va1 + I1e213His + I1e220Leu G1y205G1n + Thr206Ser + I1e208Leu + Thr211Ser +
G1y214Asn + G1y222Pro + Thr223G1u Pro204G1y + G1y205Ser + I1e208A1a + Trp212Leu +
Thr217G1n + Arg218Asp + Thr223Asn I1e208Asn + Thr211Asn + Trp212Va1 + I1e213Asp +
I1e220Aia + G1y222Pro + Thr223Pro Pro204G1y + I1e208Cys + Trp~l2Cys + I1e213G1y +
Thr217G1n + Ser219Asp + I1e220A1a G1y205Pro + I1e208Asn + Thr211Pro + Trp212Phe +
I1e220Met + Ser221G1u + G1y222Ser G1y203G1n + Pro204Ser + Thr206Ser + I1e208Va1 +
Trp212Tyr + I1e220G1y + Ser221G1u Pro204Ser + G1y205Pro + Leu209Met + Trp212Leu +
I1e213Asp + G1y215G1n + G1y222Ser ~~1y205Pro + I1e208Pr.o + Leu209Thr + Thr211Ser +
Trp%_'l2Asn + Ser216Giu + r_1e220A1a G1y203Gin + G1y205Ser + Thr206G1y ~ Trp212A1a +
G1y215Pro + Thr217Ser + Se-r2~lGlu G1y203Ser + I1e208Cys + Leu209Met + Tr_n212Ser +
G1y215Pro + Ser216G1u + Thr~23Pro G1y205Ser + Thr206G1y + Trp212G1y + Thr~l7Gly +
Arg218G1u + I1e220G1y + G1y222Asn G1y203Ser + G1y205Pro + Thr206G1y + G1y214G1n +
Ser216Asp + I1e220G1y + Thr223Ser Pro204Asn + Trp212G1n + I1e213Thr + G1y215Ser +
Ser216G1u + Thr217Asn + I1e220Asn G1y203Asn + Pro204G1y + Thr206Asn + I1e208Leu +
Thr211Pro + I1e220Leu + G1y222Pro G1y203Asn + G1y205Asn + Thr211Ser + Trp212Met +
I1e213G1u + G1y214Asn + Thr223Asn Pro204Asn + G1y205Pro + I1e208Ser + Thr211G1n +
I1e213G1n + G1y215G1n + Arg218G1u G1y203G1n + G1y205G1n + Thr206Ser + I1e208Va1 +
Trp212G1n + Thr217Pro + I1e220G1u Pro204G1y + G1y205Pro + I1e208Met + Thr211Ser +
I1e213G1y + G1y215Pro + Ser216G1u G1y203G1n + G1y205Ser + Thr206Ser + I1e208A1a +
I1e213A1a + I1e220Met + ThrL23Glu Pro204G1'y + Thr206G1n + Leu2u9Ala + Thr211G1n +
Thr217Asn + Arg218G1u + I1e220Pro G1y203G1n + Pro204G1y + G1y205Ser + Ser207Asp +
Leu209Asn + G1y214Ser + G1y215Pro G1y203G1n + G1y205G1n + Leu209Met + Trp212Thr ~
G1y214G1n + I1e220Asn + G1y222G1u G1y203Ser - Thr'-_'06Asp + I1e208Leu + Thr211Pro +
G1y214Ser + G1y215Ser + G1y222G1n Pro204G1n + Thr206G1y + I1e208Asn + Trp212G1y +
I1e213Pro + Thr217Pro + Arg218Asp G1y205Asn + I1e208Thr + Thr211G1y + I1e213His +
Thr217G1y + Arg218G1u + Thr223Asn G1y205Asn + Thr206Pro + 11e208A1a + Leu209A1a +
Trp212Leu + G1y215Pro + G1y222Asp G1y203Pro + Thr206G1n + I1e208Asn + Leu209Cys +
G1y215G1u + G1y222Pro +-Thr223Ser Pro204G1n + I1e208A1a + Trp212Asn + I1e213Ser +
G1y215Ser + Ser219Asp + I1e220Asp G1y203Ser + Thr211Ser + Trp212Met+.Arg218Asp +
Ser219Asp + G1y222Asn + Thr223Asn G1y203Ser + G1y205Pro + Thr206Pro + I1e208Leu +
Arg218G1u + Ser219G1u + I1e220A1a Pro204G1y + Thr211Asn + G1y214Pro + Ser216Asp +
Thr217G1u + I1e220G1n + G1y222Pro G1y203Asn + G1y205Pro + Leu209Va1 + Thr211Pro +
G1y215Asn + Ser216G1u + Thr217Asp Pro204G1y + G1y205G1n + Thr211Ser + Trp212Met +
G1y215Asp + Ser216G1u + Thr217Pro Pro204Asn + Thr206G1y + I1e208Met ~ Leu209Asp +
Ser219G1u + I1e220His + Thr223Pro G1y205G1n + Thr206G1u + I1e208Thr + Leu209Thr +
Thr211G1n + Trp212Cys + Ser221Asp Pro204G1y + Thr206G1u +-Leu209A1a + Trp212Met +
I1e220Va1 + Ser221Asp + G1y222Ser G1y205G1n + Thr206G1u + Thr211Asn + Trp212Ser ~
Thr2I7Gln + Ser221Asp + Thr223G1n Ser207G1u + Leu209Thr + I1e213G1n + G1y~14Gln +
G1y215Asn + Ser219G1u + I1e220G1u Thr206G1n + Ser207G1u + Leu209I1e + I1e213A1a +
G1y214Ser + i1e220Thr + Ser221Asp Pro204Asn + Thr206G1n + Ser207G1u + I1e208A1a +
Leu209Cys + Thr211G1y + I1e220G1u Thr206Pro + Ser207G1u + Thr211G1n + G1y214Pro +
G1y215Ser + Thr217G1y + Ser219G1u Pro204Asn + Ser207G1u + Leu209G1y + Thr211Asn +
G1y215Asn + Ser219G1u + I1e220Va1 Pro204G1n + G1y205Ser + Leu209Ser + Trp212G1y +
Thr217G1u + Arg218G1u + Ser219Asp G1y203G1n + Ser207Asp + Leu209His-+ G1y214Ser +
G1y215G1n + Ser219Asp + S-er221Asp Pro204G1n + G1y205Asn + Ser207G1u + Leu209G1n +
Thr211G1y + Ser219G1u + Ser221Asp G1y203Asn + Thr206G1u + I1e208G1n + Leu209G1n +
Thr211Pro + Trp212I1e + G1y222G1u G1y203Ser + Thr206G1u + Ser207Asp + Thr211Ser +
Trp212G1y + I1e220G1y + G1y222Asp G1y205Pro + Thr206Asp + Ser207Asp + I1e213Pro +
G1y214Ser + G1y222G1u + Thr223Asn G1y203G1n + Thr206Asp + Leu209Met + Trp212Leu +

WO 96128556 PCTlUS96/03005 G1y214Asn + G1y222Asp + Thr223G1u Pro204Asn + Thr206G1n + Leu209Ser + Ser216G1u +
Thr217Asp + Arg218G1u - Ser219Asp Pro204Asn + Thr206G1y + Ser207G1u + I1e208Thr +
G1y215G1n + I1e220Asp + G1y222G1u G1y203Pro + G1y205G1n + Thr206Ser + Leu209Asp +
Thr211Ser + I1e213Cys + Thr217Asp G1y205G1n + Ser207G1u + Thr211Ser + Trp212Ser +
I1e213G1n + Arg218Asp + I1e220G1u Gly2G3Ser + Pro204G1y + Thr206G1u + Ser207G1u +
I1e208Ser + Leu209Asp + Trp212A1a G1y203Ser + I1e208Thr + Leu20-9Glu + Trp212Tyr +
Thr217G1n + Ser219Asp + Ser221G1u G1y203Ser + G1y205Ser + Thr211Ser + G1y215G1n +
Thr2i7Pro + Ser219G1u + Ser221Asp Gly2C5Gln + Thr206G1y + Leu209Ser + I1e213G1y +
Ser219G1u + Ser221Asp + Thr223G1n Pro204G1n + Ser207Asp + I1e208Ser + I1e213Leu +
Arg218G1u + Ser219G1u + Ser221G1u G1y203As~: - Trp212G1r: + G1y214Pro + Ser21'6Glu +
Arg218G1u t I1e220Leu + Thr223Pro Pro204Asn + G1y205G1n + Thr206G1y + I1e213His +
G1y214Ser +-S~r216Asp + Arg218G1u I1e208Va1 + I1e213Met + G1y214Ser + Ser216Asp +
Thr217G1y + Arg218Asp + I1e220Met Pro204Ser + G1y205Asn + I1e208Leu + Leu209His +
Ser216Asp + Arg218G1u + Thr223Pro G1y203G1n + I1e208Cys + Thr211Pro + Trp212Ser +
G1y215Ser + 5er216G1u + Arg218G1u G1y203Pro.+ G1y205Ser + Leu209Iie + Thr211Asn +
Thr217G1u + Ser219G1u + I1e220His G1y205Ser + Leu209Cys + Thr211Asn + G1y215G1n +
Thr217G1u + Ser219Asp + G1y222Pro G1y203G1n + I1e208A1a + Trp212I1e + G1y215G1n +
Thr217G1u + Ser219Asp + G1y222Pro G1y203Ser + Thr206G1u + Ser207G1u + I1e208Pro +
I1e213Thr + G1y222G1u + Thr223Asp G1y203Ser + I1e208G1n + Trp212Ser + I1e213Leu +
Arg218Asp + Ser219G1u + Ser221Asp G1y203G1n + G1y205Asn + Trp212Asn + I1e213G1u +
Ser216G1u + Thr217Pro + Arg218Asp Thr206Asn + Leu209Asp + I1e213Met + Ser216G1u +
Thr217G1u + Ser219G1u + I1e220G1y G1y205G1n + Thr211Pro + Trp212Va1 + G1y214Ser +
Ser216Asp + Arg218G1u + Ser219G1u Thr206Asn + Ser207Asp + Thr211Ser + I1e213Cys +
I1e220Met + Ser221Asp + Thr223Asp G1y203Asn + I1e213Ser + Thr217Asp + Arg218G1u +
Ser219Asp + Ser221Asp + Thr223G1n Thr206Asn + Ser207G1u + Leu209Cys + Trp212Ser +
Arg218G1u + Ser221Asp + G1y222Pro G1y203G1n + Ser207Asp + G1y214Pro + G1y215Pro +
Arg218Asp + Ser221Asp + G1y222Ser WO 96/28556 PCTlUS96/03005 G1y203Asn + Thr206G1u + Leu209G1n + Arg218G1u +
Ser219Asp + Ser221G1u + G1y222Pro G1y2Q3Pro + Ser207G1u + Trp212Ser + G1y214Pro +
Ser219G1u + Ser221Asp + Thr223Asp Gl,r2~~5G1n + Thr211G1n + I1e213G1u + 5er216G1u +
Arg218Asp + Ser219Asp + I1e220A1a G1~;2~~3Asn + Pro2045er +.Ser207G1u + G1y215G1n +
Thr217Asp + -1e220Asp + Ser221Asp G1y203G1n + Gl_~205Asn + Ser207Asp + Leu209I1.e +
Thr211G1y + Thr217Asp + Arg218G1u Pro204G1y + Trp212Met + G1y214Asp + Ser216Asp +
Thr217G1u + Ser219G1u + Thr223G1n Ser207Asp + Trp212Tyr + G1y215Asn + Ser216Asp +
Arg218G1u + Ser219G1u + Thr223Pro Thr206G1y + Ser207Asp + I1e208G1n + Ser216G1u +
Arg218G1u + Ser219Asp + I1e220G1n Ser207G1u + Leu209G1n + Thr211Pro + G1y215Pro +
Ser216Asp + Arg218G1u + Ser219Asp G1y205Asn + Ser207Asp + I1e208Va1 + G1y214Pro +
Ser216Asp + Arg218G1u + 5er219G1u Thr206Asp + I1e208His + Thr2115er + Trp212Asn +
Arg218Asp + Ser219G1u + G1y222Pro I1e208G1y + Trp2i2Met + I1e213G1n + Ser216G1u +
Arg218Asp + Ser219Asp + Ser22.lAsp G1y203Asn + Thr206G1u + Leu209Asp + Thr217Pro +
I1e220Ser + G1y222G1u + Thr223Asp G1y203Asn + Thr206Asp + I1e208His + Thr2115er +
Trp212Phe + Arg218Asp + Ser221G1u Pro204Asn + G1v214Asn + G1y215Pro + Ser216G1u +
Tr.r217Asp + Arg218Asp + Ser?2lAsp Pro204G1n + G1_~~205Pro + Thr211Asn + G1y215G1n +
Ser219G1u + Ser221Asp + Thr223G1u Ser207Asp + Leu209Va1 + Trp212Cys + G1y214Ser +
Arg218G1u + I1e220G1u + Thr223Asp Thr206Asn + Ser207Asp + I1e208A1a + G1y215Pro +
Arg218G1u + Ser221G1u + Thr223Asp Pro204G1n + G1y205Asn + Thr206G1n + Leu209Cys +
Thr217Asp + Arg218G1u + Ser221G1u G1y203Pro + Thr2065er + I1e213His + Ser219G1u +
I1e220Cys + G1y222G1u + Thr223Asp G1y205Ser + Thr211Ser + Trp212G1n + G1y214Ser +
Ser219Asp + I1e220G1u + Thr223Asp Ser207Asp + Leu209G1n + I1e213G1y + Ser216G1u +
Ser219Asp + I1e220Ser + Ser221G1u Ser207Asp + Thr211Pro + Trp212G1n + I1e213Pro +
Ser216Asp + Ser219Asp + Ser221Asp Pro204G1y + G1y205Pro + I1e208G1y + Leu209G1u +
I1e213Thr + Thr217Asp + Ser221G1u Thr206G1n + Ser207Asp + I1e208Cys + Leu209Thr +
Arg218Asp + Ser219Asp + Thr223Asp G1y203Pro + I1e208Asn + Thr211G1n + I1e213Met +
Thr217G1n + Arg218G1u + Ser221Asp G1y203Asn + G1y205Asn + Thr206G1n + Thr211G1y +

WO 96!28556 PCT/US96/03005 Trp212G1n + Arg218G1u + Ser221G1u G1y203Ser + I1e208Pro + G1y215Pro + Arg218Asp +
I1e220Asn + Ser221G1u + Thr223Asn Thr206Pro + Thr211Pro + G1y214Ser + G1y215Pro +
Arg218Asp + Ser221G1u + Thr223Ser Thr206Asp + Ser207Asp + Trp212Thr + I1e213Cys +
Ser216G1u + Thr217Pro + Ser219Asp G1y205Asn + Thr206Asp + Ser207Asp + I1e213Thr +
Ser216G1u + Ser219G1u + I1e220Leu Thr20eSSer + I1e213Met + G1y214Asp + G1y215Ser +
Thr217G1u + Ser219Asp + I1e220G1u Pro204Asn + Thr206Pro + Leu209G1u + Thr217G1y +
Arg218G1u + Ser219Asp + Thr223Asp Pro204G1y + Ser207G1u + Thr211Pro + G1y214Asp +
Arg218Asp + Ser219Asp + Thr223G1n G1y205Pro + Leu209G1u + Thr211Asn + G1y214Asp +
Thr217Asp + I1e220Met + G1y222Asn Pro204Ser + Trp212His + I1e213Asn + G1y214G1u Thr217G1u + Ser219G1u + G1y222Asn G1y203G1n + Ser207G1u + I1e208Cys + G1y215Pro +
Ser219G1u + I1e220Asn + Thr223G1u Ser207G1u + I1e208G1y + I1e213Pro + Arg218Asp +
I1e220Met + G1y222Asp + Thr223G1u Thr206Asp + Ser207G1u + I1e208G1y + Thr211G1n +
Ser216Asp + Ser221G1u + Thr223Asn G1y203Asn + Ser207G1u + Trp212Ser + I1e213Asp +
G1y215Asn + Ser216G1u + Arg218G1u G1y203Asn + Leu209Ser + I1e213Va1 + Thr217Pro +
Ser219Asp + I1e220A1a + G1y222G1u I1e208Met + Thr211G1y + Trp212Leu + G1y214Pro +
G1y215Pro + Ser219Asp + G1y2~2Glu Ser207G1u + Leu209Ser + I1e213Ser + G1y214Asn +
Ser216Asp + Arg218G1u - Ser221Asp ~~1~~203Ser + Ser207Asp + ~1e208Asn + Thr211Asn +
G1y214G1u + Ser219Asp ~ I1e220Asp Ser207Asp + Thr211Asn + I1e213A1a + G1y215Asp +
Thr217Ser + Ser219G1u + I1e220G1u Pro204Ser + G1y205Asn + Thr211Pro + Thr217G1u +
Ser221Asp + G1y222G1u + Thr223G1u Ser207G1u + Leu209A1a + G1y214Pro + Ser216G1u +
Thr217Ser + Arg218G1u + I1e220Leu I1e208Va1 + Leu209G1u + Trp212Ser + G1y215G1u +
Ser219Asp + I1e220Asp + G1y222Pro G1y203G1n + I1e213Ser + G1y215G1u + Ser216Asp +
Ser219Asp + G1y222Ser + Thr223G1n G1y205Pro + Leu209Va1 + G1y214Asn + Ser216G1u +
Ser219Asp + I1e220G1u + G1y222Asp Pro204Asn + Leu209Asp + I1e213Met + G1y214Pro +
Thr217G1u + Ser221G1u + G1y222Asp G1y203Ser + Ser207G1u + Trp212Pro + G1y214Asp +
Ser216Asp + Thr217Asp + I1e220Met Thr206G1u + Thr211G1n + I1e213Pro + G1y214Asp +
Thr217G1y + Ser221Asp + G1y222Asp I1e208Cys + Leu209Ser + 'Thr211Asn + Ile2l3Ala +
G1y215Asp + Arg218G1u + I1e220G1y I1e208Thr + Leu209His-- I1e213Met + G1y214G1u +
Ser221G1u + G1y222Asp + Thr223Asp Thr206G1n + G1y214G1n + G1y215G1u + Thr217G1y +
Arg218G1u + Ser219G1u + Ser221Asp Pro204Ser + Thr206Ser-+ I1e213Cys + G1y215G1u +
Ser216G1u + Thr217G1u + Thr223Asp Thr206G1n + I1e208Met + Trp212G1y + G1y214Asp +
Ser216Asp + Thr217Pro + Ser219Asp Ser207G1u + I1e208Thr + Thr211G1n + Trp212G1n +
G1y215Asp + Ser219Asp + Ser221Asp G1y203Ser + Pro204G1n + Ser207G1u + I1e208Cys +
G1y214G1u + Ser219Asp + Ser221G1u Ser207Asp + I1e208Thr + Thr211Asn + G1y215G1u +
Ser219G1u + I1e220Met + Ser221G1u Pro204Ser + Ser207G1u + G1y214Ser + G1y215Asp +
Ser219Asp + I1e220Met + Ser221G1u Ser207Asp + I1e208Leu + Trp212I1e + I1e213Asp +
Thr217Asn + Arg218G1u + Ser221G1u Thr206G1u + Trp212Ser + I1e213Cys + Thr217G1y +
Arg218G1u + Ser219G1u + Thr223G1u Thr206Asp + Ser207G1u + Trp212Cys + I1e213Cys +
G1y215Asn + Ser216Asp + Arg218G1u Thr206G1u + Ser207G1u + G1y214G1n + G1y215Pro +
Ser216Asp + Thr217Ser + Arg218Asp Pro204Ser + G1y205Ser + Trp212Pro + Ser216Asp +
Ser219Asp + I1e220Va1 + Thr223Ser G1y203G1n + Leu209G1n + Trp212A1a + G1y215Asn +
Arg218G1u + I1e220G1u + Thr223G1u G1y203G1n + Leu209Asp + Thr211Asn + G1y214G1u +
Arg218G1u + I1e220Leu + G1y222Ser G1y203Ser + Pro204G1n + Thr211Pro + G1y215Asp +
Arg218G1u + I1e220G1u + Thr223Asn Thr206Asn + I1e208G1n + Ser216G1u + Thr217G1y +
Arg218Asp + I1e220G1y + Ser221Asp Pro204Ser + I1e208His + G1y214Pro + Ser216G1u +
Arg218G1u + Ser221G1u + Thr223Ser Pro204G1n + Leu209I1e + Trp212A1a + Ser216Asp +
Thr217Ser + Arg218Asp + Ser221Asp G1y203Pro + Pro204Asn + Ser207Asp + I1e208Cys +
Thr217G1y + I1e220G1y + Thr223G1u Pro204Ser + Ser207Asp + Leu209His + Thr211Ser +
G1y215Asn + G1y222Ser + Thr223G1u I1e208His + Thr211Pro + Trp212Leu + Thr217Ser +
Arg218Asp + Ser221Asp + Thr223G1u G1y205Asn + Thr206Asn + Ser207Asp + I1e213Thr +
Ser216Asp + Ser219Asp + G1y222G1u Thr206Asp + Ser207G1u + I1e208Leu + Trp212G1y +
Ser216G1u + Thr217G1n + G1y222Asp G1y205Asn + Thr206Asn + Ser207Asp + I1e208G1n +
Trp212Cys + Ser216G1u + Thr217G1u Ser207G1u + I1e208Asn + Trp212Thr + I1e213G1n +

Ser216Asp + Thr217G1u + Thr223G1y Ser207Asp + I1e208His + Thr211Asn + G1y215Asn +
Ser216Asp + Thr217G1u + Thr223G1y G1y203Ser + Pro204Ser + I1e213Thr + Ser216G1u +
Thr217G1y + Ser2.19Asp + Ser221G1u G1y203Asn + I1e208Ser + Thr211Asn + Ser216Asp +
Ser219G1u + Ser221Asp + Thr223G1y G1_,i203Ser + Thr206Pro + Thr211G1y + Trp212Thr +
Ser2loGlu + Se~'219G1u + Ser221Asp Trp212Ser + G1y214Pro + G1y215Asp + Thr217Asp +
Ser219G1u + Ser221Asp + Thr223Pro Thr206Ser + Ser207Asp + I1e208Pro + Leu209G1u +
G1y215Asp + I1e220Thr + Ser221G1u G1y203Asn + Trp212Tyr + G1y214Asp + G1y215Ser +
Arg218G1u + I1e220Pro + Thr223Ser Pro204Ser + I1e208Thr + Thr211G1y + Trp212His +
G1y214Asp + Thr217Pro + Arg218G1u Leu209G1u + Thr211Pro + I1e213Cys + G1y214G1u +
Arg218Asp + I1e220Asn + Ser221Asp G1y205G1n + Thr2ilAsn + G1y214Asp + G1y215G1u +
Thr217Asp + I1e220A1a +-Thr223G1u G1y205G1n + Thr211G1n + I1e213Asn + G1y215Asp +
Thr217G1u + I1e220G1u + Ser221Asp Pro204Asn + I1e213Asp + Thr217G1u + Arg218G1u +
I1e220Ser + G1y222Pro + Thr223G1u G1y205G1n + Ser207G1u + I1e208Cys + G1y214Asp +
Ser216G1u + Ser219Asp + Thr223G1y G1y203Ser + Ser207G1u + G1y215Asn + Ser216Asp +
Thr?l7Gly + Arg218G1u + G1y222G1u Thr206G1n + Leu209Met + G1y214G1u + G1y215Asn +
Ser219G1u + Ser221Asp + G1y222G1u Leu209Met + Thr211Ser + Trp212Cys + Ser216G1u +
Thr217Pro + I1e220G1u + Ser221G1u Thr206G1y + Thr211G1n + Trp212Cys + Ser216Asp +
I1e220G1u + Ser221Asp + Thr223G1n G1y203Ser + Pro204Asn + Ser207G1u + I1e213Asp +
Thr217Ser + Ser219G1u + G1y222Asn G1y205Pro + Thr206Asp + Trp212Leu + G1y214Asn +
Ser216G1u + Ser219Asp + G1y222Ser G1y203Asn + Pro204Ser + G1y205G1n + Ser207Asp +
Trp212Phe + G1y215Asp + Arg218G1u Multi-loop Double Mutation Variants Leu 96G1y + Leu209Pro Tyr104Cys + I1e220G1n Leu133I1e + Ser216G1u Leu 96A1a + Asn162G1u G1n103Asp + I1e220Thr Tyr169Ser + Ser190Asp Val 95Asp + G1y205G1n Asp 65G1u + I1e213Asn G1yl00Ser + I1e220G1u Glyl00Glu + Asn163G1n Asn162Ser + Ser207Asp Ser170G1u + I1e220Ser Ser140G1u + I1e220Leu G1n103G1u + Thr106Pro Leu133Pro + Leu209Asn G1y203Asn + G1y222Asp Ser207G1u + I1e213Leu Ser101Asp + Thr206G1y G1y20.3Ser + Ser207G1u Asn 99G1u + Trp212Asn G1y102Pro + Ser216Asp Ser219Asp + I1e220Met Gly 70Asp + Leu133Ser G1y136Asn + Ser216Asp Arg167G1u + Leu209Pro Asnl94Glu + Leu209G1y Leu209G1n + Ser216G1u Gly 68Asn + Ser221G1u Asp 65G1u + I1e107Cys Arg218G1u + I1e220A1a Asp 65G1u + G1y135Pro Gly 68Asp + Phe192His Gly 66Asn +-Leu209Ser Asn168Ser + G1y214Pro Thr 7lGly + Leu 96G1y Gly 70G1n + I1e220G1y Tyr169His + Leu209I1e Thr217Pro + Ser219Asp Gly 70Pro + I1e220Asp Leu 96G1n + Asn 99G1u Asp 65G1u + G1y136Ser Asn 99G1u + I1e213Met G1y205Pro + Ser207Asp Asn 99G1u + Leu209G1n Val 95G1n + Ser207Asp Val 95G1n + Ser219Asp Asn 67Ser + Thr217G1u G1y160G1n + Leu209Asp G1y136Asn + G1y160Asn Asn163G1n + Thr223Asn Leu209G1u + Thr217Asn G1y102Pro + Trp212G1n Arg 64Asp + G1y203Asn Asn 67G1n + Thr211Asn Asp 65G1u + Tyr137Pro G1n103Asp + Leu133I1e Leu 96G1u + I1e213G1n Asn161G1n + Arg218G1u Leu209Asn + G1y214Pro Gly 68Ser + Thr206Asn Arg167Asp + Phe192I1e Asp 65G1u + Thr211Ser Ser207Asp + Thr223G1y Ser207Asp + G1v222Ser Tyr104Leu + Arg167G1u Asp 98G1u + I1e220Asn Arg 64G1u + G1y215Asn Asn 67Asp + Asn163Ser G1y160Ser + Thr206G1n I1e213Ser+ Thr223Asp G1y215G1n + Ser219Asp Phe202Leu + Ser219G1u Ser207Asp + I1e220Cys Tyr137Ser + Ser219Asp G1yl00Asn + Leu209Thr Tyr137His + 5er170Asp Ser170Asp + I1e220Thr Thr106G1n + Ser207Asp Arg167Asp + Leu209Pro Ser105Asp + G1y136Asn Ser139Asp + Phe192Thr Ser190G1u + Pro204Ser Asn 67G1u + Leu209A1a Trp212Ser + Ser216G1u Tyr104A1a + Thr206Asp Asn161G1n + G1y222Asn Val 95Thr + Leu133Ser G1y160Ser + Asp165G1u Asn163Ser + Leu209Va1 G1y136G1u + I1e220Pro Ser191Asp + Pro204Ser Thr217G1u + I1e220Asn Leu209Met + G1y214Asp Thr 7lPro + Ser191G1u SerlOlGlu + Trp212Phe Gly 70Ser + Ser219G1u Gly 66G1n + Asn 99Ser Asn163G1n + Ser190G1u I1e107Ser + Ser190Asp I1e208Thr + Ser219Asp Ser216Asp + I1e220Leu Ser140Asp + A1a164His Asp 65G1u + Val 95Ser Phe202Met + Leu209Ser Asn 67G1n + Tyr169G1y G1y1365er + Asn162Asp G1y160Pro + Ser219Asp G1y102Pro + Ser207Asp Val 95Asn + Ser207Asp Asp 97G1u + I1e107G1n Thr206G1u + I1e220Pro Ser216G1u + Thr223G1y Asp 97G1u + Leu133Cys G1y134G1n + G1y135Asp Thr206G1n + I1e220His G1yl00Asn + Thr223Asp Gly 6&Pro + I1e220Va1 Gly 66Asn + Asn168G1u Ser190Asp + I1e213Va1 G1y135Pro + Asn162G1u Leu133I1e + G1y134Pro Asn163Ser + Arg167G1u G1y134G1n + Ser216G1u Asn194G1n + Leu209G1y Asn 99G1n + I1e21.3Met Leu 96I1e + G1y160Asp Arg167G1u + I1e220A1a Val 95A1a + Ser139Asp G1n103Ser + G1y214Ser Asn 67G1u + Thr 7lGln G1y135Pro + Ser191G1u I1e107A1a + I1e220G1u G1y214G1n + Ser219G1u G1y160Asp + Phe192Met Leu 96A1a + Asp 97G1u Asn168G1n + Leu209G1u G1n103G1u + G1y136Asn G1y215Asp + G1y222Ser Ser207Asp + Thr223Asn Ser216Asp + I1e220Cys Val 95Ser + Thr206G1u Gly 70Asn + Ser216Asp Leu209Ser + I1e220Asp A1a164Pro + I1e208Thr Tyr137Thr + Leu209G1u Thr106Asp + Tvr137Thr Thr106G1y + Aspl65Glu Asn 99Asp ~ I1e213G1y Asn161G1u + I1e220Ser G1y160Asp + I1e220Met G1y135Asp + Asn163G1n Tyr169Ser + Ser216G1u Ser140Asp + Leu209Asn Ser140Asp + Leu209G1n Ser170G1u + G1y222Pro I1e213His + Ser216Asp Leu 96Thr + G1y222Asn Asp 65G1u + Asn168G1n Tyr104Leu + Ser219G1u Thr106Pro + G1y134Pro Asn 99Asp + G1y215Asn Thr206G1u + I1e220Va1 Gly 70Pro + Leu 96GIu Thr106G1y + Ser219G1u Glyl00Glu + I1e107G1y G1y102G1n + Tyr169G1y Arg 64G1u + pro204G1y Leu133G1n + Ser207Asp G1yl00Gln + Leu133His Thr106Asp + Leu209A1a Gly 68G1u + I1e220G1n Asn 67Asp + Gly 68G1n Leu 96G1u + I1e220Leu Thr206G1n + Ser219Asp A1a164G1n + G1y222G1n Gly 66G1u + Gly 68Pro Ser140Asp + A1a166Asn Asn 67Ser + Glyl00Asp Ser219Asp + I1e220Thr Ser170G1u + G1y215Ser Thr 7lGly + Phe192I1e G1yl00Ser + Ser219Asp G1y215Pro -+ Ser219Asp Leu209Met + Ser221Asp Asn162Asp + Tyr169Met Tyr104His + Asn162G1u Asn 67G1n + Leu 96Ser Leu209Asp + I1e220A1a Gly 70Ser + I1e220G1n Gly 66Ser + Gly 70G1u Tyr169His + Thr206G1y Asn194G1n + Thr206G1y I1e107G1u + Leu209Asn Gly 66Ser + G1y222G1u Leu133I1e + Ser190G1u Ser105Asp + I1e220His G1y102Asp + Asn194G1n Leu 96Met + Ser216Asp Asn161Asp + G1y203G1n G1y136G1n + Asn161Asp Phe202I1e + Ser219Asp G1n103Asp + I1e107Thr Glyl00Gln + Ser216Asp Phe192A1a + Ser207G1u Glyl36pro + Ser138Asp Asp 98G1u + Leu209Thr Asn194Ser + Ser216G1u Val 95Asp + G1y222G1n Thr211Pro + I1e220Met Arg167G1u + Tyr169A1a Gly 66Asn + Ser138Asp Asn 67G1u + Leu209Pro Gly 70G1u + Tyr169Pro G1y102Pro + Leu209Asn Gly 68Ser + Ser207Asp Ser101G1u + I1e220Ser Tyr137Va1-+ Ser191G1u Thr211G1y + Arg218Asp Asn161G1n + Asn194G1n Thr106Asn + Leu209G1n Val 95Ser + G1y134Asp Ser191Asp + I1e220Ser Asn163Ser + Thr223G1u Leu209Thr + Arg218Asp Gly 70Asp + Val 95G1y Leu209Met + Ser216Asp Thr206Ser + Arg218G1u Leu209I1e + Thr217Asp I1e213Pro + Ser219G1u G1y205Ser + Thr206Asp Phe192G1n + Thr206G1u Gly 68Asp + Leu209Ser G1y160G1n + Thr206G1y G1y214Asp + I1e220Leu Gly 70Asn + Arg218Asp Gly 70G1u + G1y136Asn Leu133G1u + Gly2D3Pro G1y135G1n + Ser207G1u I1e107G1y + I1e213G1n Ser140Asp + I1e220Leu Ser191Asp + G1y214G1n Leu209Ser + G1y214Asp Asn 67G1u + I1e220G1n A1a166Pro + Ser219Asp G1y102Ser + I1e220Va1 G1n103Ser + Ser140G1u G1y102Asp + I1e220G1y Tyr104l1e + G1y134G1n Ser101G1u + I1e220Cys Asn162G1n + Ser207Asp Val 95Pro + Ser207Asp Asn168G1n + Leu209A1a G1yl00Gln + G1y136Asn Phe192Pro + Asn194G1u Asn 99Ser + Thr211G1n G1y215Pro + I1e220Asp Arg 64G1u + Leu133I1e Ser207G1u + I1e220Cys Asp 97G1u + Thr206Asn Thr106Asp + Tyr137Met Ser138G1u + Thr223Pro Ser138Asp + I1e220A1a Ser191Asp + Leu209Asn Leu133I1e + Ser170G1u I1e107Cys + Ser139G1u Asn161G1n + Ser207Asp Thr206Asn + Ser216G1u Asn 67G1n + Ser207Asp Leu209Met +Ser219G1u Gly 68Ser + Asp 97G1u G1y136G1u + G1y222Asn Thr106Pro + Leu209Va1 G1n103G1u + I1e213A1a Leu-96G1n + Ser219G1u I1e107Asp + A1a164G1n Tyr169Asp + Thr211Pro Leu133G1u + Leu209Va1 Asp 97G1u + Asn161Ser I1e213G1y + Ser216Asp Thr206Pro + Ser207Asp A1a164G1n + Leu209Cys Ser170G1u + Leu209A1a G1y160Pro + Leu209Cys Asn 67Asp + Asn163G1n Thr206G1y + Ser216Asp Leu133Thr + Ser216G1u Asn163Asp + Leu209Thr Val 95Met + Phe202Ser G1y136Ser + G1y222Ser Leu209Met + I1e220Met G1n103Ser + Asn163G1u I1e107His + Arg218Asp Phe192A1a + G1y215G1n Leu209Thr + Ser216G1u Tyr169G1u + Thr217Ser Ser101Asp + Leu209A1a Leu209A1a + Ser221Asp Gly 66Asp + Thr106G1n Val 95G1u + I1e213Cys Ser105Asp + Thr211G1n Asp165G1u + Asn168Ser Gly 66Asn + I1e220Thr Ser219G1u + I1e220Pro G1y 68G1u + I1e220Met Val 95His + Leu209Met Gly 66Ser + Leu209Met Ser140G1u + G1y214G1n G1y160Asn + Leu209Asp Thr206Asp + I1e220Met G1y102Asn + Thr206G1u G1y134Pro + Phe192Pro Leu133Asp + Phe202Ser Asn162G1u + I1e220His Leu133G1y + Leu209Met Asn194Ser + Ser219Asp Gly 70G1n + Thr217Pro Asp 97G1u + Thr217G1n Glyl00Asp + Leu20-9Cys Ser216Asp + T1e220Thr Arg167G1u + Asn194Ser Gly 66G1n + Ser170G1u Ser101Asp + Tyr137A1a Asn 67G1n + Asn161Asp Asn168Asp + Leu209I1e Asn 67G1n + Gly 68G1u Asnl6iAsp + Thr217Pro Thr206Asp + Leu209Met Asp 97G1u + Leu209MeL
Asp 97G1u + Tyr104His Asp 98G1u + G1y102Pro Tyr104A1a + Ser191G1u G1yl00Ser + Arg167G1u Ser207Asp + Leu209Ser Asn194Ser + Ser207Asp Leu 96Va1 + I1e220Cys Asn163G1n + Ser191Asp Asn162G1n + Asp165G1u Gly 66G1n + Thr 7lAsn Tyr137Leu + Phe192Asn Gly 66Asn + Ser207G1u Asp 97G1u + Leu133Ser Thr206G1n + Ser216Asp Leu 96Pro + I1e213G1u Asp165G1u + G1v214Asn Ser-190G1u + Thr217G1y Tyr104Met.+ I1e22OGlu Asp 65G1u + Thr223Ser Glyl00Asn + Ser170G1u G1y134Ser + Leu209Cys A1a164G1y + Ser207Asp Thr 7lAsn + I1e220Asp I1e107Cys + Ser219Asp Thr106G1y + Asn162Asp Asn161G1u ~ Leu209Thr G1y214G1n + Ser221Asp A1a164G1n + Ser207G1u Asn 67Ser + Ser216Asp Leu209A1a + Thr217Ser A1a166G1n + Ser216Asp Leu209G1n + I1e220G1u Multi-loop Triple Mutation Variants Leu 96G1y + Leu209Pro -i- Ser216G1u Arg 64G1u + Tyr104Cys + I1e220G1n Tyr169Ser + Ser190Asp + I1e220Thr G1y100G1u + Asnl&2Ser + Asn163G1n G1n103G1u + Leu133Pro + Leu209Asn Gly 70Asp + Leu133Ser + G1y136Asn Gly 68Asn + Leu209G1n + Ser216G1u Asp 65G1u + G1y135Pro + I1e220A1a Gly 66Asn + Asn 67G1u + Leu209Ser Thr 7lGly + Leu 96G1y + G1y214Pro Gly 70G1n + Leu209I1e + I1e220G1y Tyr169His + Thr217Pro + Ser219Asp Asn 67Ser + Val 95G1n + Thr217G1u G1y136Asn + G1y160G1n + Leu209Asp G1y136G1u + Asn163G1n + Thr223Asn Arg 64Asp + G1y203Asn + Trp212G1n Gly 68Ser + Leu209Asn + G1y214Pro Argi67Asp + Phe192I1e + Thr206Asn Ser207Asp + G1y222Ser + Thr223G1y G1y160Ser + Asn163Ser + Thr206Gin Glyl00Asn + Tyr137Ser + Ser219Asp Asn n7Asp + G1yl00Ser + Tyr137His Ser105Asp + G1y136Asn + Phe192Thr Vai 95Thr + Leu133Ser + Asn161G1n Ser140G1u + Asn163Ser + Leu209Va1 Pro204Ser + Thr217G1u + I1e220Asn I1e107Ser + Ile2D8Thr + Ser219Asp Asp 65G1u + Val 95Ser + Leu209Ser Asn 67G1n + Tyr169G1y + Phe202Met G1y136Ser + G1y160Pro + Asn162Asp Val 95Asn + I1e107G1n + Ser207Asp G1y135Asp + Thr206G1n + I1e220His Gly 66Pro + G1y100Asn + I1e220Va1 G1y135Pro + Asn162G1u + I1e213Va1 Leu133I1e + G1y134Pro + Arg167G1u G1y134G1n + Asn163Ser + Ser216G1u Asn194G1n + Ser207G1u + Leu209G1y.
Arg 64G1u + Asn 99G1n + I1e213Met Tyr104Ser + Arg167G1u + I1e220A1a Val 95A1a + G1n103Ser + Ser139Asp I1e107A1a + G1y214G1n + I1e220G1u G1n103G1u + G1y136Asn + Asn168G1n Thr206Pro + Ser216G1u + I1e220Cys Asn c~7Asp + A1a164Pro + I1e208Thr Asp 65G1u + Asn168G1n + Gly~?2Asn Tyr104Leu + Leu209Ser + Ser219G1u I1e107Pro + Asn194G1u + Thr206G1y Leu133Asn + Leu209I1e + I1e213Asp Gly 70Pro + Thr106G1y + Ser219G1u Asp 65G1u + G1y102G1n + Tyr169G1y G1yl00Gln + Leu133G1n + G1y160Asp A1a164Ser - Ser216Asp + Thr217G1y Gly 68G1u + Leu209A1a + I1e220G1n Gly 66G1u + A1a164G1n + G1y222G1n Thr 7lGly + Phe192I1e + G1y215Ser Asn 67G1n + Leu 96Ser + Leu209Asp Arg 64G1u + Gly 70Ser + I1e220G1n Gly 66Ser + Tyr169His + Thr206G1y Ser170G1u + Thr206G1y + G1y215Ser Gly 66Ser + Ilel07Glu + Leu209Asn Ser105Asp + Asn194G1n + I1e220His Leu 96Met + Asn161Asp + G1y203G1n G1y136G1n + Asn161Asp + Phe202I1e Glyl00Gln + Phe192A1a + Ser216Asp Thr211Pro + I1e220Met + G1y222G1n Gly 68Ser + Thr106G1n + Ser207Asp WO 96!28556 PCTIUS96/0300~
Asn161G1n + Thr211G1y + Arg218Asp Thr106Asn +-Asn194G1n + Leu209G1n Asp 65G1u + Tyr169Va1 + I1e220Va1 Gly 70Asp + Val 95G1y + Leu209Met G1y205Ser + Thr206Asp + I1e213Pro Phe192G1n + Thr206G1u + Leu209Ser G1y160G1n + Thr206G1y + G1y214Asp Gly 70Asn + Arg218Asp + I1e220Leu G1y 70G1u + G1y136Asn + G1y203Pro I1e107G1y + I1e213G1n+ Ser219Asp G1y102Ser + A1a166Pro + I1e220Va1 Tyr104I1e + G1y134G1n + I1e220Cys Val 95Pro + Asn162G1n + Ser207Asp G1y100G1n + G1y136Asn + Asn194G1u Asn 99Ser + Thr211G1n + I1e220Asp Arg 64G1u + Leu133l1e + I1e220Ser Asp 97G1u + Thr206Asn + I1e220Cys I1e107Asn - Thr206Asr_ + Ser207G1u Asn 67G1n + Leu 96G1n + I1e213Asp Gly 68Ser + Leu209Met + Ser219G1u Tyr169Asp + Leu209Va1 + Thr211Pro Thr206Pro + I1e213G1y + Ser216Asp A1a164G1n + Ser207Asp + Leu209Cys Asn 67Asp + G1y160Pro + Leu209Gys Asn163G1n + Thr206G1y + Ser216Asp Val 95Met + Phe202Ser + Leu209Thr G1y13oSer + I1e220Met + G1y222Ser G1n103Ser + Asnln3Glu + Leu~09Met I1e107His + Phel92Aia + Arg218Asp Ser101Asp + Leu209A1a + Thr217Ser Gly 66Asn + Asp165G1u + Asn168Ser Gly 66Ser + Leu209Met + G1y214G1n G1y134Pro + Ser140G1u + Phe192Pro Leu133G1y + Ser207G1u + Leu209Met Gly 70G1n + Asn163Asp + Asn194Ser G1y100-Asp + Leu209Cys + Thr217G1n Gly 66G1n + Ser101Asp + Tyr137A1a Asn &7Gln + Asn168Asp + Leu209I1e Asn161Asp + Leu209Met + Thr217Pro G1yl00Ser + Ser2Q7Asp + Leu209Ser Leu 96Va1 + Asn194Ser + Ser2U7Asp Asn163G1n + Serl9lAsp + I1e220Cys Gly 66G1n + Thr 7lAsn + Ser216Asp Gly 66Asn + Tyr137Leu + Phe192Asn Asp 97G1u + Leu133Ser + I1e220Met Leu 96Pro + Thr206G1n + I1e213G1u Asp165G1u + G1y214Asn + Thr217G1y Asp 65G1u + G1y100Asn + Thr223Ser G1y134Ser + Ser170G1u + Leu209Cys Thr 7lAsn + I1e107Cys + Ser219Asp Thr106G1y + Asn162Asp + Leu209Thr Asn 67Ser + A1a164G1n + Ser207G1u A1a166G1n + Leu209A1a + Ser216Asp Leu 96G1y + Leu133Ser +-Ser190G1u Ser207Asp + Leu209Thr + Ile22gPro Tyr137Thr + Ser139G1u + G1y215Pro Thr206G1y + Leu209Asp + Thr211Asn Thr206Asp + I1e208Va1 + I1e220Leu Leu 96Cys + Ser207G1u + I1e220Leu I1e107Leu + G1y136G1u + I1e220G1y Thr106Asn + Ser139G1u + A1a164Thr Ser191G1u + Leu209Ser + I1e220G1y Ser140G1u +-G1y215Ser + G1y222Pro Leu 96Ser + A1a164G1y + I1e220His Asp 97G1u + Tyr104Asn + Tyr137G1n Gly 70G1n + Asp 98G1u + Leu133Thr Gly 66Ser + Thr 7lGly + Phe202Ser SerinlAsp + Thr206G1y,+ I1e213G1y Tyr169I1e + Serl'70G1u + Thr211G1y G1y160Ser + I1e213Va1 + Ser219G1u Leu209G1y + I1e220G1y + Thr223Pro Thr106G1n + A1a154G1n + Phe192G1u Asn 99G1u + Leu209G1n + I1e220Met G1y160G1n + Asn194G1u + G1y215G1n Ser105Asp + Phe202Pro + Leu209G1n Gly 66G1u + Leu133Thr + Leu209Asn A1a164Thr + Leu209Met -~ I1e220Thr Thr 7lGly + G1y214Asp + G1y222Asri Val 95Pro + Thr206G1y + I1e213Asp Leu 96Asn + G1yl00Asn + Asn194Ser Arg 64G1u + G1yl00Asn + I1e220Met Gly 66Ser + Arg218Asp + G1y222Ser Asp 65G1u + Asn162G1n + I1e220Thr Asn 99Asp + Thr206Ser + I1e220Ser Leu 96Ser + Tyr169His + Ser216Asp Asp 98G1u + Tyr169His + G1y215Asn Tyr104Leu + Ser207Asp + Leu209Ser Asn161Ser + Phe192His + Thr206Pro Leu 96Ser + Ser170Asp + Leu209I1e Thr106G1n + Asn163G1n + Ser207Asp Gly 70G1n + Asn 99Ser + Asn161Ser Asn 99Ser + A1a164Asp + Thr217Pro Ser139G1u + Thr206Asn + I1e213Leu Leu 96Asn + Thr217Asn + I1e220His G1n103Ser + Leu209Pro + Ser219G1u Asp 65G1u + Leu 96Cys + G1n103Asn Asn 67G1n + Ser207Asp + G1y214Asn Gly 68Pro + Thr211Ser + Thr223Asp Tyr137G1n + Ser138G1u + I1e220Asn Asp 65G1u + Phe192Va1 + G1y215Ser Thr 7lGln + G1y203G1n + Thr206Asp Arg 64Asp + Asn 99Ser + Thr106Ser G1y135G1n + Asn168Ser + Phe192Asp Leu133A1a + Leu209A1a + Ser219G1u A1a164Thr + Asn168Ser + .I1e213Pro Ser207G1u + G1y214G1n + I1e220Asn Phe192Tyr + Leu209Pro + G1y215G1n Asn163G1n + Thr206G1y + Leu209Asn Leu 96Pro + Asp 98G1u + Asn1.63G1n SerlOlAsp + Asn168G1n + Tyr169Va1 G1y136Pro + Asn162G1u + I1e220Thr Leu209Cys + G1y214G1n + I1e220Cys Arg 64Asp + Leu209I1e + I1e220His G1y134Pro + G1y135Ser + Ser219G1u A1a164Thr + Leu2-09Thr + Ser219G1u Trp212Phe + Ser219G1u + I1e220G1u Leu 96G1u + Asp 97G1u + I1e220Leu Leu 96G1u + Asp 97G1u + G1y222Pro Asn161Asp + Asn162G1u + G1y214Ser G1y160Asp + Asn161G1u + I1e220Ser Tyr137Va1 + Thr206G1u + Ser207Asp Gly 68Pro + Arg218G1u + Ser219G1u Leu209Thr + Arg218G1u + Ser219Asp Tyr104G1n + Ser216Asp + 'I'hr217As~
Leu209G1u + Thr217Asn + Ser219Asp Leu209Asp + Ser219G1u + I1e220Ser Asn194G1n + Ser207Asp + Ser221G1u A1a164G1y + Ser207Asp ~- I1e220Asp Leu 96Va1 +-Ser207Asp + I1e220Asp Leu 96G1u + G1y102G1u + I1e213G1n G1y 68Asn + Ser207G1u + Ser219G1u Ser207Asp + Ser219G1u + I1e220Cys Leu 96G1n + Ser207G1u + Ser219G1u G1y102Pro + Ser207Asp + Ser219Asp G1n103Ser + Ser207Asp + Ser219G1u Val 95G1n + Ser207Asp + Ser219Asp Ser207Asp + I1e213Pro + Ser219G1u Asp 65G1u + Asn 67Asp + Alalo4His Asp 65G1u + Asn 67G1u + G1yI36Ser Asn162G1n + Asp165G1u + Arg167G1u Leu209Met + G1y214Asp + Ser216G1u Ser138Asp + Ser170Asp + I1e220Met Ser138G1u + Asn168G1n + Ser170G1u Arg 64G1u + Gly 66Asp + Thr217Pro Asp165G1u + Asn168Asp + Thr211Asn Leu 96G1u + Asp 98G1u + G1y222Ser Thr206Ser + Ser216Asp + Arg218G1u A1a164Ser + Ser216G1u + Arg218G1u Asnl6lAsp + Asn162Asp + Thr223Asp Leu209I1e + Thr217Asp + Ser219G1u Ser101Asp + G1n103Asp + Leu133I1e Asn 67G1u + Asp 97G1u + Leu209A1a Leu133I1e + Ser190G1u + G1y222G1u Ser207Asp + Ser219G1u + G1y222Asp A1a164Asp + Asn168G1u + Leu209Pro Asp 65G1u + Gly 68G1u + Asp 97G1u Asn163Ser + Thr206Asp + Ser219G1u Arg 64G1u + Asn 67Asp + G1y215Asn Tyr137Asp + Ser139G1u + Arg167Asp Ser140Asp + Ser170G1u + G1y222Ser Tyr137G1u + Ser140Asp + Leu209G1n Arg 64G1u + Asp 97G1u + Tyr104His Val 95G1u + Ser105Asp + Thr211G1n G1y100Asn + Asn161G1u + Ser221Asp Asp 65G1u + Asn 99G1u + Trp212Asn Asp 65G1u + Asn 99Asp + Thr217Ser Ser190G1u + Ser207G1u + I1e220G1u Gly 68G1u + G1y136Pro + Ser216G1u Val 95G1u + Asp-98G1u + G1y102Pro Asn162Asp + Asp165G1u + Ser191G1u Val 95Cys + Ser138Asp + Arg167G1u Leu209Asp + Ser216G1u + I1e220A1a Glyl00Glu + G1n103Asp + I1e107G1y Glyl00Asp + G1y102Pro + G1n103G1u Ser216Asp + Ser219G1u + I1e220Thr G1y102Pro + Ser216Asp + Ser219Asp Tyr104G1n + Ser216G1u + Ser219G1u Asn 99G1n + Ser216G1u + Ser219Asp Thr20aGln + Ser216Asp - Ser219Asp Ser207Asp + Ser216Asp + Thr217Asp G1y134Asp + Asn162G1u + Phe192G1u G1y102G1u + Ser105Asp + Leu133G1u Ser216Asp + Ser219Asp + Ser221Asp Asn163Asp + Ser170G1u + Asn194Asp Tyr137G1u + Asn162G1u + Arg167G1u Ser105Asp + G1y135Asp + Tyr169Asp Val 95Asp + Asp 98G1u + Ser105G1u G1y136Asp + Ser140Asp + G1y203G1n Ser207Asp + Ser216G1u + Ile22QAsp Thr106G1y + Tyr137Asp + Asp165G1u Asp 65G1u + G1yl00Glu + G1n103Asp Ser101G1u + Arg167G1u + Asn168G1u Asn 67Asp + Ser191Asp + Phe192G1u Asp 98G1u + Asn 99G1u + Ser216Asp Asp 98G1u + Asn 99Asp + Ser216Asp Arg 64G1u + Asp 65G1u + Asp165G1u G1y102G1u + G1n103G1u + Ser221G1u Leu133G1u + Thr206Asp + Ser207G1u Ser139Asp + Ser216Asp + Thr217Asp Asp165G1u + G1y215Asp + Ser216Asp Asp 97G1u + Leu209G1u + Ser219G1u Ser191G1u + Leu209G1u + Ser219G1u Thr106Asp + Leu209Asp + Ser219Asp Asn 99G1u + I1e213Asp + Ser216G1u Ser207G1u + Leu209G1u + I1e213Asp Asn 67G1u + Asp 98G1u + Leu209Asp Ser101Asp + Ser207Asp + Ser221Asp G1y135Asp + Ser207Asp + Ser219G1u Asn162G1u + Ser207Asp + Ser219G1u Ser139G1u + Ser207G1u + Ser219G1u G1y134G1u + Ser170G1u + Ser216Asp Asp165G1u + Arg167G1u + Ser207Asp Asp165G1u + Arg167Asp +-Ser219Asp Asp 65G1u + Ser138G1u + Ser140Asp Asp 97G1u + Arg167G1u + Tyrl69Asp Ser207G1u + I1e213G1u + Ser2l6Glu Thr ,~Prc + Ser191G1u + Ser207G1u Ser191Asp + Ser207Asp + G1y214G1n Gly 66G1u + Gly 70G1u + Ser216G1u Asn163Asp + Ser191Asp + I1e220G1u ' Asp 97G1u + Ser207G1u + Leu209G1u Tyr104Asp + Ser207G1u - Leu209G1u Ser190Asp + Thr206G1u + Ser216Asp Asp 97G1u + Leu133G1u + Asn161Ser Asn168Asp + Ser170Asp + Ser190Asp Asp 98G1u + Ser221Asp + Thr223G1u Asn o7Asp + Asp 97G1u + G1y135G1u Asn 67Asp + Asp 97G1u + Ser219Asp Ser105G1u + G1y160Asp + G1y222Asp Asp 98G1u + G1y160Asp + Asn163G1u Ser139G1u + Ser170G1u + Arg218Asp G1n103G1u + Ser207Asp + G1y222G1u Ser207G1u + Ser216Asp + G1y222Asp G1y135G1u + G1y160G1u + Ser216G1u Tyr104G1u + Asn163Asp + Ser190G1u Ser190G1u + Asn194Asp + Ser219Asp Tyr104Met + Ser190G1u + Iie220G1u Asn 67Asp + Ser216Asp t Ser219G1u Leu133Asp + Asnle~2Glu + Phe202Ser Asn162G1u + Ser191Asp + Leu2D9Glu Asp 65G1u + Leu 9GAsp + Asn162Asp Arg 64Asp + G1y136G1u + Argi67Asp Asn o'7Asp + Gly oBGlr. - Ser216G1u Asn 67G1u + -Ser216G1u ~ I1e220Cys SerlOlGlu + Thr2D6Asp T Leu20-9Glu Asp 65G1u + Leu209G1u + Ser216G1u Ser101G1u + I1e107G1u + Ser219G1u Gly 68Asp + Ser170G1u + Ser216Asp G1y136Asp + Ser139G1u + Ser216G1u G1y135Asp + Leu209Ser + Thr223Asp Leu133G1u + Asn161G1u + G1y214G1u Arg 64G1u + Ser191G1u + Ser221Asp Asn 67G1u + G1y214Asp + Ser219G1u Thr106G1u + Ser219G1u + G1y222G1u Asp 98G1u + I1e220Asp + Thr223G1u Arg167Asp + G1y215G1u + Arg218Asp Tyr104Asp + Ser139G1u + Ser219Asp I1e107G1y + Asn161Asp + I1e220Asp G1y160G1u + Ser216Asp + Ser219Asp Asp165G1u + Ser216G1u + Ser219Asp Asp 98G1u + Ser207Asp +-Thr223Asp Ser139Asp + Asn168G1u + Ser207G1u Ser105Asp + Ser138-Glu + I1e220G1u Ser105G1u + Ser138Asp + Ser219Asp Ser105Asp + Ser138Asp + G1y222Asp I1e213Pro + Ser216G1u + I1e220G1u SerlOlGlu + G1y214G1u + Arg218G1u G1y13~Glu + Asp165G1u + Ser190Asp Asp 65G1u ~ Leu209Cys + Ser216Asp Ser138Asp + Asn161G1u + Thr206G1u G1y13&Asp + Ser140Asp + Ser219G1u Gly 68G1u + G1y134G1u + Ser219Asp Gly 68Asp + Asn194G1u + Ser219Asp Gly 68G1u + Asn161G1u + Ser219Asp Gly 68Asp + I1e107Asp + Ser219G1u Gly 68G1u + Ser139Asp + Ser219Asp Asp 98G1u + G1y135Asp + Ser139G1u G1y135G1u ,- Ser139G1u + Leu209G1u Alalei4Glu + Ser190Asp + Ser219G1u G1n103Asp + Tyr137Asp + G1y160Ser Gly 70G1u + G1y102Asp + Tyr169Pro Gly 70G1u + Ser219G1u + G1y222Asn Gly 70G1u + Leu209Met + Ser221Asp I1e213Ser + Ser219Asp + Thr223Asp G1y135Asp + Ser140G1u + G1y215Asp G1y102G1u + Ser170G1u + Thr206Asn GlylG=:lu + G1y135Asp + Thr206G1u Gly 70G1u + Asp165G1u + I1e220Asp Phe192Asp + Ser2Q7Asp + I1e213G1u A1a164G1u + Arg21r8Asp + Thr223Asp G1y135Asp + Ser190G1u + I1e220G1u Asn 99G1u + Leu133Asp + Asp165G1u Asp165G1u + Ser219G1u + Thr223Asp Asn 67G1u + Asn162Asp + Ser216G1u Multi-loop Quadruple Mutation Variants Tyr104Cys + Leu133I1e + Ser216G1u + I1e220G1n G1y102Asn + G1y160Ser + Alalb-4Gly + Arg218Asp Asn 67G1u + Thr206G1n + Leu209Met + I1e220Met G1n103G1u + Thr106Pro + Leu133Pro + Leu209Asn Val 95His + G1y136Asn + G1y160G1n + G1y222G1u Arg 64Asp + G1y102Pro + G1y203Asn + Trp212G1n Val 95Thr + Leu133Ser + Asn161G1n + G1y222Asn Gly 66G1n + Gly 70Ser + Asn 99Ser + Ser219G1u Asp 65G1u + Gly 68Asn + Tyr169Vai + I1e220Va1 Phe192G1n + G1y205Ser + Thr206G1u + Leu209Ser Leu133Va1 + Asn162Asp + Leu209Va1 + I1e220A1a Leu 96Thr + Leu209I1e + I1e220Va1 + Thr223Ser Glyl00Glu + G1y102Asn + G1y134Ser + I1e208Pro GlyI00Ser + Asn194G1n + Ser207G1u + I1e220Thr Asn 67G1n + A1a164G1n + Tyr169ASp + I1e220Thr G1y160Ser + Ser191G1u + I1e220Ser + G1y222Ser Thr206Pro + Ser207Asp + Leu209Cys + I1e213G1y Val 95Met + G1y136Ser + Phe202Ser + G1y222Ser G1n103Ser + Asn163G1u + Leu209Met +-I1e220Met WO 96!28556 PCT/US96/03005 I1e107His + Phe192A1a + G1y215G1n + Arg218Asp Gly 66Asn + Asp165G1u + Asn168Ser + I1e220Thr Glyl00Asn + Ser140G1u + Leu209Met + G1y214G1n Gly 70G1n + Asn163Asp + Asn194Ser + Thr217Pro Asn161Ser + Phe192His + Thr206Pro + Ser219Asp Val 95Cys + Leu 96Ser + Ser138Asp + Leu209I1e Gly 68G1n + SerI0lAsp + Thr106Pro + I1e220Met G1yl00Pro + G1y102G1n + Leu209Cys + G1y215Asp G1yl00Asn + G1n103Ser + Ser207Asp + Leu209Va1 Thr106G1n + I1e107Asn + phe192Va1 + Thr223G1y Tyr137G1n + Ser138Asp + Leu209Va1 + Trp212Asn Leu133A1a + Asn162Asp + Tyr169Va1 + Leu209I1e G1y102Ser + G1y134G1n + G1y136Asn + Ser207G1u Glyl00Gln + G1y134Pro + G1y160Asn + Ser219G1u Tyrl3-7Met + Asn194G1n + Ser207Asp + G1y215Pro A1a164Asn + Ser190Asp + Phe202Ser + I1e213Asn Gly 68Pro + Asp 98G1u + A1a166His + Asn168Ser Iie107Pro + G1y136Pro + Ser207G1u + G1y222G1n Arg c~4Asp + Tyrl6-9Gly + Phe192Pro + G1y205Ser Val 95Thr + Tyf104Cys + G1y134Asp + I1e208Asn I1e107His + G1y135G1u + Phe192G1y + Thr217Pro I1e107G1y + Ser191G1u + Leu209Met + Thr223G1n Gly 68G1n +Val 95A1a + Ser207G1u + Leu209Asn Gly 66Asn + Asp 97G1u + Asn161Ser + G1y214Asn Tyr137Met + Leu209Ser + Ser216Asp + I1e220Pro Val 95Asn + Leu209Va1 + Ser219Asp + Thr223Pro G1y135Asn + Ser140G1u + A1a164Thr + I1e220Leu Gly 70Ser + G1y135Asp + Thr21'7Gln + I1e220Thr Gly 66Ser + Ser140Asp + Leu209Pro + I1e213Leu Val 95Asn + Leu 96I1e + Thr217Pro + I1e220G1y Leu 96Met + G1y203Asn + Ser219G1u + I1e220G1y Tyr104Cys + Tyr169Ser + Leu209Thr + Ser216G1u Gly 70G1n + Ser138Asp + Thr206Asn + I1e220G1n Asn163G1n + G1y214Pro + I1e220Asn + Thr223G1u Gly 68Asn + Ser138Asp + Asn168Ser + I1e220Met Asn161G1n + Phe192His + Thr217Asp + I1e220A1a G1n103Ser + Phe192Asn + Arg218G1u + I1e220Thr A1a164G1y + Asn168G1n + Ser207Asp + I1e208Met Thr106G1y + Ser139Asp + Phe192Thr + G1y215Asn Leu209His + I1e213Ser + G1y214Asn + Ser219G1u Gly 66Asn + Asn 99Ser + SerlOlGlu + Asn194G1n Tyr137G1u + Thr206Pro + Trp212Thr + I1e220His G1y135G1u + Tyr169Cys + -Thr206G1n + I1e220Ser Thr206Pro + Leu209Ser + Ser216G1u + I1e220Va1 Gly 66Asn + Gly 68Asn + Ser105Asp + Leu209Pro , Ser170G1u + Thr206G1y + Leu209G1n + I1e220Cys Tyr104Thr + Asn163Ser + Leu209His + G1y222Asn Thr106G1u + Tyr137G1n + Asn162Ser + Leu209A1a Leu 96Thr + Ser207Asp + G1y2145er + I1e220G1y Asn 99Ser + Phe192Pro + Trp212Va1 + Ser216Asp G1y136Ser + Tyr137Asp + Leu209Va1 + I1e220G1y Gly 66Ser + G1y203Asn + Ser219G1u + I1e220Met A1a164Thr + Thr206Asn + Thr217Ser + Ser219G1u WO 96!28556 PCT/US96/03005 G1y102Pro + Thr106Asn + Asn161G1u + Tyr169Ser ThrI06G1y + G1y134G1n + Thr206Asn + Ser207G1u Giy 66Asn + G1n103Asn + Thr106G1n + I1e220G1n Arg 64Asp + Asn 67G1n + I1e107A1a + Asn162Ser Th- 7lGly ~- Thr106Ser + Asnl63Ser + I1e220Asn Asr: 67G1n A1a164Ser + Ser207Asp + I1e220Thr G'_,% 68Ser - Asn163G1n + Arg167Asp + I1e208Thr T~~r~37Ser ~ Asn168G1u + Leu209Asn + I1e213A1a G1y134Ser + G1y135Pro + A1a164His + Asn168Ser Asn163G1n + A1a164Asp + Leu209G1y + G1y215Pro G1y102Pro + G1y136Pro + Leu209Ser + I1e220Pro Tyr104Cys + G1y136Asn + A1a164Asn + Leu209His Asp 65G1u + Leu 96Va1 + G1y136Asn + Tyr169Pro Asn 67Asp + G1yl00Pro + Tyr104Leu + Leu209A1a Val 95His + G1y135G1n + Ser191G1u + I1e220A1a Asn163Ser + Asn194Ser +-Ser207G1u + Leu209Ser Gly 66G1u + Leu 96I1e +Tyr104Thr + I1e213A1a G1ni03Asr_ + Ser138G1u + G1y160Ser + Phe202Cys Gly 68Pro-- Thr106Ser + Asn194Asp + Thr211Ser G1y100G1n + Tyr169Thr + Thr211Pro + Thr217Ser Gly 66Asn-+ Val 95Cys + Ser139Asp + Asn194G1n G1yi35Pro + Asn161G1u + Asn162Ser + Leu209A1a Gly 66Ser + Thr 7lSer + Val 95Thr + I1e107Asn Asn168G1n + Leu209Pro + Ser219G1u + I1e220G1n Gly 66Pro + Tyr137Asn + Tyr169I1e + Thr223Asp Ser101G1u + I1e107Pro + Leu133Cys + Leu209Pro Asn 67Asp + Gly 68Ser + Asn161Ser + Thr223Ser Asn 99G1n + G1y102Asn + I1e107Met ~ Trp212G1y Tyrl37A1a -_- Asn194G1n ~ I1e213Asp + G1y214Asn Leu 96A1a - heu209Met + G1y222Asr. + Thr223G1n Leu 96Va1 + Asn162Ser ~- AsnlG3Gln + Ser170G1u . Asr_ 67Ser T Leu133G1n + Leu209G1n + Ser221Asp Val 95G1n + A1a164Thr + Ser207G1u + Leu209His Glyl00Gln + Asn168G1n + Ser219Asp + I1e220Asp Asp 97G1u + Asp 98G1u + I1e213A1a + Thr217G1y G1n103Asn + G1y135Ser + G1y215G1u + Ser216G1u Asn194Ser + Leu209G1y + G1y215G1u + Ser216G1u Gly 70G1n + Tyr104Cys + Leu209G1u + Ser219G1u I1e107Va1 + Thr206Asp + Ser207G1u + Ser221G1u Leu133G1y + Ser207G1u + Leu209Met + Ser221G1u Val 95Thr + Tyr137His + Ser207G1u + Ser219G1u Gly 68G1n + Asn162Ser + Ser207Asp + Ser219G1u Tyr137A1a + Ser207G1u + I1e213Pro + Ser219Asp G1yl00Asn + Tyr137Ser + Ser207Asp + Ser219Asp Tyr137G1y + Ser207G1u + G1y215Asn + Ser219G1u Asp 65G1u + Asn 67Asp + Phe202Ser + Thr206G1y G1y160Ser + Asn161G1u + Asn194G1u + Thr217Asn Asr_ 67G1n + Ser207Asp + Ser219Asp + Ser221G1u G1y102Asn + G1y134G1n + Ser138G1u + Ser170Asp Asp 65G1u + Gly 68Asp + G1y135Pro + Phe192His Phe192Asn + Thr206G1u +Ser207G1u + Ser219G1u G1y160Pro + Thr206Asp + Ser207G1u + Ser219Asp Leu 96Asn + Thr206G1u + Ser207Asp + G1y222G1u WO 96!28556 PCT/US96/03005 Asp 65G1u + Asp 98G1u + Phe192Leu + I1e213G1n Gly 66G1n + Asn162Ser + Ser207G1u + Leu209Asp Gly 66Ser + --G1y136G1n+ Ser190Asp + Thr206Asp Asn 67G1n + I1e107His + Asp165G1u + Asn168Asp -Phe202Thr + Ser207Asp + I1e220G1u + G1y222G1u Asn 67Ser + Val 95G1n + Leu209Asp + Thr217G1u Asn163Asp + Ser191Asp + Asn194G1u + I1e220Met Ser207G1u + Leu209G1u + I1e213Met + Ser221Asp Asn 67G1u + Leu 96Asp + Asp 97G1u + Leu209A1a Thr206Asp + Leu209Asp + Ser219Asp + I1e220Pro Ser216G1u + Thr217G1u + Ser219G1u + G1y222G1n Ser139G1u + Ser170G1u + Phe192His + Leu209G1n G1y102Asn + Pro204Ser Ser207Asp + G1y222G1u Thr106G1n + Ser207Asp + Leu209Ser + G1y222Asp G1yl00Ser + Ser207Asp + I1e220Asn + G1y222Asp Thr106Asn + Ser190-Glu+ Ser207Asp + Ser221G1u Leu209Met + G1y214Asp + Thr217G1u + I1e220Asn A1a164G1n + Ser207Asp + Arg218Asp + I1e220Va1 Tyr169G1y + Leu209Asp + Ser216G1u + Ser219Giu Arg167G1u + Asn194Asp + Leu209Ser + I1e220Pro G1y102Pro + I1e213Asp + Arg218Asp + Ser219G1u Asp 98G1u + G1y102Asp + Asn161G1n + Leu209Ser I1e107G1y + G1y134Asp + Ser138Asp + Tyr169Ser Gly 68G1u + G1y134Pro + Phe192Pro + Ser216G1u Asp 65G1u + G1y214G1u + I1e220Thr + G1y222Pro Ser190Asp + Ser207Asp + I1e208Met + I1e220Va1 G1y134Asn + Phe202His + Arg218Asp + Ser221Asp Leu 96A1a + I1e213G1u + Ser216G1u + Ser219Asp G1y160G1n + Ser207Asp + Arg218G1u + G1y222Asp Gly 70G1u + I1e107His + Leu133G1u + Phe192Thr G1y136Asp + Ser170Asp + Asn194Asp + G1y222Ser Tyr104G1u + Leu133Asp + I1e213Ser + G1y215G1n G1y100G1n + Ser207G1u + Ser216Asp + Ser219G1u G1y100Asn + Tyr137A1a + Leu209Asp + Ser216G1u Asn162Ser + Ser216G1u + Ser219Asp + I1e220Asn Val 95Ser + Leu 96Pro + Ser216Asp + Ser219Asp G1y102Pro + Ser216Asp + Ser219Asp + I1e220Met G1y135G1u + G1y136G1n + Arg167Asp + Asn194G1u Asn 99G1n + Ser207G1u + Ser216Asp + Thr217Asp Asp 65G1u + SerlOlGlu + G1y102G1u + Leu133Thr Asn163G1n + Ser216G1u + Ser219Asp ~ Ser221Asp Gly 66G1n + Asn163Asp + Ser170G1u + Asn194Asp Gly 66Pro + G1y134Asp + Asn161G1u + Ser191Asp Leu133Thr + Asn161Asp + Ser219Asp + I1e220Asp Ser105Asp + G1y135Asp + G1y136Ser + Tyr169Asp Asn 67G1n + Asn163G1u + Asn194G1u + Ser221Asp Ser207G1u + I1e213Pro + Ser216G1u + I1e220G1u Gly 68Asp + Leu 96G1u + Thr206G1y + G1y214G1u Asn 99G1u + Ser101G1u + G1y136G1u + Asn163G1n , Ser138G1u + Ser139Asp + Asn163Asp + I1e220Met Ser101G1u + Trp212Phe + Ser219G1u + I1e220G1u A1a164G1u + Asn168G1n + Ser219G1u + I1e220Asp Ser101G1u + G1y160G1n-+ Arg167G1u + Asn168G1u Arg157G1u + Asn168G1u + Asn194Ser + Ser207Asp Arg167G1u 1 Asn168Asp + Asn194Ser +_I1e220G1u Le~~ 96Thr - Arg167Asp + Asn168G1u + Ser216G1u Thr106Asp + Leu209Asn + Ser221Asp + G1y222Asp Asp 65G1u + Tyr137His + Asn161G1u + Asn162Asp Asn 67Asp + Ser191Asp + Phe192G1u + Pro204Ser Arg 64Asp + Gly 68Ser + G1y222Asp + Thr223G1u Ser139G1u - Ser140G1u + Arg218Asp + Thr223Pro Leu 96Met + Asp 98G1u + Asn 99Asp + Ser219Asp Arg 64Asp + Asp 65G1u +G1n103Asp + Leu20911e Tyr137G1u + Ser138G1u + Ser216Asp + Thr217Asn G1y136Asp + Thr206Asp + Ser207G1u + I1e220Met Ser101Asp + A1a164Ser + Thr206Asp + Ser207G1u Gly 66G1n + Tyr104Asp + Ser105Asp + Ser219Asp Ser138Asp + Thr217G1n + Arg218G1u + Ser219Asp Arg 64Asp + Leu209G1n + Ser216G1u + Thr217G1u Ser105G1u + Leu209Asp + Ser219G1u + Thr223G1n Ser101Asp - Leu209Asp + Ser219G1u + I1e220Ser Asn163Asp + Phe192Leu + Asn194G1u + Ser207Asp Asr~163Asp - Asn194G1u + Ser207G1u + Trp212His A1a164Asp + Asn194G1u + Ser207Asp + I1e220Leu A1a164G1u + Asn194G1u + Ser207G1u + I1e220Ser G1yl00Asp + A1a164G1u + Asn194Asp + Thr206Ser Leu 96Pro + Ser207G1u + .;er216G1u + Ser22,lAsp Val 95Asp + Phe192Tyr + Ser207Asp + Ser221Asp A1a164Asp + Ser207Asp + G1y215G1n + Ser221Asp Asp 65G1u + Leu209G1n + Ser216Asp + Arg218Asp I1e107Cys + Ser139Asp + Ser207G1u + Ser219Asp Tyr137G1u + Ser207Asp + Ser219G1u + I1e220Ser -Val 95G1n + Tyr104Asp + Ser207Asp + Ser219Asp Asn163G1u + Ser207G1u + Ser219G1u + G1y222Ser Asn168G1u + Ser207G1u + Ser219G1u + G1y222Asn G1y135Asp + G1y203Ser + Ser207G1u + Ser219G1u Leu 96Thr + Ser139G1u + Ser207Asp + Ser219Asp G1yl00Glu + Ser207Asp + Ser219G1u + I1e220Cys G1n103G1u + Tyr104Pro + G1y136G1u + I1e220Asn G1n103G1u + Ser105G1u + Ser219Asp + I1e220Cys G1n103G1u + Ser105Asp + Tyr137Thr + Thr206G1u G1y160Asn + Ser207Asp + G1y214G1u + 5er216Asp Asp 97G1u + SerlOlAsp + Ser207Asp + I1e220Thr Thr106Asp + Tyr137Met + Leu209G1u + Arg218G1u G1y136G1n T Ser138Asp + Ser170G1u + Ser216G1u Gly 70Asp -~ Leu133Ser + Ser138Asp + Ser170Asp Asn163G1u + Tyr169Cys + Ser219Asp + G1y222Asp Arg 64G1u + Gly 66Asp + Asn168G1n + Arg218Asp Ser105Asp + G1y136Asn + Ser139Asp + Phe192Thr Tyr137Leu + Ser191G1u + Ser207G1u + G1y222Ser Ser191Asp + Ser207Asp + Leu209His + I1e220Thr Asn 99Ser + Ser191G1u + Ser207Asp + I1e220Asn Gly 66Asp + Asn 99Asp + Thr106G1n + Ser216Asp Asp 97G1u + Phe202Thr + Arg218G1u + I1e220Asp Gly 70G1n + Asn 99Asp + Ser207G1u + Leu209G1u Thr106G1u + Ser207G1u + Leu209Asp + G1y214G1n Asp 65G1u + Tyr137Pro + Asp165G1u + Asn168Asp Asn 99G1n + Asp165G1u + Asn168Asp + G1y215G1u Asn 67G1u + Asn161G1u + I1e220A1a + G1y222G1u Gly 66Asp + Leu 96G1y + Leu209G1u + I1e220Asp Asp 98G1u + Leu209A1a + Ser219G1u + Ser221Asp Asn 67G1u + Alal64Gln + Ser219G1u + Ser221Asp G1y100G1u + G1y136Asn + Ser216Asp + Arg218G1u Iie107Asp + Thr206Ser+ Ser216Asp + Arg218G1u A1a164G1y + Asn194Asp + Thr217Asp + Ser219G1u _ Asp 98G1u + Asn163Asp + Asp165G1u + Trp212Tyr Ser139G1u + G1y215G1u + Thr217Asp + G1y222Pro Ser101G1u + Ser105G1u + Phe192Va1 + Leu209His Tyr104Asn + Leu209G1u + Ser216Asp + G1y222Asp Gly 70G1u + Tyr104A1a + Thr206Asp + Ser219G1u Leu 96G1u + Asn 99Asp + A1a164G1n + G1y214G1u Ser139Asp + Asn161G1u + A1a164G1u + I1e220G1y Asn 67Ser + Gly 70Asp + Leu 96G1u + Ser221G1u Asn 67Asp + Asp 97G1u + Asn163Ser + Ser221G1u Ser170G1u - Ser207Asp + I1e220Ser + G1y222Asp Leu 96G1y + Asn163Asp + Thr206Asp + Ser219G1u Asn 67Asp ~ Ser190Asp + G1y215Ser + G1y222Asp Tyr137Cys + Arg167Asp + Ser170Asp + Ser219Asp G1y135G1u + Ser138Asp + G1y214G1u + G1y222Ser Asp 65G1u + Asp 97G1u + Asn194G1u + G1y205Asn G1y102Ser + Ser105Asp + Leu133G1u + Thr223Asp Gly 70Asp + Asp 98G1u + Leu209Met + Ser216Asp Ser138G1u + A1a164Ser + Thr206G1u + Ser219G1u Gly 68Pro + Asp 97G1u + Thr206Asp + Ser219Asp Ser105G1u + Ser140G1u + Arg218G1u + Thr223G1n Asn 99Asp + G1y102G1u + Ile''13Va1 + Ser219G1u Asn 99Asp + G1y102Asp + G1y136Ser + Ser138Ast~
Gly 70G1u + Va1 95G1u + G1yl00Asn + G1y222G1u Asn 67Giu + Ser216G1u + Ser219Asp + I1e220Cys Asn 67Asp + A1a164Pro + Ser216G1u + Ser219Asp Asn 99Asp + Asn161G1u + Ser190Asp + G1y215Asn Val 95Asp + Asn161G1n + Ser190G1u + Ser221Asp Gly 68Asp + G1y100G1u + G1y222Ser + Thr223G1u G1y134G1u + Tyr169Leu + Ser207G1u + Arg218G1u Thr106Asp + Ser207G1u + Arg218Asp + I1e220Cys Arg 64G1u + Asn163Ser + Ser207Asp + Arg218Asp Ser139Asp + Ser207Asp + Arg218Asp + I1e220Thr Leu133Asp + G1y134Pro +-Asn162Asp + Tyr169Asn Val 95Asp + G1y134Giu + Asn194G1u + Thr211G1y I1e107Asn + Ser140G1u + Ser170G1u + Ser219Asp Tyr137Cys + Ser140G1u + Ser170Asp + Ser219G1u Tyr137Thr + Ser140G1u + Ser170G1u + Thr206G1u Tyr137Asn + G1y160G1u + Asp165G1u + Ser216G1u Asn~ 99G1u + G1y160Asp + Asp165G1u + Leu209G1n Asp 97G1u + Tyr104G1u + Leu133Ser + I1e220Leu Asn 67G1u + Val 95G1y + Pro204Ser + Ser216Asp Asn 99Asp + Asn162Gin + Ser191Asp + G1y222G1u G1yl00Glu + Thr106G1y + Asn162Asp + Tyr169G1u Asp 65G1u + Asn 99Asp + G1y135Asn + G1y160Asp WO 96!28556 PCT/US96/03005 Asp 65G1u + Asn 99G1u + Trp212Asn + Ser219Asp G1y134G1u + Asn161Asp + Thr217Pro + Ser219Asp Asp 97G1u + G1n103G1u + Asn163G1n + I1e220G1u Asn161G1u + Asp165G1u + Ser207Asp + Thr223G1n Ser140G1u + A1a164His + Thr217Asp + I1e220Asp Gly 70Ser + G1y102Asp + Leu133Asp + Ser191Asp SerlOlGlu + I1e107G1u + Thr217Pro + Ser219G1u Tyr137G1u + Asn162G1u + Ser190Asp + I1e213G1n Gly 68Asp + Ser170G1u + Ser216Asp + I1e220Va1 G1n103Asp + G1y135Asp + Leu209Met + Ser216Asp Tyr104Va1 + G1y136G1u + Ser139G1u + Ser190Asp G1y102Pro + Asn161G1u + Asp165G1u +.G1y214G1u Gly 70G1u + Tyr169Cys + Ser190G1u + Ser207G1u Asn168G1u + Serl9OGlu + Pro204Ser + Ser207G1u Ser190Asp - Ser2C7Asp + Leu209Ser + Ser216G1u Asn 57G1u + Ser101G1u + Tyr137Asp + G1y215G1n Arg 64G1u + Ser191G1u + I1e220His + Ser221Asp Arg167Asp + Ser191Asp + I1e213A1a + Ser221G1u Arg167G1u + T'hr206Ser + Ser219Asp + G1y222G1u SerlOlGlu + Asn161G1n + Asn162Asp + Ser190Asp Ser101Asp + G1y136Pro + Ser216Asp + Ser219G1u Tyr104G1n + Thr106G1u + Ser216G1u + Ser219G1u Gly 68Pro + Leu133G1u + Ser216G1u + Ser219G1u G1y160G1u + Leu209Va1 + Ser216G1u + Ser219G1u Ser191G1u + Leu209G1n + Ser216Asp + Ser219Asp Asn 67G1n + Arg167G1u + Ser216G1u + Ser219Asp Asn 99G1n + Ser170Asp + Ser216G1u + Ser219Asp Ser138G1u + Asn162G1n + Ser216G1u + Ser219G1u G1y135G1n + Asn168G1u + Ser216Asp + Ser219Asp G1yl00Glu + Thr206Ser + Ser207G1u + Thr223G1u Asn163G1n + G1y214Asn + Ser216G1u + I1e220Asp Glyl00Pro + Thr211Asn + Ser216Asp + I1e220G1u Thr206Pro + Leu209Thr + Ser216G1u + I1e220Asp G1y102Asp + G1y160Asp + Ser190G1u + Trp212Ser Leu133Cys + G1y134Asp + Asn162Asp + Ser216Asp Asp 65G1u + Thr106G1n + Ser216G1u + G1y222G1n Asp 98G1u + Glyl00Ser + Asn168Asp + I1e213G1u Ser101Asp + Tyr137A1a + Asn161G1u + Thr206G1u Gly 70Asp + G1y136G1u + Seri40Glu + I1e220Pro Gly 68G1u + Tyr104G1u + Leu209Va1 + Ser219Asp Gly 68Asp + G1y160G1n + Asn194G1u + Ser219Asp Asn163G1n + Ser190G1u + Ser216G1u + I1e220G1u Arg 64Asp + G1n103Asn + Leu133Asp + G1y214G1u Arg 64G1u + Tyr104Ser + Leu209G1u + G1y214Asp Gly 70Asp + Ser101Asp + Ser140G1u + I1e220Va1 Gly 70Asp + Leu 96Thr + Leu209Asn + Ser216G1u I1e107Asp + Ser138G1u + A1a166G1n + Ser219Asp Asn161G1u + Ser207Asp + Leu209I1e + Thr217Asp Gly 68Asn + Asn163G1u + Arg167G1u + Ser219Asp Asn 99Ser + G1n103Asp + G1y136G1u + Thr206G1u G1n103G1u + Leu133G1n + G1y136G1u + Ser216G1u G1y102Asp + Leu133Thr + Ser207Asp + Thr217G1u Asp 97G1u + Ser207Asp + Thr217G1u + Thr223Asn WO 96!28556 PCT/US96/03005 SerlQlGlu + Ser191Asp + Ser207G1u + Leu209Cys I1e107Ser + Ser191G1u + Ser207Asp + Ser216Asp lhr 7lPro + S-er191G1u + Ser207G1u + Ser216G1u A1a164G1n + Ser191G1u + Ser207G1u + Ser216Asp Ser170G1u + Asn194Asp + Ser207G1u + I1e220Va1 ' Ser170Asp + Asn194G1u + Trp212Ser + Ser216G1u Arg-64G1u + Asn 99G1u + Ser170Asp + Leu209Thr _ Asp 97G1u + Leu133G1u + Asn161Ser + Ser216Asp Tyr137Pro + Ser140Asp + Phe192Asp + Ser207G1u Asn 99Ser + G1n103G1u + Ser170Asp + Ser219Asp G1n103~1u + Asn163G1u + Ser170G1u + I1e220His Asp 65G1u + Asn 99Ser + Ser101G1u + Ser191Asp Ser105G1u + G1y135Asp + Thr206Asp + Thr217G1n Leu 96Cys + Asn161G1u +-Arg167G1u + Arg218Asp Arg 64G1u + Glyl00Glu + Asn161Ser + Leu209G1u Val 95Met + Asp 97G1u + Ser105G1u + I1e220Asn Gly 68G1u + Val 95G1u + Asn168Ser + Leu209A1a Asn 67G1u + Asn161Asp + Ser216Asp + G1y222Asn Asn 67Asp + Leu133His + Arg167G1u + Ser216G1u Ser105Asp + G1y160Ser + Ser170Asp + Thr206G1u Gly 66Pro + Asn162Asp + Ser170G1u + Ser219G1u G1yl00Glu + G1y102Asn + Tyr104Asp + Ser207Asp Glyl00Glu + Tyr104Asp + G1y160G1u + G1v215c;7n Multi-Poop Quintuple Mutation Variants Leu133G1n + G1y136G1u + Thr206G1n + Leu209Met +
I1e220Met G1y203Asn + G1y205G1n + Ser216G1u + Thr217G1y +
I1e220G1y Val 95His + A1a164Ser + Ser170G1u + Phe192Met +
Thr223G1y Leu133Asn + Asn161G1n + Asn163G1n + Leu209Met +
I1e220Leu Asn 67G1n + G1y136Ser + Asn162Asp + Tyr169G1y +
Phe202Met I1e107Asn + Leu209G1n + I1e213G1y + Ser219G1u +
I1e220His Gly 66Ser + Thr 7lGly + Tyr169His + Phe202Ser +
I1e220Asp G1y135Ser + A1a166Ser + Ser207G1u + I1e213G1y +
G1y222Ser Asp 98G1u + Thr106Pro + Tyr137Cys + Trp212I1e +
I1e220Va1 ' G1y134Asn + A1a166Pro + Phe192Met + Thr206G1y +
Ser219G1u Thr106G1u + G1y205Asn + Leu209Cys + G1y215Pro +
I1e220Thr Asn163G1n + Asn168G1n + Phe192Cys + Thr206G1n +
Leu209Ser Leu 9661-y + I1e107G1y + Ser140Asp + Leu209A1a +

Thr223G1y Asn 67Ser + Val 95His + G1y135G1n + Ser191G1u +
I1e220A1a I1e107G1y + Tyr169Ser + Asn194Ser + Ser207G1u +
Leu2Q9Ser Tyr137His + Leu209Met + I1e220Va1 + G1y222Asn +
Thr223G1n Val 95A1a + Tyr137Pro + A1a164G1u + Asn168G1n +
' Thr206Asn Leu 96G1n + Tyr137Leu + Tyr169His + Leu209Asp +
I1e220Pro Gly 66Pro + Gly 70Asn + Leu133Pro + Phe202Pro +
Ser207G1u Val 95A1a + Leu 96Met + Tyr104Asn + I1e107A1a +
Ser216Asp Gly 70Ser + I1e107G1y + Leu133Met ~ Phe192His +
Asn194Asp Gly 68Pro + Val 95Ser + G1y100Asn + Tyr137Cys +
I1e220Pro Gly 66Ser + G1y102G1n + A1a164G1n + Ser219G1u +
G1y222Asn G1y135G1n + Ser190G1u + Leu209Cys + G1y215G1n +
I1e220Va1 Gly 68Pro + Asn163Asp + Phe192Pro + Leu209G1y +
I1e220Thr Asn 67G1n + Gly 70Asn + I1e107Leu + G1y136Asn +
Asn162G1u Gly 70Pro + I1e107His + Ser190G1u + Pro204Asn +
Leu209G1n Leu 96I1e + Asp165G1u t Phe202G1y + Leu209Asn +
Thr211G1y Gly 66Pro + Gly 70Asp + fiyr169Asn + Leu209His +
G1y215Ser Tyr104Met + Asn194G1u + Leu209Cys + Thr217Pro +
I1e220G1n Gly 70Asn + Leu 96I1e + Tyr104His + Asn161Asp +
I1e220G1n Val 95Thr + G1y102Pro + Thr206Ser + G1y214G1u +
G1y222Ser Thr106G1n + Asn163Ser + I1e213Pro + Ser216G1u +
I1e220Ser L-eu 96Cys + G1y136Asn + Ser170G1u + Thr206G1n +
I1e220G1n Gly 66G1n + Ser105G1u + G1y135Pro + G1y205Asn +
Thr206Ser G1y102Pro + Tyr104I1e + Thr211G1y + Ser216G1u +
Thr223G1n Tyr104A1a + G1y203Asn + Leu209G1y + Arg218Asp +
I1e220Cys Gly 66Pro + Val 95Thr + Asn161Ser + Asn162G1n +
I1e220Cys G1y102Asn + G1y136G1n + Phe202Met + I1e208Va1 +
I1e213G1u WO 96!28556 PCT/US96/03005 G1~. 66Ser+ Gly 68Ser + G1y134G1n + A1a164G1y +

Asp165G1u Gly 66Pro A1a164Asp + Tyr169Ser + Thr206G1n +

Thr217Asn Leu 96I1e + G1y136G1n + Asn161Ser + Thr206G1n +

Ser207Asp Asn o'7Asp+ Tyr104Asn + G1y135G1n + Tv_r169I1e+

Leu209His Gly 70Pro - G1y136Pro + Ser190Asp + Leu209Ser +

G1y214Pro Asp 65G1u + Tyr137Va1 + A1a164Asn + Phe192A1a +

Thr206Ser Leu 96Thr + G1n103Ser + Tyr104Asn + Asp165G1u +

I1e220His Gly 68Asp + Gly 70Pro + Asn 99Ser + G1y214Ser +

Thr223Ser Leu 96Ser + Leu133A1a + Tyr137Met + Leu209I1e +

Ser221Asp G1y134G1n + Asn194G1n + Thr206Ser + Leu2-09Met+

I1e213Leu Gly 70G1u + Leu133Cys + G1y135G1n + Thr206G1y + -G1v222Asn r G1y134Ser + A1a166Gln + Thr206G1y +
+ Tyr169Cys Ser221Asp G1y160Ser + Thr206Asp + I1e213G1n + I1e220Pro + -Thr223G1y Tyr104Asn + G1y135Pro + Leu209I1e + Ser219G1u +

I1e220Va1 Asn 67G1n + Thr106Asn + G1y160Giu + Tyr169Asn +

Leu209Aia .

Val 95G1u + G1y100Pro + ~i:~~06Asn+ Leu209Thr +

I1e220A1a Asp 98G1u + G1y102Ser + A1a164G1n + Thr206Ser +

I1e220Asn G1n103Asn + G1y136Pro + Ser138Asp + Leu209G1y +

I1e220G1n Asn 67G1n + Thr106Asn + G1y136G1n + Thr206Ser +

I1e220Asn Asn 67G1n - G1y134G1n + Tyr169Cys + Ser219Asp +

I1e220Asp A1a166His + Thr206G1u + Ser2Q7Asp + I1e213Cys +

I1e220His Gly 70Pro + I1e107G1y + Leu209G1n + I1e220G1u +

Ser221Asp Asn 99Ser + G1y160Ser + G1y215G1u + Ser216G1u + . -Thr217G1n G1y102G1n + I1e107Cys + A1a164Ser + Leu209Asp + -Ser219Asp .

G1y134Asn -r-Tyr137G1n + Leu209G1u + Ser219Asp +

I1e220Met-G1y102G1n + G1y160G1n + Leu209Asp + Arg218Asp +

Ser219G1u Gly 70Asn + Ser2C7Glu + Leu209Va1 + Ser219G1u +

WO 96!28556 PCT/US96/03005 I1e220G1u ._la~.e4Asn - Ser207Asp~ + Thr217Pro + Ser219G1u +

I1e220Asp -v-a =. 9 5 T h r . Al a 1 6 4 P r o + S + i l a 2 1 3 Va l +
' a r 2 0 7 G l a Ser221Asp '~'a~ 95Asn + G1y102Pro + G1y160Pro + Ser207Asp +

Ser219Asp Asn 57G1n + Ser207G1u + Leu209Va1 + Ser219Asp +

I1e220A1a Asn 67G1n + G1y136G1n + Ser138G1u + Ser140Asp +

Leu209Met Tyr104Asn - er138G1u + Ser140Asp + Leu209Thr +
S

I1e220Va1 Asp 97G1u - Asn 99Asp + G1y102G1n + Tyr137Ser +

I1e220Cys Asn161Asp + Asn163G1n + Tyr169A1a + Asn194Asp +

Leu209A1a Thr206G1u -~-Ser207Asp + Leu209His + Ser219Asp +

Thr223G1y G1y102Ser + G1y136Asn + Thr206G1u + Leu209Thr + -G1y222Asp Thr 7lGly + G1y102Asn + Ser219Asp + I1e220Cys +

Ser221G1u Thr1065er + G1y160Asn + G1y205Asn + Ser219Asp +

Ser221Asp Val 95G1n + Leu133G1n + G1y215Asn ~ Thr217Asp +

Ser219Asp Leu209Ser + Ser219G1u + I1e220G1y + Ser221G1u +

G1y222Asp G1n103Ser + G1y203Pro + Ser216G1u + Arg218G1u +

Ser219As~

Val 95Ser + G1y135G1n + Ser138Asp + Tyr169G1u +

I1e~13Thr %~sn o7Gln - Glyl~OPro + Ser207G1u + Leu209Cys +

G1y222G1u G1y135Asp + Ser138G1u + G1y203G1n + I1e213Met +

G1y222Asn Asn162G1n + Ser207Asp + Leu209Pro + Ser219G1u +

G1y222Asp Tyr137Asn + A1a164Asp + Arg167Asp + Leu209I1e +

I1e220Met Leu 96G1n + G1y102Asn + G1y135Asp + Asn168Asp +

I1e220A1a Val 95Pro + Asn 99Asp + G1y102Asp + G1y160G1n +

Leu209Asn Asn 99Ser + Iie107G1y + t~sn162G1n + Leu209Asp +

Ser221G1u Gly 66Pro + Asn162Asp + Ser191Asp + Thr206Asn +

Thr223Asn G1n103Asp + Tyr104Asn + Ser105G1u + Ser140Asp +

Leu209Cys Leu 96Ser + G1y136Ser + Asn168G1u + Asn194Asp +

Thr206Asn Gly 68G1u -Val 95A1a + G1y136Asn + G1y203Asn +

Ser216Asp Asp 65G1u +Thr106Asn + G1y134G1n + G1y214Asp +

Ser2I6Glu Gly 68G1n +Val 95Prc + Asn162G1n + Ser190G1u +

Ser207Asp A1a166Thr +Ser190G1u + Ser207G1u + Leu209G1n +

Thr217G1y G1y100Pro +G1y160G1n + Ser190G1u + Ser207Asp +

Leu209Pro Asn 67G1u +Ser101G1u + Tyr104Fiis + Phe192Ser +

Leu209Thr .

Ser191G1u +Phe202His + Leu2Q9Met + Ser221Asp +

Thr223G1n G1y134Asp -Ser139Asp + Ser140G1u + I1e213Met +

Thr223Asn Tyr137Asn +Tyr169I1e + Thr206Asp + Ser219Asp +

Thr223Asp Tyr104Cys +Leu133I1e + Ser207G1u + Ser216G1u +

Arg218Asp Gly 68Asn +Tyr137Ser + Ser207G1u + Ser216Asp +

Ser219G1u Tyr137Ser +Asn168Ser +-G1y214Asp + Ser216Asp +

Ser219G1u I1e107His +Asn162Ser + Ser216G1u + Ser219Asp +

I1e220Asn Asn 67G1n +Asn163Ser + Ser216Asp + Ser219G1u +

Thr223Ser Leu 96Asn +Leu133Pro + Thr206G1n + Ser216Asp +

Ser219Asp Ser191G1u +Thr206Asp + G1y214Asn + Ser219Asp +

Thr223Asn Gly 66Ser --Ser105G1u + Thr106Pro + Ser138G1u +

Leu209I1e G1y136Asp +G1y160G1u + Asn163Ser + I1e220Ser +

G1y222Asp I1e107Met +Asn162Asp + Ser190Asp + Ser221Asp +

G1y222Ser Gly 70G1n +G1y214Ser + Ser216G1u + Ser219G1u +

Ser221G1u G1y136G1u +Asn162G1u + G1y214Ser + G1y215Asn +

I1e220Leu Gly 68Asp +Leu209Thr + Arg218G1u + Ser221Asp +

Thr223Pro Glyl00Ser +Phe192G1u + G1y215Asn + Ser219Asp + , I1e220G1u G1y134G1n +Phe192G1u + I1e213G1y + Ser219Asp +

I1e220G1u Thr106G1y +Asn162Ser + Asn194Asp + Thr206Asp +

Ser221Asp G1n103Asn +Asn161Asp + Ser207Asp + Thr217Ser +

I1e220G1u Gly 68Asn +Phe192Asp + Ser207Asp + Leu209Asp +

Ile22QAsn Gly 68Asp + Phe192Tyr + Leu209G1y + Ser219Asp +

I1e220G1n Arg 64Asp + Asp 98G1u + Tyr137Va1 + Phe192Va1 +

G1y214Asp Tyr 104G1y+ I1e107G1u + G1y160G1u + Ser170G1u +

Leu209Cys Val 95Thr + I1e107G1y + Ser191G1u + Ser219G1u +

I1e220Asp Asp 65G1u + Gly 66Asp + G1yl00Asn + Glv_102Ser +

Ser190G1u Asp 65G1u + Gly 66Asp + G1y134G1n + I1e220Asp +

Thr223G1n Asp 65G1u + Gly 66Asp + Leu209G1u + I1e213A1a +

I1e220A1a Asn 99G1n + Ser190G1u + Ser191Asp + Ser219G1u +

I1e220His Gly 70Asp + SerlOlAsp + Tyr137Cys + G1y160Ser +

I1e220His Tyr137Ser + Ser138Asp + Ser139G1u + I1e~13Met +

Ser216Asp G1y135Ser + Ser138G1u + Asn194Ser + Ser219G1u +

I1e220G1u Thr 7lPro + Asp 97G1u + Thr106Ser + Ser219G1u +

I1e220G1u Arg 64Asp + Gly 66G1n + Thr206G1y + Ser219G1u +

I1e220Asp Val 95A1a + G1n103Ser + Ser139Asp + Arg167G1u +

I1e220A1a Ser1 01Asp + G1y102G1u + Ile?'_3Vai+ Thr217Ser +

Thr223G1u Asn 99Asp + Glyl00Glu + Tyri37Thr + Asn163G1u +

G1y215G1n Gly 66Ser + I1e107G1u + Leu209Asn + Ser221G1u +

G1y222G1u Asn 67G1u + Thr 7lGln + Asn161Asp + Asn162G1u +

G1y214Ser G1yl 00Asp + Ser101G1u + Thr106G1n + I1e213Cys +

Ser221Asp Arg 64Asp + Asp 65G1u + Gly 68Ser + Leu 96G1n +

Asn168Asp G1y1 60Pro + Thr206G1u + Ser207G1u + G1y215G1u +

G1y222Pro G1y1 02Ser + Tyr137Asp + Tyr169His + Thr206G1u +

Ser207Asp Gly 70Asp + Asn 99Ser + Tyr104G1u + Ser105Asp +

Thr217G1n Leu 96G1u + G1y135Ser + Thr217Ser + I1e220G1u +

Ser221G1u Asp 97G1u + Asp 98G1u + G1y205Pro,+ G1y215Ser +

Ser219G1u Gly 68G1n + Ser138Asp + Leu209I1e + Arg218Asp +

Ser219Asp WO 96!28556 PCT/US96/03005 Asn 67Ser + + Ser216G1u + -G1y136Asp +
G1y215Pro Thr217Asp Asn 99G1n - Ser101G1u + Asn161G1n + Asn162Asp +

Asn163Asp T;~r~37Gln- Tyr159Thr + Ser207Asp + G1y215Asp +

Ser216Asp Le:~ 9oCys-~Ser190Asp + G1y215G1u + Ser216G1u + -I1e220Cys G1y160Pro - Phe192Asn + Ser207G1u + G1y215Asp +

Ser216G1u Thr 7lAsn + Phe192Va1 + Ser207Asp + G1y215Asp +

Ser216Asp G1y134G1u + Asn168Asp + Trp212I1e + I1e220Asn +

Thr223G1u G1n103Asp + G1y134Ser + Ser139G1u + Asn162Ser +

Ser170Asp G1n103Asp + Thr106Ser + Ser138G1u + Leu209Va1 +

G1y215G1rL

Asr~ 67G1n- G1n103Asp + G1y135Gin + Ser138G1u +

Leu209Asn Asp 97G1u + I1e107G1n + G1y136Ser + Ser207Asp +

Ser221Asp Tyr137I1e + Ser138G1u + Asn194Ser + Ser207Asp +

Ser221Asp G1n103Asp + G1y135Asn + G1y136Pro + Ser207G1u +

I1e220G1u G1y102G1n + G1y135G1u + G1y203Ser + Ser207G1u +

I1e220G1u Tyr104I1e + AsnlE3Asp + Arg167G1u + Tyr169Cys +

I1e208Cvs Gly 65G1u ,-Asp 97G1u + Tyr104G1u + G1y136G1n +

Leu209Met GIyIC2Ser + I1e107Cys + Arg167Asp + Ser207Asp +

Ser219Asp Glyl00Asn + G1y134Asp + Ser207G1u + G1y214Ser +

Ser219Asp Arg 64Asp + Val 95Ser + Thr206Ser + Ser207Asp +

Ser219G1u Asnl94Asp - Ser207G1u + Ser219Asp + I1e220Met +

Thr223Asn Ser140G1u + Ser207G1u + Leu209A1a + G1y215G1n +

Ser219G1u Tyr104Ser + G1y160G1u + Ser207G1u + G1y215Ser +

Ser219Asp Asp 65G1u + Thr206Ser + Ser207G1u + G1y215Ser +

Ser219Asp G1n103Ser + Asn168G1u + Pro204G1n + Ser207G1u +

Ser219Asp Leu133Thr + Ser140G1u + Asn161Ser + Ser207Asp + .

Ser219G1u Leu 96Pro + Ser140G1u + Ser207G1u + Leu209Thr +

Ser219G1u Asnl6lAsp + Asn168G1n + Ser170Asp + Ser191Asp + -Phe192G1n G1n103G1u -;- Thr106G1u + Leu209G1y + I1e213His +
Ser219G1u Asp 65G1u + Asn 67Asp + Thr106Asn + Asn194G1u +
I1e208Asn Asp 65G1u + Asn 67G1u + Leu 96A1a + G1y102Ser +
- Tyr169Asp Asn 99Ser + Asp165G1u + Arg167Asp + I1e213G1y +
Ser219Asp Tyr104l1e + Tyr137I1e + Asp165G1u + Arg167G1u +
Ser219Asp Ser190G1u + Phe192G1u + Leu209Cys + Ser219G1u +
I1e220A1a Ser138G1u + Ser140G1u + Thr206G1y + I1e220His +
Ser221G1u Val 95Pro + I1e107Ser + Ser138Asp + Ser140G1u +
Ser219Asp Asp 65G1u - G1y102Ser + Ser138G1u + Ser140Asp +
A1a166G1y Val 95Cys + SerlOlGlu + Ser138G1u + Ser140Asp +
Phe192Ser Asp 97G1u + Asn 99Asp + Ser139Asp + I1e213Thr +
Thr223G1y Asp 97G1u + Asn 99Asp + Ser140G1u + A1a166G1n +
Phe192Thr Leu 96Met + G1y136G1u + Asn161Asp + Asn168G1n +
I1e220Pro G1n103Asp + Ser105G1u + Asn162Ser + Ser207G1u +
Thr223G1n Arg 64G1u + G1n103G1u + Ser105Asp + Asn163G1n +
Leu209Cys Tyr137A1a + Ser170G1u + Phe192Met + G1y214G1u +
Ser216Asp G',~ 70Ser + SerlCSGlu + G1y214G1u + Ser216G1u Thr223Asn G1n103Ser + Asn168Asp + G1y205Ser + G1y214Asp Ser216Asp G1y135G1u + Tyr169G1u + Ser207Asp + Leu209Met +
I1e220Cys Asn 99G1n + G1y136Asp + G1y160Ser + Ser170Asp +
Ser216Asp G1y136Asp + Tyr1695er + Ser170Asp + I1e213G1u +
Thr217Asn Asp 97G1u + SerlOlAsp + Ser139G1u + Asn162Ser +
I1e220Va1 Asp 97G1u + Ser101G1u + I1e213Met + Thr217G1y +
Arg218Asp Thr106Asn + I1e107Pro + Asn194G1u + Ser219Asp +
Ser221G1u Leu 96Met + Asn 99Asp + Ser101G1u + Ser139Asp +
Leu209Thr Asn 99Asp + SerlOlGlu + G1n103Ser + Ser139Asp +
I1e220Pro Gl% 6eGlu + Asp 97G1u + G1y160Asn + Asn163Ser +
I1e220Asp Arg 64Asp + Gly 66Asp + I1e107G1y + Ser207G1u +
I1e220Va1 Gly 70G1n + G1n103Asp + Leu133I1e + Ser140Asp +
Leu209Thr Asn 67G1u + Gly 70G1u + Asn 99G1n + Tyr104Met +
Leu209Asp ' Tyr104G1n + I1e107His + Ser191Asp + Thr206G1u +
Arg218Asp Asni~lGlu + A1a164G1n + I1e213Asp + Ser216Asp +
Thr217Asn Thr106G1n + G1y136Pro + Thr206Ser + Ser207G1u +
Thr217G1u Asp 65G1u + Leu 96G1y + Asp 98G1u + Thr106Asp +
I1e220G1n Asp 65G1u + Asp 98G1u + I1e107G1y + G1y135Asn +
G1y136Asp G1y136Asp + Ser138Asp + Ser207G1u + Leu209Asn +
G1y222Asn Thri06Gln + G1y136Asp + Ser138Asp + Leu209Cys +
Ser_216G1u Val 95A1a + Asp 97G1u + Asn168Ser + Ser207Asp +
Leu209G1u I1e107G1u + Thr206Ser + Ser207G1u + Leu209G1u +
Thr211G1y Gly 70Pro + Leu 96Va1 + G1y102G1u + Ser207G1u +
Leu209Asp Val 95His + Asn194Asp + Ser207Asp + Leu209Asp +
Thr217Asn Ser140Asp + A1a164Pro + Ser207Asp + Leu209Asp +
I1e220G1y G1n103Asp + Tyr104G1n + G1y160Ser + Asp165G1u +
Asn168Asp Asp 65G1u + Gly 66Ser + Gly 70G1u + G1y136Asn +
Ser207Asp Gly 68Asp + Phe192Asn + G1y205Asn + Thr217Asp +
Ser221Asp G1y134Asp + G1y136Asp + Ser207G1u + Leu209I1e +
I1e220Cys G1yl00Asn + G1y136G1u + G1y160G1n ~~ Ser219Asp +
Ser221G1u G1y102Ser + Ser170G1u + G1y215Asn + Ser219G1u +
Ser~2lGlu Gly 66Ser + Asn163G1n + Arg167G1u + Ser219G1u + , Ser221Asp G1yl00Glu + G1y136Asn t Leu209Ser + Ser219G1u +
Ser221Asp Leu133A1a + G1y134Asp + I1e213Cys + Ser219G1u +
Ser221G1u G1y102Asn + G1n103G1u + Thr206Ser + Ser216G1u +
Arg218Asp Asp 98G1u -~ Thr206G1y + I1e213A1a + Ser216Asp +

Arg218G1u Asn163Asp + I1e208Asn + I1e213Asn + Ser216Asp +

Arg218Asp Gl~.~ 66Ser + Gly 70G1u + Tyr169His + Thr206G1y +

' I1e220Asp Arg 64G1u + Asn 67G1u + I1e107G1y + Leu209Va1 +

I1e220G1u G1n103Giu + G1y134Ser + Se.r138G1u+ Arg167Asp +

Thr223Asn Glyl00Pro + Tyr104Pro + Leu133G1n + Phe192Asp +

Ser207G1u Gly 68Asn + Serl3gGlu + Asn163G1u + ~1rg167Asp+

Thr206G1y Gl,,r 70Ser + G1n103Asp + Asn162Ser + Ser170Asp +

Leu209Met Asp 98G1u + Glyl00Ser + Ser101Asp + Asn161Ser +

I1e220Asp Tyr137Pro + Ser139Asp + Asn161G1u + A1a164G1u +

I1e220G1y Gly 6&Asp ~ Asn 99G1u + Tyr104Met + Ser207G1u +

Leu209Pro Gly 66Asp + Asp 97G1u + Tyr104G1n + I1e208Va1 +

Leu209Asp I1e107Asp + Phe192Met + Ser207Asp + G1y222G1u +

Thr223G1n Arg 64G1u + Leu 96A1a + A1a164Asn + Ser207G1u +

G1y222Asp Leu 96His -;-Ser207G1u + Ser216G1u -;-Thr217Asn +

G1y222G1u Tyr169His + Ser170G1u + Ser207Asp + I1e220Ser +

G1y222Asp Gly 68Pro + Leu133G1u + Leu209Asp + G1y214G1n +

Ser221G1u Ser101Asp + Arg167Asp + Ser170G1u + G1y215Asn +

Thr223G1y Asp 65G1u + Asp 97G1u + Ser207Asp + G1y222Ser +

Thr223Ser Asp 65G1u + Asp 97G1u + Asn 99G1n + G1y135G1u +

Thr206G1y Tyr104Met + G1y160Asp + A1a164G1u + Leu209Asp +

I1e220Thr Leu 96Ser + Asn162G1u + Asp165G1u + Tyr169Cys +

Arg218G1u Asp 65G1u + I1e107Thr + Asn162G1u + Asp165G1u +

Thr217Pro Val 95G1u + Ser101Asp + Asn162G1u + Phe192Thr +

G1y215G1n G1n103Asn + Tyr104His + Thr206G1u + Ser216G1u +

Thr223G1u G1y135Asp + Asn168Asp + Tyr169Asn + Ser191G1u +

G1y205Pro G1y102G1u + Ser105G1u + Asn161G1n + Ser207G1u +

I1e220Met Leu133G1u + Phe192Pro + Thr206G1u + Thr217Pro +

Ser219G1u Asn 99Asp + Ser105G1u + Leul3-3Va1+ Ser140G1u +

G1y214Ser Asn 99G1u + G1y102Asp + Asn162G1n + Phe192Pro + "

Ser219Asp IlelG7Va1 + Ser190G1u + Asn194G1u + Leu209T>~,r+
-Ser216G1u _ G1yl00Glu + Leu133G1u + A1a166Thr + Ser216Asp +

Thr217Pro Arg 64Asp + A1a164G1n + Ser191Asp + Thr206G1u +

Leu209Cys Leu 96Va1 + G1y134Asp + G1y136Pro + Ser191Asp +

Thr206G1u Tyr104Asn + Asp165G1u + Phe192Asp + Ser207Asp +

I1e220G1n SerIGIAsp + A1a164Ser + Thr206Pro + Ser207G1u +

Arg218Asp-Asp165G1u + Ser207G1u + Leu209Asn + Trp212G1y +

Arg218Asp Glyl00Asn + Tyr104Pro + Ser138G1u + Ser207G1u +

Arg218G1u Ser140G1u + Ser170G1u + Thr211G1n + G1y215Asn + -Ser221Asp Glyl00Glu + Ser140Asp + Ser170Asp + Thr206Ser +

Leu209His Ser140G1u + Tyr169Pro + Ser170G1u + Arg218Asp +

I1e220Pro G1n103Ser + Tyr104Asp + Leu209G1u + Ser221Asp +

G1y222Ser Asp 97G1u + I1e107Cys + Asn162Asp + Ser191Asp +

Leu209A1a Asp 97G1u + Tyr104G1u + I1e107G1y + eu133Ser +
L

I1e220Leu Leu 96G1u + Leu133Asp + G1y134Asn + G1y215Ser +

Ser216G1u Asn 67G1u + Leu 96G1u + Thr106Pro + I1e107Cys +

Ser207G1u Arg 64G1u + Asp 97G1u + G1y102Asn + Asp165G1u +

Phe192Tyr Gly 66Ser + G1y134Asp + Asn161Asp +-Ser207G1u +

Thr217G1y Asp 65G1u + Gly 70Asn + Glyl00Pro + Leu209Asp +

Ser216Asp Gly 68G1u + Ser101Asp + Leu209Thr + Trp212A1a +

Ser219Asp Thr106G1y + Asn163Asp + Leu209Pro + Ser219G1u +

Thr223G1u Asp 65G1u + G1n103Ser + G1yI34Glu + G1y136Asn +

Ser138G1u G1n103Ser + I1e107His + Asn161Asp + Asp165G1u +

Ser207G1u Gly 66Pro + Ser140G1u + A1a164His + Thr2I7Asp +

I1e220Asp Arg 64Asp - Ser101Asp + Tyr104Leu + I1e107G1u +

I1e220Asn --=~ ~4Glu - ~~ai 95Met + G1y136Ser+ Ser190G1u + _ I1e213Asp Giy135G1u - G1y160Pro + Leu209G1n + I1e220Leu +

Thr223G1u Asp 65G1u + Val 95G1n + Ser170G1u + Phe192Met +

G1y214G1u G1y100Asn + Ser190Asp + Ser207Asp + Leu209Ser +

Ser216G1u Val 95G1y + Arg167Asp + Ser190Asp + Ser207G1u +

I1e220Asn Asn 67G1u - Ser101G1u + Leu133Ser + Asn163Asp +

Thr223Ser Asn 67G1u + Asn 99Ser + Ser101G1u + Tyr104A1a +

Ser207Giu G1y134Asn + Ser138Asp + Arg218Asp + I1e220His +

Ser221G1u G1y 66G1n + Val 95Pro + Ser138G1u + Arg218G1u +

Ser221Asp Asn 67Asp + Thr 7lSer + G1y135G1u + Asn161Asp +

A1a164His Asp 65G1u + Gly 66Pro + Ser191G1u + I1e213Ser +

Ser221G1u Gly 70Pro + G1y134Pro + Ser191Asp + Ser216Asp +

Ser221Asp G1yl00Glu + Tyr104G1u +- Asn163G1n+ I1e213Pro +

I1e220Ser G1y134G1u + G1y136Pro +- Asn162G1n+ Ser216Asp +

Thr223Asp Thr 7lAsn + Ser105Asp + Leu209Met + Ser219Asp +

G1y222G1u G'_y 66Asn + G1y102Asn + Tyr169Asp + Ser216Asp +

Ser219Asp G1y136Pro + Asn161G1u + Ser216G1u + Ser219G1u +

I1e220Ser Ser140G1u + I1e208Met + Ser216G1u + Ser219G1u +

I1e220Asn G1y160Asp + Thr206G1n + G1y214Ser + Ser216G1u +

Ser219G1u Asn 99Ser + Ser190G1u + Ser216G1u + Ser219G1u +

I1e220His Ser139Asp + Thr206G1n + Ser216G1u + Ser219Asp +

G1y222Asn G1y102Asn + Ser190Asp + Ser216G1u + Ser219Asp +

G1y222G1n G1n103Ser + Asn163Asp + Trp212Met + Ser216Asp +

Ser219Asp Thr 7lGly + G1y136G1u + Asn194G1n + Ser216Asp +

Ser219G1u Asp 97G1u + Leu209Met + Ser216G1u + Ser219Asp +

I1e220Va1 WO 96!28556 PCT/US96/03005 Asn 99Gin + Asp165G1u + Leu209Met + Ser216G1u +
Ser219G1u Thr106G1u + Asn168G1n + =_=u209Met + Ser216Asp +
Ser21=:~lu Leu 96G1n + Asn161G1n + Tyr169Asp + Ser216Asp + ' Ser219G1u Leu 96Met + Asn161Asp + G1y203G1n + Ser216Asp +
Ser219G1u Asn 99G1u + Thr206Ser + Ser207G1u + Leu209Va1 +
Thr223G1u Ser207G1u + Leu209Met + Ser216Asp + I1e220Leu +
Thr223Asp Val95A1a + G1y214Asn + Ser216Asp + I1e220Asp +
G1y222Pro Ser101Asp + Tyr104G1n + G1y136Asn + G1y160G1u +
Ser190Asp Gly 70Asp + G1y135G1u + Arg167Asp + Asn168G1n +
I1e220G1n Asp 65G1u + Leu 96Met + G1y136G1u + Asn162Asp +
Leu209A1a -Val 95Asp + Asn 99G1u + Asn194Ser + G1y214Pro +
Thr223Asp G1y136G1u + A1a164Ser + Asp165G1u + Tyr169Pro +
Ser216Asp Arg 64G1u + G1y135Asn + Asn162G1n + Ser191Asp +
G1y214Asp Arg 64Asp + Asnl62Gln + Thr206G1n + Ser207G1u +
G1y214G1u Leu 96A1a + G1n103Ser + Asn162G1u + Arg167Asp +
G1y215G1u Asn161Ser + Asn162G1u + Arg167G1u + Thr20r~Pro +
Ser219Asp A1a1~4His + Asp165G1u + Tyr169Pro + Ser170Asp +
G1y215G1u Val 95A1a + G1n103G1u + Ser138Asp + G1y160Asn +
Ser219Asp I1e107G1u + Leu133Asn + Ser138Asp + A1a164G1y +
Ser191G1u I1e107G1u + Leu133Ser + Asni63Glu + Arg167G1u +
Leu209His Ser101G1u + Leu209Asp + G1y215G1n + I1e220Asn +
G1y222G1u Ser139Asp + Asn162G1n + Tyr169G1n + Leu209Asp +
G1y222Asp Thr106G1u + G1y136Ser + Ser170G1u + Asn194G1u +
Thr206G1n Tyr104A1a + Leu133Asn + Asn162Asp + Ser207Asp +
Thr217Asp Tyr169Gys + Ser191G1u + Ser207G1u + Ser216Asp +
G1y222G1n Val 95G1n + Tyr104Leu + Tyr137G1u + Ser191G1u +
Ser207G1u Gly 66Asp + G1yl00Asn + SerlOlGlu + Ser139G1u +

Tyr169G1n Gly 66G1u + Val 95Ser + Ser101G1u + Asn163Ser +
Ser191Asp Arg 64G1u + Val 95A1a + Asn 99G1u + A1a164G1u +
' G1y214Ser Arg 64G1u + Asn 99Asp + Ser105Asp + G1y160Pro +
Leu209G1y Asn 67Asp + Val 95Asp + G1y102Pro + Ser207G1u +
G1y214Ser SerlOlAsp + Ser105Asp + Asn162G1u + Tyr169G1n +
I1e220Ser Ser139Asp + G1y160Ser + A1a166Thr + Phe192G1u +
Ser207Asp Val 95G1y + G1y136Pro +-Asn161G1u + Arg167Asp +
Ser207Asp Asp 65G1u + Ser101G1u + G1y102Asn + Leu133Thr +
Ser170Asp G1=,:136G1n + Asn161Asp + Arg167Asp + Asn194Ser +
Ser216G1u G1y135Asp + A1a164Asp + Ser216G1u + T1e220Leu +
G1y222Asn Asp 97G1u + Asn 99G1n + G1n103Asn + Ser105G1u +
A1a166Ser Asn 99G1n + Asn163G1u + I1e213Leu + I1e220His +
Ser221Asp G1y135G1u + Asp165G1u + Phe192G1n + Ser207G1u +
G1y215Ser Asn162Asp + Ser170G1u + Asn194Ser + Leu209G1n +
Ser219G1u Glyl00Asp + Thr106Asp + A1a164G1u + Phe192Thr + -Asn194G1n Ser105G1u + Asn162G1u + Leu209His + Ser221G1u +
G1y222G1n Gly 68Asn + G1y135G1u + G1y160G1n + Ser216G1u +
I1e220Asp Asp 97G1u + A1a164G1y + Leu209Met + Ser216Asp +
I1e220G1u Gly 68Asp + Tyr137Asp + Asn194G1n + Leu209Cys +
Ser221Asp I1e107Asp + G1y136G1n + Ser139Asp + Ser216Asp +
I1e220Thr Glyl00Pro + SerlOlGlu + G1y136G1u + Ser191Asp +
Phe192A1a Gly 70G1u-+ G1y160Pro + A1a164Thr + Ser170G1u +
Ser216G1u .
Arg 64Asp + Tyr104His + Asp165G1u + Leu209G1n +
Thr223G1u Leu133A1a + Ser138Asp + Leu209Cvs + Thr217G1u +
Ser221Asp Ser140G1u + G1y203Asn + Thr217G1u + I1e220Met +
Ser221Asp Asp 97G1u + Thr106Asn +-G1y134G1u + Asp165G1u +
Thr211G1n G1y102Pro + Ser140Asp + Asn194Asp + G1y215Pro +
Ser221Asp Arg157Asp + Leu209Asn + Ser219Asp + I1e220Va1 +
Thr223G1u Gl_~' SBAsp ~ G1y102Asp + G1y136Ser +-Se-r170Asp +
Phe192Leu Multi-loop Sextuple Mutation Variants Vai 95His + A1a154Ser + Ser170G1u + Phe192Met +
Leu209Thr + I1e220Pro Leu133Asn + Asn161G1n + Asn163G1n + Leu209Met +
I1e220Leu + Thr223G1y G1y134Asn + Tyr159A1a + Thr211Pro + Ser215Asp +
I1e220Met + G1y222G1n G1y136G1u + G1y203G1n + Thr206Pro + Leu209Thr +
G1y215Pro + Iie220Met Glyl00Ser + G1y135Asn + A1a164Thr + G1y214G1u +
Thr217G1n + I1e220Leu Thr105Ser + Thr206Pro + Ile2b8Cys + Leu209Ser +
Ser215Asp + I1e2205er Gly 68Asn + Leu 96His + G1y102Asn + Leu209Cys +
Trp212A1a + I1e220Va1 Gly 70Pro + Val 95G1n + G1y102G1n + Asn152Asp +
G1y214G1n + Iie220His Asn 67G1n + Leu 96Va1 + Leu133A1a + A1a164Thr +
Leu209A1a + Ser219Asp G1yl00Asn + Leu133Asp + Asn151G1n + Thr205G1y +
I1e213Leu + G1y222G1n Leu 96Asn + Glyl00Gln + Thr106Asn + Ser140G1u +
Tyr159His + I1e220Asn Gly 70Pro + Asn 99Ser + G1y135G1n + Tyr159G1y +
Thr205Ser + Ser215Asp SerlOlGlu~+ G1y102G1u + Tyr169Met + Thr206Asn +
Leu209I1e + I1e220G1n Val 95A1a + Asn168G1n + G1y214Asp + G1y215G1u +
Ser216G1u + I1e220Pro Leu 96Pro + Thr105G1y + I1e107G1n + Asn163G1u +
Phe192G1u + Leu209G1y Asn194Ser + Phe202G1y + Ser207G1u + Arg218G1u +
Ser219Asp + I1e220Asp Asp-65G1u + Asp 97G1u + Asp 98G1u + Asn168Ser +
Tyr169Thr + Thr211Ser Asn 67G1n + G1y215Ser + Ser219G1u + I1e220Pro +
Ser221G1u + G1y222G1n G1y102Asn + G1n103Ser + Tyr159I1e + Thr206G1y +
Ser215G1u + Arg218Asp Asn 67G1u + Gly 70G1u + Leu 96Pro + Asn 99Ser +
Asn162G1n + Phe192Pro Asn 67Asp + Gly 68G1n + Gly 70Asp + Leu133Va1 +
Leu209G1y + Thr217Asn WO 96!28556 PCT/US96/03005 Gly 68Ser + Leu133G1u + G1y135Asp + Ser170_Glu +
Ile2'_3Asn + I1e220Ser '~al 9~Asp + Asp 97G1u + G1y100Asp + G1y135G1n +
Leu209G1n + Thr223Ser ' Leu 96Cys + G1y134Asp + G1y160G1u + Asn161G1u+
Asn162G1u + Leu209Pro Asp 65G1u + Leu 96G1u + Asp 97G1u + Asn 99Asp +
I1e220Ser + G1y222Pro Val 95Pro + Leu209Ser + Ser219G1u + I1e220G1y +
Ser221G1u + G1y222Asp Gly 66Asn + Asp 98G1u + Ser101Asp + Tyr104Cys +
Asn194G1n + I1e220Asn I1e107Thr + Tyr137His + Phe202Lau + Ser216G1u +
Arg213Asp + Ser219G1u G1y134Pro + Tyr137Pro + Ser2J7Glu + Leu209Cys +
Ser219Asp + G1y222G1u Leu133Thr + Asn163Ser + Ser207Asp + I1e213Met +
Arg218Asp + Ser221Asp Arg 64Asp + Asp 98G1u + Thr106Ser + G1y160Asn +
I1e213Pro + I1e220Asn Gly 70G1n + Leu 96Asn + Tyr169Met + Thr206G1u +
Leu209Cys + Thr223Asp Gly 68Pro + A1a164G1u + Arg167G1u + Pro204Asn +
Thr206G1n + I1e220Ser Asn 99Ser + G1y135Asp + Tyr137Met + Ser138G1u +
Asn168Asp + Thr211Asn Gly 68Pro + Tyr104G1u + Ser138G1u + Thr217Ser +
I1e220G1n + Thr223Pro Gly 66Ser + Asn163G1n + Thr206Asp + Leu209G1y +
Ser219Asp + I1e220Met Gly 68Asp + Thr106Pro + Thr206G1y + Leu209G1u +
Ser219G1u + I1e220Asp Tyr137Leu + Tyr169Cys + Phe192Asn + Ser207Asp +
Arg218G1u + I1e220Leu Gly 68Asn + Val 95G1u + G1yl00Glu + Leu133Cys +
G1y134Pro + Thr217Ser G1y 68Asp + G1yl00Gln + Thr206Pra + Ser219G1u +
I1e220G1u + Ser221G1u Tyr137Asn + A1a164G1n + Ser207G1u + Leu209G1u +
Arg218Asp + G1y222G1u G1y135Pro + Ser191Asp + Asn194G1n + Thr206G1u +
Ser207G1u + Ser219G1u Val 95Asp + Ser105Asp + G1y160Asn + Leu209G1y +
G1y215Pro + I1e220A1a Gly 68Asp + G1yl00Ser + G1y134Asn + Ser207G1u +
Ser219G1u + Ser221G1u Asn 67G1n + G1n103Ser + Thr206G1u + Leu209G1u +
Ser216G1u + Ser219Asp Tyr104I1e + .Thr206G1u + Ser216Asp + Arg218Asp +
Ser219G1u + Thr223Ser Gly 68G1n + Val 95Pro + Leu133A1a + Asn162G1n +
Ser190G1u + Ser207Asp Asn163Ser + Thr206Asp + Ser207G1u + Leu209Ser +

WO 96!28556 PCT/US96/03005 Ser216G1u + Ser219Asp Asni6lSer + Thr206Ser + G1y214Asp + G1y215Asp +
Thr~l7Glu + I1e220G1u Asn 99Ser + Ser138Asp + Ser139Asp + Ser140Asp +
Ile2I3Gly + Ser216Asp Gly 70G1u + Leu 96Asn + Thr206Asn + Ser207G1u +
Ser219Asp + Ser221Asp Tyr104His + Asn161Asp + Ser170Asp + Phe192His +
Asn194Asp~+ G1y222Pro Leu 96G1u + Asp 97G1u + Ser101Asp + Leu209Thr +
Ser219Asp + I1e220Asn Ser207Asp + I1e208A1a + Leu209Thr + Ser216Asp +
Ser219G1u + I1e220Leu Glyl00Glu + Leu133G1u + G1y135G1u + Asn161G1u +
A1a164G1y + Leu209Thr Leu 96Asn + G1y136Ser + Leu209His + G1y214G1u +
Ser216Asp + Ser219G1u Tyr104Leu + I1e107G1u + Tyr137G1u + Ser138Asp +
Asn168G1n + Leu209I1e - --Tyr137G1u + Phe192Met + Ser207G1u + Ser219Asp +
Ser221G1u + Thr223Pro Tyr137Asp + Asn168Asp + Ser170G1u - Phe192Asn +
Ser207Asp + I1e220Asn Val 95Ser + G1y102Ser + Asn162G1n + Leu209Ser +
Ser216G1u + Ser219Asp Giy 665er + G1y102G1n + Ser216G1u + Ser219Asp +
I1e220Va1 + G1y222Asn Asp 97G1u + Asp 98G1u + Ser101G1u + G1y134Pro +
Asn162G1u + Thr206G1n '~al 95Pro + 5er101Asp + G1y135Asn + Ser207Asp +
Leu209Asp + I1e220Asp Asp 98G1u + G1y203Ser + Ser207G1u + Leu209Pro +
Arg218G1u + Ser219Asp Thr106Pro + G1y135Asp + Ser140Asp + Asn162G1u +
Ser170G1u + G1y214G1n Gly 68G1n + I1e107Asn + Asn162Ser + Ser191Asp +
Ser207Asp + Ser219G1u G1y102Ser + G1y136Asn + Thr206G1u + Leu209Thr +
Arg218Asp + G1y222Asp Asp 65G1u + G1n103Ser + Ser207G1u + Leu209G1n +
Arg218Asp + Ile2ZOGlu Gly 66Pro + Asn 99G1n + G1y136Asp + Arg218Asp +
I1e22~Glu + Ser221Asp Tyr104Asp + G1y134G1u + G1y135Asp + Thr211Ser +
Ser219G1u + I1e220Va1 , G1yl00Gln + Tyr104Ser + Ser139G1u + Ser191G1u +
Phe192Asp + Thr206G1u Ser138G1u + Thr206Pro + Leu209Thr + Ser216G1u +
Arg218Asp + Ser219Asp Asp 97G1u + Leu209G1n + Ser216Asp + Arg218Asp +
Ser219G1u + I1e220A1a G1y102Asp + Leu133Thr + Asn163G1n + Ser216G1u +
Thr217G1u + Ser219Asp Asp 98G1u + Asn 99Asp + G1y102Asp + Thr106Ser + -I1e107G1n + Ser138Asp Asn 67Asp + Gly 70G1u + Ser170Asp + Phe192Pro +
Leu209Thr + I1e213G1u G1n103Asp + G1y134Asn + Asn162G1u + Phe192G1u +
I1e220Leu + G1y222Asp Asp 65G1u + Asp 97G1u + G1yl00Asp + Tyr169Met +
Ser191G1u + I1e220Cys Asr~ 67Asp + Asp 98G1u + A1a164Pro + I1e208Thr +
Leu209Ser -- I1e220Asp Giy136Asn + Ser191Asp + Thr206Pro + Arg218Asp +
Ser219Asp + G1y222Asp Asp 98G1u + G1y136Asp + Ser138G1u + Ser140Asp +
G1y203G1n + I1e220Met Leu133I1e + G1y134Pro + G1y135Pro.+ Asn162G1u +
Arg167G1u + I1e213Va1 G1y102Asn + Ser140Asp + Asn161G1u + Asn162Asp +
Ser190G1u + G1v205Asn Ser139G1u + G1y160G1u + Asn161G1n + Thr206Pro +
Ser221G1u + G1y222G1u Gly 66Asp + Asn 67Asp + I1e107G1y + Asn194G1n +
Ser207Asp + I1e220G1u Tyr104G1y + G1y136Asp + Ser170Asp + Ser190G1u +
Asn194Asp + G1y222Ser Gly 68Asp + I1e107Asp + G1y135Asn + G1y215G1u +
Ser216G1u + I1e220G1y Ser101G1u + Ser207Asp + Leu209Met + G1y215Pro +
Thr217G1u + Ser219G1u Asp 65G1u + G1yl00Glu + G1n103Asp + Asn161G1n +
Phe192His + I1e220Ser Gly 68G1n + Ser138Asp + Ser139G1u + Alale~4Gly +
Asn194G1u + Phe202G1n Asn 99G1u + Thr106G1n + G1y214G1n + G1y215Asn +
Ser219Asp + I1e220G1u Gly 66Pro + Asn163G1u + A1a164G1n + Phe192His +
Ser219Asp + I1e220Asp G1yl00Gln + Tyr104Pro + Tyr137His + Arg167Asp +
Asn168Asp + Ser207Asp Asn 99Ser + G1yl00Ser + Asn161Asp + Asn162Asp +
Leu209Asn + Ser216G1u G1yl00Asp + Ser101G1u + Asn168G1n + Leu209Met +
I1e213Ser + Ser219Asp Val 95His + G1yl00Gln + Ser139G1u + Ser140Asp +
Ser207G1u + Thr217Ser G1y100Asp + G1y134G1n + Ser139Asp + Ser140Asp +
Asn161Ser + Leu209Va1 Val 95Cys + Glyl00Glu + Thr106Asn + Ser139Asp +
Ser140G1u + G1y222Ser Ser105Asp + Asn194G1n + Thr206Asp + Ser207Asp +
Leu209Va1 + G1y215Ser Asn163G1u + Phe192Pro + Trp212Va1 + Ser216Asp +
Thr217Asp + G1y222Pro Val 95Asn + G1n103Asn + Leu209G1n + G1y215Asp +

Ser216G1u + Ser221Asp Gly 66G1n G1y134G1u + Ser170Asp + Leu209Thr +
+

I1e220Asp + G1y222Asp Gly 6&Gln Thr106G1u + I1e107G1u + Asn163Asp +
+

Asn194G1u , + Pro204Asn G1y102G1u G1n103Asp + Phe192I1e + Ser207G1u +
+

Leu209Pro + I1e220Asp Arg &4Glu Asp 65G1u + A1a164Asn +--Ser207G1u + ' +

Leu209His + Ser219Asp Arg 64Asp Asp 65G1u + G1y135Pro + Tyr137Met +
+

Ser207G1u + Ser219Asp Tyr104G1y Ser105G1u + Phe192G1n + Ser207Asp +
+

Ser221Asp + G1y222G1n Ser105G1u Tyr137I1e + A1a164His + Thr206Asn +
+

Ser207G1u + Ser221G1u Asn 67Gin Asp 98G1u + Asn161G1n + Asn168Ser +
+

Ser207G1u + I1e220Asp G1y134Ser Ser139Asp + Thr206Asp + I1e213A1a +
+

Arg218G1u + Ser219Asp Asp 98G1u G1yl00Gln + G1n103Asn + G1y134Asp +
+

Ser139G1u + Ser170G1u Leu 96G1y Asp 98G1u + Thr106Asn + Leu209G1u +
+

Ser216Asp + Ser219Asp Gly 70G1u Asp 97G1u + Asn 9gGlu + G1y135Ser +
+

Asn194G1u + Leu209Thr Thr106G1y Arg167G1u + Ser207G1u + G1y214G1n +
+

G1y215Pro + Ser219G1u A1a164G1u Thr206G1y + Ser207Asp + Leu209His +
+

Ser219Asp + I1e220G1n G1y160Ser Asn162G1n + Ser170G1u + Thr206Pro +
+

Ser207Asp + Ser219Asp Tyr104Cys Asn162G1n + Tyr169Asp + Ser207G1u +
+

Leu209A1a + Ser219Asp Asn 67G1n Asp165G1u + Ser207G1u +.Leu209Va1 + -+

Ser219Asp + I1e220A1a Gly 66G1u Thr106G1n + Phe192Asp + Asn194Ser +
+

Thr206G1u + Ser207Asp G1y134Asp A1a164Ser + Ser170Asp + Ser207Asp +
+

I1e208Met + Ser219Asp Gly 66G1n G1y102Asn + G1y136Asp + Ser139G1u +
+

Asn168Asp + Ser219Asp Asp 97G1u Asn 99Asp + Thr206Asn + Ser207Asp +
+

Ser219Asp + I1e220Met I1e107Va1 Asn162Asp + Ser191Asp + Ser207Asp +
+

Ser219G1u + G1y222Pro Gly 66Ser Asn 67G1u + Ser138Asp + Ser140G1u +
+

Leu209Cys + I1e213A1a Gly 70Ser + Asn162G1n +
+ Asp 98G1u + G1y100Asp Ser207G1u + Ser219G1u Asp 97G1u + Thr206G1u +
+ Asn 99G1u + Tyr104G1y Leu209A1a + Thr217G1n Arg 64G1u Gly 66G1u + Asn162Asp + Tyr169Ser +
+

Leu209His + G1y214G1u Gly 70G1n + Leu133Asp + G1y1.36G1u + Asn194Asp +
I1e213Cys + Gl_,:'_'22Asp Gly 70Gin + G1n103Asp + Ser105Giu + Thr106Pro_ +
Ser219Asp + I1e220Ser G1n103G1u -r Ser105G1u + Asn162Gin + G1y205Pro +
Trp212Asn + Arg218G1u Asp 97G1u + Asn 99G1n + G1y136Asp + G1y160Ser +
Ser170Asp + Leu209Met Ser105G1u + I1e107G1u + Ser207G1u + I1e213Thr +
G1y215Ser + I1e220Asn Ser190Asp + Phe192I1e + Ser216G1u + Ser219G1u +
I1e220G1u + G1y222Ser .
Asp 65G1u + G1y102G1n + Ser207Asp + I1e213Asp +
Ser216G1u + I1e220Asn G1n103Asp + Leu133Asn + Phe192Asp + I1e213Va1 +
I1e220Asp + Ser221Asp Leu 96Cys + I1e107G1y + Asn162Asp + Asn194Asp +
Ser216Asp + I1e220His Ser101Asp + Ser105G1u + Thr106Asn + Ser138Asp +
Ser170G1u + Leu209Ser Gly 70Ser + Tyr169Va1 + Phe192Asp + Ser216G1u +
Ser:'l9Glu + I1e220Asp G1y136G1n + Asn161Asp + Asn163G1n + Arg167Asp +
Asn194G1u + Ser216G1u Asp 97G1u + Asp 98G1u + Asn161G1u + A1a164Asp +
I1e213A1a + Thr217G1y I1e107Cys + Argl~7Asp + Asn168G1u + Ser207G1u +
I1e220Thr + G1y222G1u Arg167Asp + Tyr169His + Ser170G1u + Leu209Va1 +
Ser219Asp + I1e220G1u Gly 70G1n + Phe192Asp + Ser216G1u + Arg218G1u +
I1e220Va1 + G1y222G1u Arg 64Asp + Gly 70Asn + Phe192A1a + Ser207Asp +
Leu209G1u + Thr223G1y Gly 68Asn + Asp 98G1u + Ser207Asp + Leu209Asp +
I1e220Asn + Thr223G1n G1y100G1n + Leu133I1e + Asp165G1u + A1a166His +
Tyr169Asp + Ser191G1u Asn163Asp + Ser191Asp + Thr206G1y + Leu209Asp +
Thr211Asn + I1e220G1u Asn 99G1n + G1y160G1u + Tyr169Va1 + Ser191Asp +
Ser216Asp + G1y222Asp Asn 67G1u + Asp 97G1u + Ser105Asp + A1a164Thr +
Thr206Ser + I1e220Va1 Arg 64Asp + Gly 70G1u + Val 95Thr + Asp 97G1u +
Asn162Asp + Thr223G1n Leu 96Ser + G1n103G1u + Ser105G1u + G1y134Asp +
Asn194Ser + Ser219Asp G1yl00Gln + Leu133G1n + A1a164G1n + Arg167G1u +
Ser219Asp + Ser221G1u Val 95G1n + Tyr137Asn + Ser140G1u + Pro204Asn +
Ser219Asp + Ser221Asp G1y134Ser + Ser190G1u + Ser207G1u + Leu209G1n +

Thr2i7Glu + I1e220His Gly 70Ser + G1y134Ser + Asn162Ser + Ser170Asp +
Ser216G1u + Arg218Asp Asp 97G1u + Tyr137Thr + Tyr159G1n + Ser216G1u +
Arg218G1u + G1y222G1n Asn 67G1u + Gly 70G1u + Asn 99G1n + Tyr104Met +
G1y136Asp + A1a164Asn Asn 67G1u + Gly 68Asp + Gly 70G1n + Ser191G1u +
I1e213Ser + I1e220G1u Thr106G1y + Asn162G1u + G1y215G1u + Thr217Asp +
Ser219G1u + I1e220Asn Arg 64Asp + Asp 65G1u + I1e208G1n + Leu209Asp +
Ser216Asp + I1e220Va1 Ser101G1u + Leu133G1u + Thr206Asn + Ser207G1u +
Thr217Asn + Ser219Asp Tyr104G1u + Ser138G1u + Asn162G1u + Asn194G1u +
Thr211Ser + Trp212Ser Asp 98G1u + Asn 99G1n + G1y102G1u + Ser190G1u +
Ser191G1u + I1e220Pro Val 95Asp + G1y134G1n + Tyr169Pro + Phe192Asn +
Ser221G1u + Thr223Asp Asn 67G1u + Leu 96G1u + G1y160Ser + Asn162Ser +
Thr206G1y + Thr217Asp Gly 68Asp + Val 95G1u + Asn 99G1u + Tyr169A1a +
G1y215Asp + Thr217Ser Val 95Ser + G1y135Ser + Asn161Asp + I1e213Asp +
Ser216Asp + Ser219Asp Arg 64G1u + Asp 98G1u -+- G1y136Ser + G1y160G1n +
Leu209Asp + I1e220Asp Asp e~SGlu + Gly 66Ser + Asr_194G1n + Ser207Asp +
Ser216Asp + Ser219Asp Asn 99Ser + Ser101Asp + Tyr104Asp + G1y134Ser +
Ser207Asp + I1e220Asp Arg 64G1u + Asp 98G1u + Iie107Asn + Thr206Ser +
Ser219Asp + Ser221G1u Val 95Pro + A1a164Asp + Asp165G1u + Ser170Asp +
I1e213Asp + I1e220Va1 G1y135Ser + Asnl&lGln + Asn162Asp + Tyr169Asp +
G1y215G1n + Ser221G1u Tyr137His + Ser140Asp + Asn161G1n + Ser170Asp +
Ser207Asp + Leu209Asp Leu 96I1e + Ser138G1u + A1a164His + Asn194Asp +
Thr206Asp + G1y222G1u Asn161G1u + Ser207Asp + G1y214Asn + Ser216Asp +
Arg2lSGlu + G1y222G1n Leu 96Asn + Ser170Asp + I1e208Asn + G1y215G1u +
Arg218G1u + Ser219Asp G1y134Asp + Ser139Asp + Ser140G1u + Thr206Asp +
I1e213Met + Thr223Asn Asp 65G1u + Asp165G1u + Phe192Pro + Leu209G1y +
G1y215G1u + Ser216Asp Asn 99Asp + Ser101G1u + I1e107G1u + Ser191G1u +
Asn194G1n + I1e220Leu Asp 65G1u + Ser190G1u + Asn194G1n + Ser207G1u +
G1y215Pro + Ser219G1u G1y134Asn + Ser138G1u + Ser139G1u + Ser216G1u +
Ser219Asp + I1e220Pro Tyr137Cys + A1a164Thr + Ser190G1u + I1e208His +
Ser216G1u + G1y222G1u Asp165G1u + Ser207Asp + Ser216G1u + Arg218Asp +
I1e220Pro + G1y222Pro Val 95Met + Ser139G1u + Ser140Asp + A1a164Asn +
Ser216Asp + Ser219Asp Val 95G1y + G1y102Ser + Ser105Asp + Ser138G1u +
Asn163G1u + A1a164G1u Asp 65G1u + Val 95G1y + Asp 97G1u + G1y160Ser +
Asp165G1u + I1e220Ser Thr 7lGln + Ser101Asp + Thr106G1u + Tyr169Pro +
Ser207G1u + Ser219Asp Tyr137I1e -r- Asp165G1u + Tyr169Cys + G1y214Asp +
Arg218G1u + Ser219Asp Asn 99Ser + A1a164Asp + Ser170Asp + Asn194G1u +
Thr206G1n + Ser216Asp Asp 65G1u + G1y134Asp + G1y160Asn + Asn161G1u +
Asn168Asp + I1e220Asn Asp 65G1u + Phe192Ser + Ser216G1u + Ser219G1u +
I1e220Met + Ser221G1u Gly 68Asn + Leu133G1n + Asni68Asp + Ser207G1u +
Ser216G1u + Ser219Asp G1y135G1u + Ser207G1u + Ser216Asp + Thr217Asn +
Ser219G1u + I1e220Pro Gly 68Asn + Asn163G1u + Ser207Asp + Ser216G1u +
Ser219G1u + I1e220Asn G1y102G1u + Tyr137Cys + Thr206Ser + Ser207G1u +
Ser216Asp + Ser219Asp Leu133Ser + Arg167G1u + Ser170Asp + Ser191Asp +
Thr206Asp + Leu209His Gly 66G1n + G1y136G1u + Ser138G1u + Thr206G1n +
Arg218G1u + Ser221Asp Asn 67G1u + Ser139Asp + I1e208Va1 + Leu209Ser +
Ser216Asp + Thr217Asp Arg 64Asp + Val 95His + G1y134G1u + Ser138G1u +
Arg167G1u + Leu209Cys Arg 64Asp + Phe192Va1 + Thr206Asp + Leu209A1a +
Ser219G1u + G1y222Ser Leu 96A1a + Ser140Asp + Phe202G1n + Thr206Asp +
Leu209A1a + Ser219G1u Leu 96I1e + G1y102Pro + G1y134Asp + Thr206Asp +
Leu209A1a + Ser219Asp G1y102G1u + Ser105Asp + I1e107Cys + Ser140Asp +
Asn194Asp + I1e220A1a G1yl00Glu + Ser105Asp + Tyr137Pro + Ser140G1u +
G1y203Asn + G1y222Pro Thr106Asn + Asn162G1u + Asn168Asp + Ser216G1u +
Arg218Asp + I1e220Asn Asp 98G1u + Leu133G1y + G1y136Asp + Ser140G1u +

Asn168G1u + Phe192Ser Val 95A1a + Asp165G1u + Asn168Ser + Ser207Asp +
Leu209G1u + Ser216Asp G1y134G1u + A1a164Asp + Asn168G1u + Phe192Cys +
Ser219G1u + -1e220Pro Giy e~8Asn + Ser191Asp + Phe192Asn + G1y215G1u +
Ser219G1u + Ser221G1u G1n103G1u + I1e107Asn + Ser140G1u + Ser190G1u +
Ser191G1u + Leu209Thr Tyr104A1a + Leu133Asn + Asn162Asp + Thr206G1u +
Ser207Asp + Thr217Asp G1y160Asn + Asn194Asp + Ser207Asp + I1e213Va1 +
Ser216Asp + Thr217Asp Leu 96I1e + A1a164G1n + Asp165G1u + Ser207G1u +
Arg218G1u + I1e220Met Gly 66G1u + I1e107A1a + G1y160Asn + Phe192Va1 +
Ser207G1u + Arg218G1u G1yl00Gln + Tyr104Asp + Glyl3-4Asp + A1a164Ser +
Phe192Leu + Ser216Asp Arg167G1u + Leu209Thr + Ser216G1u + Ser219G1u +
Ser221G1u + G1y222Asn Gly 68Asn + Asp 98G1u + Leu209G1y + Ser216Asp +
Ser219Asp + Ser221G1u Asp 65G1u + Ser207G1u + Leu209Asp + G1y215Asp.+
G1y222Ser + Thr223Asn G1y134Ser + Ser140G1u + Asn168G1n + Ser191G1u +
Asn194G1u + Ser221Asp Leu 96I1e + Thr106G1u + G1y160Asp + Ser219G1u +
G1y222G1u + Thr223Ser Ser101Asp + Ser105G1u + Asn161G1n + Ser207G1u +
I1e213Asn + Ser221G1u G1n103Ser + G1y135Asp + Ser190G1u + Thr206Ser +
Ser219G1u + I1e22-OGlu Asp 65G1u + G1yl00Asp + Ser105Asp + Ser140G1u +
Phe192Thr + I1e220His Asp 65G1u + Ser101G1u + A1a164Thr + I1e213G1u +
Thr217Asn + Ser219G1u Ser105Asp + G1y134Asp + Asn168Ser + Ser191Asp +
Phe192G1u + I1e220Cys Leu 96Asp + I1e107Asp + G1y214Ser + Ser216G1u +
Ser219G1u + I1e220Va1 Asn162G1n + Asp165G1u + Arg218Asp + Ser219G1u +
I1e220Thr + Thr223Asp Thr 7lGly + Asn151Asp + Asn168Asp + Ser207G1u +
Leu209Asp + Thr223Gln Asp 65G1u + Asp 98G1u + Leu133Ser +-Asp165G1u +
Leu209Met + Gl_y215G1u Arg 64Asp + Asn 99G1u + Asp165G1u + p~ a166Asn +
Asn194Ghu + I1e220Cys Asn 99Ser + G1y136Asp + Asn163Asp + Asp165G1u +
Phe202Cys + Ser216G1u Val 95Pro + Leu133Thr + G1y136G1u + Asp165G1u +
Ser216Asp + Arg218G1u Leu133Pro + G1y134Asn + Asn194Asp + Thr206G1u +
G1y214G1u + Ser221Asp G1'_v~ 70Asp + Asp 97G1u + Tyr104Asn + Pro204G1y +
Leu209Asp + Ser216G1u G1n103Asp + G1y135Asp + Ser140Asp + Asn168G1n +
G1y214Asp + I1e220Leu Asp 65G1u + Thr 7lSer + Asn 99G1u + Ser138Asp +
Phe192Tyr + Leu209G1y Tyr104Va1 + I1e107G1u + Thr206Asp + Ser216Asp +
Ser219G1u + I1e220Leu Leu133A1a + Arg167G1u + Leu209G1n + Ser216G1u +
Thr217G1u + Ser221G1u Asn 67G1u + Asp 98G1u + Tyr137A1a + Asp165G1u +
Thr206Pro + Iie220Asp Ser101G1u + I1e107Asp + Asn161G1u + Asp165G1u +
I1e220Thr + Thr223G1n Tyr104Asp + Thr106Asn + Tyr1.37G1u + G1y160G1n +
Asn162Asp + G1y215Ser Asn 67Asp + Asp 97G1u + Tyr104Thr + Leu133Asp +
A1a164G1u + I1e220A1a Asp 97G1u + Asn 99Ser + Ser105Asp + Thr206Asp +
Ser207G1u + Trp212Thr Asn 67G1n + Tyr104I1e + Ser105G1u t Asn163G1u +
I1e220G1u + Ser221G1u Asp 98G1u + Asn 99G1n + Leu133Asp + Phe192G1u +
Asn194G1u + G1y205Pro Asn 99Asp + I1e107Ser + Tyr137Met + Asn161G1u +
Asp165G1u + I1e213Asn Gly 70Asn + G1n103Ser + Ser138G1u + A1a164Asp +
Ser170G1u + Ser219Asp Ser101G1u + Thr106Asn + G1y135G1u T Leu209Ser +
Thr217Asn + Ser219G1u Gly -68G1u + G1y102G1n + Gly'35Ser - Ser190Asp +
Ser207=isp + I1e220A1a Asp nSGlu + Val 95G1u + Glyi00Gln + Ser10SAsp_ +
Asn194Ser + Leu20-9Glu Asp 65G1u + Tyr137His + Asn161G1u + Asn162Asp +
Tyr169Thr + Ser207Asp Asn 67Ser + G1y134Pro + Asn194Asp + G1y214Asp +
Arg218Asp + Ser221G1u Asp 65G1u + Tyr137Asn + Thr211Pro + Arg218Asp +
I1e220Va1 + Ser221Asp Asp 65G1u + G1yl00Ser + Tyr104A1a + Ser207G1u +
Thr217Asp + G1y222Asp Asp 98G1u + Asn 99G1n + G1n103G1u + G1y136Ser +
Leu209Asp + Arg218G1u Asp 65G1u + Val 95Pro + Seri0lAsp + G1n103G1u +
Asn168Ser + Ser219G1u Tyr104Va1 + G1y134Pro + Tyr137Asp + A1a164G1u +
Thr211G1y + G1y215Ser SerlOlAsp + G1y135Pro + Ser190Asp + Ser191Asp +
Ser216Asp + G1y222G1n Asn 99G1u + Ser101G1u -t- G1n103Ser + G1y160Pro +

Iie213Asp + G1v222G1u y ~BSer + Ser138G1u + Ser139G1u + G1y160G1u +

Asni~3Ser + Ser219G1u Arg 64G1u Ser101GIu + Ser138G1u + Ser139G1u +
--Asn161Ser + Thr206Asn Gly 66Asn Gly 68Asp + Tyr104Leu + Ser138Asp +
+

Ser139Asp + Ser207Asp Asp 65G1u G1n103G1u + Tyr137Cys + Thr206Asn + ' +

Ser219G1u + I1e220G1u Leu 96Asp Tyr137Asp + Leu209Thr + I1e213G1y +
+

Ser219Asp + I1e220G1u Leu 96Asp Asp 97G1u + Ser140G1u + Phe192A1a +
+

Thr217Asp + G1y222Pro Ser101G1u Leu133Cys + Arg167Asp + Asn168G1u +
+

Leu209A1a + Ser216G1u Val 95G1y Thr106G1u + Arg167Asp + Asn168G1u +
+

Ser191Asp + I1e220Va1 Gly 66Ser Asp 97G1u + Tyr169Thr + Ser170Asp +
+

Thr217G1u + Arg218Asp Arg 64Asp I1e107His + G1y135Asp + G1y136Asp +
+

I1e213Thr + Ser219Asp Asp 97G1u Leu133Thr + G1y134G1u + Arg218G1u +
+

Ser219Asp + Thr223Ser Asp 98G1u G1y102Ser + Leu133Met + Ser139Asp +
+

Ser140Asp + Ser216G1u Tyr137I1e Ser139G1u + Ser140G1u + G1y160G1n +
+

Leu209Asp + G1y214Asp Leu -96A1a Asp 98G1u + Asn 99G1u + Asp165G1u +
+

Leu209Thr + Ser216G1u Asn 99G1n Ser138Asp + Asn161G1u + G1y215G1u +
+

Ser216Asp + I1e220G1y Ser105Asp Thr106Pro + G1y136Pro + G1y160G1u +
+

Leu209Asp + Ser219Asp G1n103G1u A1a166Thr + Asn194G1u + Leu209Asp +
+

Ser219G1u + I1e220Cys Tyr104Met Thr106Ser + Ser139Asp + Thr206Pro +
+

Ser219Asp + G1y222G1u I1e107G1n Asp165G1u + Asn168G1u + Ser191Asp +
+

Leu209Met + Ser219G1u Gly 68Ser Thr106G1u + I1e107G1y + Asn162G1u +
+

Ser207Asp + I1e220Asp Arg 64Asp G1y160Asp + Ser207G1u + I1e208G1y +
+

I1e213Leu + I1e220Asp Tyr137Asp Ser170Asp + Phe192Ser + Ser207G1u +
+

Leu209Pro + G1y214G1u Tyr104Cys G1y135Asp + Ser138Asp + Thr206Asp +
+

G1y214G1n + Arg218G1u Arg 64Asp Asp 97G1u + Ser139Asp + Asn168G1n + .
+

Thr206Asn + G1y214Asp Asp 65G1u Leu133Asn + Ser170G1u + I1e208Thr +
+

Ser216G1u + Arg218Asp Ser101Asp Ser138G1u + Thr206Pro + Ser2b7Glu +
+

Ser219G1u + I1e220Cys Giy134G1u + Ser207G1u + Leu209Ser + G1y214G1u +
Ser219G1u + I1e220A1a Arg 64Asp + Gly 68Asn + G1y135Asp + G1y136G1n +
Ser207Asp + Ser219Asp ~er101Asp - Ser138G1u + Ser207Asp + Leu209I1e +
Ser2i9Glu + G1y222G1n Gly 56Asp + Ser105Asp + A1a164G1n + phe192A1a +
Ser207G1u + Ser219Asp Arg 64G1u + Gly 6~Asn + Asn 99Ser + Ser138G1u +
Ser207Asp + Ser219G1u Arg 64Asp + I1e107Thr + I1e213G1y + Ser216Asp +
Arg218Asp + G1y222G1u G1n103G1u + I1e107His + G1y135Pro + G1y136Asp +
Arg167Asp + Ser219G1u Gly 68G1u + Asp 97G1u + ThrlO6Glu + Phe192Met +
Ser207G1u + I1e213A1a Alaln4Glu + Asn168Ser + Ser191G1u + Ser216Asp +
Ser219G1u + I1e220Thr Gly 68G1n + Asn102G1u + Ser190Asp + Leu209Thr +
Ser219Asp + G1y222G1n Val 95Cys + I1e107Asp + Tyr137A1a + Asn194G1u +
G1y214Asp + Ser216G1u Asp 65G1u + Gly 70G1n + Leu209G1y + Ser216G1u +
Ser221Asp + Thr223G1u Ser101G1u + G1y205G1n + Leu209G1u + Ser216Asp ~+
Thr217Ser + I1e220Thr Tyr137Thr + Ser139G1u + Leu209Asp + G1y215Pro +
Ser216G1u + Thr217G1n Asn 99G1u + G1y136Pro + Ser140G1u + Ser170G1u +
Asn194G1u + I1e220Met Gly 66G1n + Asp 98G1u + G1y100G1u + Ser105G1u +
I1e220Asp + Thr223Asn Asp 98G1u + G1yl00Glu + Ser105Asp + G1y136Ser +
Asnln8Glu + G1y214Pro Val 95Asn + Asp 97G1u + Ser101Asp + Asp165G1u +
Phe192His + I1e220G1u G1y135G1u + Asn163Asp + Phe192Tyr + Asn194G1n +
Ser219G1u + Ser221G1u Asn 99G1u + Ser101Asp + Ser140Asp + Asn161G1n +
Ser190Asp + G1y215Asn Gly 68G1n + G1y100Asp + G1n103Asp + Tyr137G1y +
G1y160Asp + Leu209Cys G1yl00Asp + G1n103Asp + G1yI60Gln + Leu209Asn +
Trp212Ser + Ser221G1u G1y135Asp + Ser138Asp + Asn162G1n + Leu209Va1 +
Ser216G1u + Thr223Asp Arg 64Asp + Gly 66G1u + Leu 96Thr + Thr106Ser +
Asn161G1u + Arg218G1u Tyr137Ser +.Asn161Asp + Ser170Asp + Ser190G1u +
Leu209Asp + G1y215G1n Leu133A1a + Ser139Asp + Thr206G1n + Leu209A1a +
Ser216Asp + Ser219G1u Asn 67Ser + Val 95Thr + Ser140G1u + Thr206Pro +

Ser216G1u + Ser219Asp Val 95Asn Ser138G1u + I1e208G1n + + Leu209Va1 +

Ser216G1u + Ser219Asp Giy 70ASp Asn 99G1n + SerlOlGlu + + Leu133Asn +

Leu209G1n + Ser216Asp Tyr104G1n I1e107His + Ser138Asp Ser191Asp +
+ +

Thr206G1u + Arg218Asp Thr106G1u Ser138G1u + Thr206G1y Ser219G1u +
+ +

I1e220Thr.+
Ser221G1u Tyr137G1u Asn161G1u + Leu209Thr I1e213Asp +
+ +

Ser216Asp + Thr217Asn Arg 64G1u Asn 67G1u + Ser170Asp Trp212His +
+ +

I1e213A1a + Arg218G1u Val 95Asp I1e107G1y + G1y134Asp Ser138Asp +
+ +

Asnl6lGln + Ser190G1u Asp ~SGlu Asp 98G1u + G1y160Se~+ Asp165G1u +
+

Phe192G1y + Ser219G1u Asp 97G1u Leu133His + G1y136G1u+ Ser138Asp +
+

Leu209G1y + Ser216Asp Asp 65G1u Ser138G1u + Tyr169Met Ser207Asp +
+ +

Leu209G1u + G1y215Pro -Leu 96G1n Asp 97G1u + Ser138G1u Thr206Pro +
+ +

Ser207G1u + Leu209Asp Gly 70G1n Asp 97G1u + Ser190Asp Thr206G1u +
+ +

Ser216Asp + I1e220Asn Arg 64G1u Gly 66Pro + Gly 70Ser Asp165G1u +
+ +

Tyr169G1u + Ser191Asp G1y136Asp Asn163Asp + Phe192Pro Ser216G1u +
+ +

Arg218Asp + I1e220Va1 Gly 66Asp Tyr137G1y + Asn162Ser A1a164G1u +
+ +

Arg167Asp + Ser216G1u G1y100Asn G1y102Asp + Tyr104His Asp165G1u +
+ +

Ser216Asp + Arg218Asp G1y102G1u Leu133Va1 + Ser140G1u Thr206G1u +
+ +

I1e220G1u + Thr223Pro Ser105G1u Leu133Asn + A1a164Asn Asp165G1u +
+ +

Thr217G1u + Ser219Asp Gly 70Asp Val 95Thr + G1y134Asp Ser139G1u +
+ +

Asn161G1u + Asn194Ser G1yl00Ser G1y135G1u + Ser140G1u Asn161Asp +
+ +

I1e213Cys + Arg218G1u G1y136Asp Tyr137G1y + Ser140Asp Asn162G1u +
+ +

Thr211G1y + G1y214Asp Val 95G1y Tyr104A1a + Ser138G1u Asn163Asp +
+ +

Arg167Asp + Ser219Asp , Asp 65G1u Ser191Asp +
+ Gly 70Asn + Val 95Ser +

Thr206G1u + Ser216Asp Tyr104Leu Asn162Asp +
+ Leu133Cys + G1y134Asp +

Tyr169Pro + Ser216Asp Val 95G1u Tyr137A1a +
+ Asn 99Asp + Tyr104A1a +

Leu209Asp + I1e220Cys Gly 66G1u G1y215Asn +
+ Thr106Ser + Leu209G1y +

Ser216Asp + I1e220His Thr 7lPro + Glyl'J2Glu + G1y134Asp + Ser139Asp +
Iie220Pro + Ser221Asp Leu 96Ser + Asp 98G1u + Ser101Asp + G1n103Ser +
G1y160Asp + Ser216G1u Leu 9eiCys + A1a164Asn + Tyr169Pro + Thr217Asn +
Arg218Asp + G1y222Asp Tyr104Asn + G1y136G1u + Tyr137His + Ser140G1u +
Ser216G1u + I1e220Cys Asp 98G1u + G1y135Asp + Ser138G1u + Asn163G1u +
Phe192Cys + I1e220Ser Gly 66G1u + Asn 99G1u + G1y102Pro + Thr106Asn +
Ser139G1u + Ser221Asp Gly 68G1u + Tyr104A1a + A1a164G1n + Asn168Asp +
Ser219G1u + Thr223Asn Asn 99G1u + G1y102Pro + Ser140Asp + Ser207G1u +
Leu209Asn + G1y222Asp Ser105Asp + Thr106G1n + G1y160Asp + Ser191Asp +
Phel92His + G1v203G1n Asp 98G1u + Tyr104Thr + Asn162G1n + Arg167Asp +
Ser170G1u + Ser216G1u Asn 99Asp + G1y102Ser + Tyr137Asp + Asp165G1u +
I1e220A1a + Thr223G1y Asp 98G1u + Thr106Pro + G1y135Asp + Ser138G1u +
Phe192Asp + I1e220G1n Arg 64G1u + Val 95A1a + Leu 96Asn + A1a164G1u +
Leu209A1a + G1y214G1u Arg 64G1u + G1y136Asn + A1a166Ser + Leu209His +
G1y214Asp + Ser221G1u Asn 67Asp + G1y102Asn + A1a164G1u + Asn168G1u +
Ser207Asp + I1e220Leu G1y135G1u + G1y136G1n + Ser139Asp + G1y160Asn +
A1a164Ser + Thr206G1u Ser105Asp + Asn163G1n + Thr206G1u + G1y215G1u +
Arg218G1u + Iie220Cys Gly 68G1n + Ser101Asp + Ser139G1u + Asn162Asp +
Asp165G1u + Leu209G1n G1yl00Ser + Ser140Asp + Asn162G1u + Asp165G1u +
Pro204G1n + Ser207G1u Thr106G1u + I1e107A1a + G1y136G1u + Ser140Asp +
Asn168G1n + Ser219Asp Arg 64G1u + Val 95Thr + Asp 98G1u + Tyr104Thr +
Ser140G1u + Ser191Asp II. Cleaning Compositions In another embodiment of the present invention, an effective amount of one or more of the enzyme variants are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal.
Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in .
form (e.g., liquid. tablet).
The cleaning compositions also comprise, in addition to the Proteinase K variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme. the term "cleaning composition material", as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the Proteinase K
variant used in the composition. the specific selection of cleaning composition materials are readily made by considering the surface material to be cleaned, the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use). The term "compatible", as used herein, means the cleaning composition materials do not reduce the proteolytic activity of the Proteinase K variant to such an extent that the protease is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.
As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. Preferably the cleaning compositions comprise from about 0.0001 % to about 10% of one or more enzyme variants of the present invention, more preferably from about 0.001 % to about 1 %, more preferably still from about 0.01 % to about 0.1 %. Several examples of various cleaning compositions ' wherein the enzyme variants may be employed are discussed in further detail below. All parts, percentages and ratios used herein are by weight ' unless otherwise specified.
As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, oral cleaning l'_I
compositions, denture cleaning compositions and contact lens cleaning compositions.
A. Cleaning Compositions for Hard Surfaces. Dishes and Fabrics The enzyme variants of the present invention can be used in a variety of detergent compositions where high sudsing and good insoluble substrate removal are desired. Thus the enzyme variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like. Such compositions can be in the form of liquids, granules, bars and the like. Such compositions can be formulated as modem "concentrated"
detergents which contain as much as 30%-60% by weight of surfactants.
The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surtactants are typically present at levels of from about 5% to about 35% of the compositions.
Nonlimiting examples of surfactants useful herein include the conventional C11-C1g alkyl benzene sulfonates and primary and random alkyl sulfates, the C10-C1 g secondary (2,3) alkyl sulfates of the formulas CH3(CH2)x(CHOS03)-M+)CH3 and CH3(CH2)y(CHOS03'M+) CH2CH3 wherein x and (y+1 ) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizing cation, especially sodium, the C10-C 1 g alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C 10-C 1 g alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C10-C 1 g alkyl polyglycosides, and their corresponding sulfated polyglycosides, C12-E18 alpha-sulfonated fatty acid esters, C12-C1g alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxylpropoxy), C12-C18 betaines and sulfobetaines ("sultaines"), C10-C1g amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. (Use of such surfactants in combination with the aforesaid amine oxide and/or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C1g N-methyl glucamides disclosed in US
Patent 5, 194,639, Connor et al., issued March 16, 1993 , A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C1p-C16 alkolamides can be incorporated into the compositions, typically at about 1 % to about 10%
levels. The C10-C14 monoethanol and diethanol amides illustrate a typical , class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2, MgS04, and the like, can be added at levels of, typically, from about 0.1 % to about 2%, to provide additionally sudsing.
The liquid detergent compositions herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable.
Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-propanediol, ethylene glycol, glycerine, and 1,2-propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50%
of such carriers.
The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11Ø Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight.
Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like.
Other conventional builders are listed in standard formularies.
Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001 % to about 1 % by weight. Various detersive and fabric care enzymes are well-known in the laundry detergent art.
Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1 % to about 10% by weight.
Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1 % to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials.
Enzyme stabilizers may also be used in the cleaning compositions.
Such enzyme stabilizers include propylene glycol (preferably from about 1 to about 10%), sodium formate (preferably from about 0.1 % to about 1 %) and calcium formate (preferably from about 0.1 % to about 1 %).
1. Hard surface cleaning compositions As used herein "hard surtace cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like. Hard surface cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about .01 % to about 5%, more preferably still from about .05% to about 1 % by weight of active enzyme of the composition. In addition to comprising one or more of the enzyme variants, such hard surface cleaning compositions typically comprise a surtactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface.
The surfactant component, when present, may comprise as little as 0.1 % of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, more preferably from about 1 % to about 5°:° of surfactant.

Typically the compositions will contain from about 0.5% to about 50%
of a detergency builder, preferably from about 1 % to about 10%.
Preferably the pH should be in the range of about 8 to 12.
Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary.
Solvents may be included in the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2.2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, preferably from about 3% to about 11 %.
Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions.
The hard surtace cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 7-12 Liquid Hard Surface Cleaning Compositions Example No.

Component 7 8 9 10 11 12 Asn67Ser 0.05 0.50 0.02 0.03 0.10 0.03 Va195His - - - - 0.20 0.02 Na2DIDA*

EDTA** - - 2.90 2.90 - -Na Citrate - - - - 2.90 2.90 NaCl2 Alkyl-benzene 1.95 - 1.95 - 1.95 -sulfonate NaCl2 Alkylsulfate - 2.20 - 2.20 - 2.20 NaCl2(ethoxy)*** - 2.20 - 2.20 - 2.20 sulfate C12 Dimethylamine - 0.50 - 0.50 - 0.50 oxide Na Cumene sulfonate 1.30 - 1.30 - 1.30 -Hexyl Carbitol*** 6.30 6.30 6.30 6.30 6.30 6.30 Water**** balance to 100%

*Disodium N-diethyleneglycol-N,N-iminodiacetate **Na4 ethylenediamine diacetic acid ***Diethyleneglycol monohexyl ether ****All formulas adjusted to pH 7 In Examples 7-10, the Proteinase K variants recited in Tables 2-36, among others, are substituted for Asn 67Ser, with substantially similar results.

In Examples 11-12, any combination of the ProteinaseK variants recited in Tables 2-36, among others, are substituted for Asn67Ser and Va195His, with substantially similar results.

WO 96!28556 PCT/US96/03005 Examples 13-18 Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew Example No.
Component 13 14 15 16 17 18 GIy134Asn + Ser140Asp 0.50 0.05 0.60 0.30 0.20 0.30 Leu96Gly + Leu209Pro - - - - 0.30 0.10 Sodium octyl sulfate 2.00 2.00 2.00 2.00 2.00 2.00 Sodium dodecyl sulfate 4.00 4.00 4.00 4.00 4.00 4.00 Sodium hydroxide 0.80 0.80 0.80 0.80 0.80 0.80 Silicate (Na) 0.04 0.04 0.04 0.04 0.04 0.04 PerFume 0.35 0.35 0.35 0.35 0.35 0.35 Water balance to 100%
Product pH is about 7.
In Examples 13-16, the Proteinase K variants recited in Tables 2-36, among others, are substituted for GIy134Asn + Ser140Asp, with substantially similar results.
In Examples 17-18, any combination of the Proteinase K variants recited in Tables 2-36, among others, are substituted for GIy134Asn +
Ser140Asp and Leu96Gly + Leu209Pro, with substantially similar results.
2. Dishwashin4 Compositions In another embodiment of the present invention, dishwashing compositions comprise one or more enzyme variants of the present invention. As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms. The dishwashing composition embodiment of the present invention is illustrated by the following examples.

Examples 19-24 Dishwashing Composition Example No.
Component 19 20 21 22 23 24 AIa164Asp 0.05 0.50 0.02 0.40 0.10 0.03 Ser191Asp + Phe192Met - - - - 0.40 0.02 + Asn194G1n C 12-C 14 N-methyl-glucamide 0.90 0.90 0.90 0.90 0.90 0.90 C 12 ethoxy ( 1 ) sulfate 12.00 12.00 12.00 12.00 12.00 12.00 2-methyl undecanoic acid 4.50 4.50 4.50 4.50 4.50 4.50 .
C12 ethoxy (2) carboxylate 4.50 4.50 4.50 4.50 4.50 4.50 C12 alcohol ethoxylate (4) 3.00 3.00 3.00 3.00 3.00 3.00 C12 amine oxide 3.00 3.00 3.00 3.00 3.00 3.00 Sodium cumene sulfonate 2.00 2.00 2.00 2.00 2.00 2.00 Ethanol 4.00 4.00 4.00 4.00 4.00 4.00 Mg++ (as MgCl2) 0.20 0.20 0.20 0.20 0.20 0.20 Ca++ (as CaCl2) 0.40 0.40 0.40 0.40 0.40 0.40 Water balance to 100%
Product pH is adjusted to 7.
In Examples 19-22, the Proteinase K variants recited in Tables 2-36, among others, are substituted for AIa164Asp, with substantially similar results.
In Examples 23-24, any combination of the Proteinase K variants recited in Tables 2-36, among others, are substituted for AIa164Asp and Ser191Asp + Phe192Met + Asn194G1n, with substantially similar results.

3. Fabric cleaning compositions In another embodiment of the present invention, fabric cleaning compositions comprise one or more enzyme variants of the present invention. As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form.
a. Granular fabric cleanin4 compositions The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01 % to about 1 by weight of active enzyme of the composition. In addition to one or more enzyme variants; the granular fabric cleaning compositions typically comprise at least one surtactant, one or more builders, and, in some cases, a bleaching agent.
The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.
Examples 25-28 Granular Fabric Cleaning Composition Example No.
Component 25 26 27 28 IIe208A1a 0.10 0.20 0.03 0.05 Trp212Phe - - 0.02 0.05 C13 linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 Phosphate (as sodium 23.00 23.00 23.00 23.00 tripolyphosphates) Sodium carbonate 23.00 23.00 23.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.20 8.20 8.20 Chelant (diethylaenetriamine- 0.40 0.40 0.40 0.40 pentaacetic acid) Sodium sulfate 5.50 5.50 5.50 5.50 Water balance to 100%
In Examples 25-26, the Proteinase K variants recited in Tables 2-36, among others, are substituted for IIe208A1a, with substantially similar results.
In Examples 27-28, any combination of the Proteinase K variants recited in Tables 2-36, among others, are substituted for IIe208A1a and Trp212Phe, with substantially similar results.

Examples 29-32 Granular Fabric Cleaning Composition Example No.
Component 29 30 31 32 ~ Asn194G1u + Leu209G1y 0.10 0.20 0.03 0.05 GIy1 OOAsn + GIn103Ser + Ser207Asp - - 0.02 0.05 + Leu209Va1 C12 alkyl benzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00 2-butyl octanoic acid 4.00 4.00 4.00 4.00 C12-C14 secondary (2,3) alkyl sulfate, 5.00 5.00 5.00 5.00 Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Water and minors balance to 100%
In Examples 29-30, the Proteinase K variants recited in Tables 2-36, among others, are substituted for Asn194G1u + Leu209G1y, with substantially similar results.
In Examples 31-32, any combination of the Proteinase K variants recited in Tables 2-36, among others, are substituted for Asn194G1u +
Leu209G1y and GIy100Asn + GIn103Ser + Ser207Asp + Leu209Va1o, with substantially similar results.

Examples 33-36 Granular Fabric Cleaning Composition Exampl e No.

Component 33 34 35 36 Arg64Asp + GIy70Gln + Thr71 Gly 0.20 0.03 0.05 -0.10 Phe192Asp + Asn194Ser - - 0.02 0.05 C13 linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 Phosphate (as sodium 23.00 23.00 23.00 23.00 tripolyphosphates) Sodium carbonate 23.00 23.00 23.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.20 8.20 8.20 Chelant (diethylaenetriamine- 0.40 0.40 0.40 0.40 pentaacetic acid) Sodium sulfate 5.50 5.50 5.50 5.50 Water balance to 100%

In Examples 33-34, the Proteinase K variants recited in Tables 2-36, among others, are substituted for + GIy70Gln+ Thr71 Gly, with Arg64Asp substantially similar results.

In Examples 35-36, any combination of the Proteinase K variants recited in Tables 2-36, among others,substitutedfor Arg64Asp +
are GIy70Gln + Thr71 Gly and Phe192Asp with substantially + Asn194Ser, similar results.

WO 96!28556 PCTIUS96/03005 Examples 37-40 Granular Fabric Cleaning Composition Example No.

Component 37 38 39 40 AIa164His + AIa166G1y 0.10 0.20 0.03 0.05 IIe213Pro + Ser216G1u + IIe220G1u- - 0.02 0.05 C12 alkyl benzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00 2-butyl octanoic acid 4.00 4.00 4.00 4.00 C12-C14 secondary (2,3) alkyl 5.00 5.00 5.00 5.00 sulfate, Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Water and minors balance to 100%

In Examples 37-38, the Proteinase K variants recited in Tables 2-36, among others, are substituted for AIa164His + AIa166G1y, with substantially similar results.

In Examples 39-40, any combination of the Proteinase K variants recited in Tables 2-36, among are substituted for AIa164His others, +

AIa166G1y and IIe213Pro + Ser216G1u + IIe220G1u, with substantially similar results.

Examples 41-42 Granular Fabric Cleaning Composition Example No.

Component 41 42 Linear alkyl benzene sulphonate11.4 10.70 Tallow alkyl sulphate 1.80 2.40 C14-15 alkyl sulphate 3.00 3.10 C14-15 alcohol 7 times ethoxylated4.00 4.00 Tallow alcohol 11 times ethoxylated1.80 1.80 Dispersant 0.07 0.1 Silicone fluid 0.80 0.80 Trisodium citrate 14.00 15.00 Citric acid 3.00 2.50 Zeol ite 32.50 32.10 Malefic acid acrylic acid copolymer5.00 5.00 Diethylene triamine penta methylene1.00 0.20 phosphonic acid , GIy203G1n + Thr211 Gly + IIe213Leu0.30 0.30 + GIy214Asn Lipase 0.36 0.40 Amylase 0.30 0.30 Sodium silicate 2.00 2.50 Sodium sulphate 3.50 5.20 Polyvinyl pyrrolidone 0.30 0.50 Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Peroxidase 0.1 0.1 Minors Up to 100 Up to 100 WO 96/28556 PCTlUS96/03005 Examples 43-44 Granular Fabric Cleaning Composition Example No.

Component 43 44 Sodium linear C12 alkyl benzene-sulfonate6.5 g.0 Sodium sulfate 15.0 18.0 Zeolite A 26.0 22.0 Sodium nitrilotriacetate 5.0 5.0 Polyvinyl pyrrolidone 0.5 0.7 Tetraacetylethylene diamine 3.0 3.0 Boric acid 4.0 -Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Asp98Glu + Tyr104Leu 0.4 0.4 Fillers (e.g., silicates; carbonates;Up to 100 Up to 100 perfumes: water) Example 45 Compact Granular Fabric Cleanin Composition Component Weight Alkyl Sulphate g_0 Alkyl Ethoxy Sulphate 2.0 Mixture of C25 and C45 alcohol 3 and oxylated 6.0 7 times eth Polyhydroxy fatty acid amide 2.5 Zeolite 17.0 Layered silicate/citrate 16.0 Carbonate 7.0 Malefic acid acrylic acid copolymer 5.0 Soil release polymer 0.4 Carboxymethyl cellulose 0.4 Poly (4-vinylpyridine) -N-oxide 0.1 Copolymer of vinylimidazole and vinylpyrrolidon0.1 e _ 0.2 GIy136Ser + Tyr137Met + Ser138G1u 0.5 Lipase 0.2 Cellulase 0,2 Tetracetylethylene diamine 6.0 Percarbonate 22.0 WO 96/28556 PCT/iJS96/03005 Ethylene diamine disuccinic acid 0.3 Suds suppressor 3.5 Disodium-4,4'-bis (2-morpholino -4-anilino-s-triazin-6-0.25 ylamino) stilbene-2,2'-disulphonate ' Disodium-4,4'-bis (2-sulfostyril) biphenyl0.05 Water, Pertume and Minors Up to 100 Example 46 Granular Fabric Cleaning Composition _ Component Weight Linear alkyl benzene sulphonate 7.6 C16-C1g alkyl sulfate 1.3 .

C14-15 alcohol 7 times ethoxylated 4.0 Coco-alkyl-dimethyl hydroxyethyl ammonium1.4 chloride Dispersant 0.07 Silicone fluid p.g Trisodium citrate 5.0 Zeolite 4A 15.0 Malefic acid acrylic acid copolymer 4.0 Diethylene triamine penta methylene phosphonic0.4 acid Perborate 15.0 Tetraacetylethylene diamine 5.0 Smectite clay 10.0 Poly (oxy ethylene) (MW 300,000) 0.3 Ser219Asp 0.4 Lipase 0.2 Amylase 0.3 Cellulase 0.2 Sodium silicate 3.0 Sodium carbonate 10.0 Carboxymethyl cellulose 0.2 Brighteners 0.2 Water, perfume and minors Up to 100 Example 47 Granular Fabric Cleaning Composition Component Weight Linear alkyl benzene sulfonate 6.92 Tallow alkyl sulfate 2.05 014-15 alcohol 7 times ethoxylated 4.4 012-15 alkyl ethoxy sulfate - 3 times 0.16 ethoxylated Zeol ite 20.2 Citrate 5. 5 Carbonate 15.4 Silicate 3.0 Malefic acid acrylic acid copolymer 4.0 Carboxymethyl cellulase 0.31 Soil release polymer 0.30 Ser190G1u + Ser191 Glu + Phe192Leu +
Asn194Ser 0.2 Lipase 0.36 Cellulase 0.13 Perborate tetrahydrate 11.64 Perborate monohydrate g.7 Tetraacetylethylene diamine 5.0 Diethylene tramine penta methyl phosphonic0.38 acid Magnesium sulfate 0.40 Brightener 0.19 Perfume, silicone, suds suppressors 0.85 Minors Up to 100 b. Liduid fabric cleaninct compositions Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzymevariants of the present invention, preferably from about 0.005%
to about 5%, more preferably from about 0.01 % to about 1 %, by weight of active enzyme of the composition.

Such liquid fabric cleaning compositionsadditionally comprise typically an anionic surfactant, a fatty acid, a detergency builder water-soluble and water.

The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 48-52 Liquid Fabric Cleaning Compositions Example No. y Component 48 49 50 51 52 IIe220Asp 0.05 0.03 0.30 0.03 0.10 Thr206Asp + IIe220Met - - - 0.01 0.20 C12- C14 alkyl sulfate, Na 20.00 20.00 20.00 20.00 20.00 2-butyl octanoic acid 5.00 5.00 5.00 5.00 5.00 Sodium citrate 1.00 1.00 1.00 1.00 1.00 C10 alcohol ethoxylate (3) 13.00 13.00 13.00 13.00 13.00 Monethanolamine 2.50 2.50 2.50 2.50 2.50 Water/propylene glycol/ethanol (100:1:1 ) balance to 100%

In Examples 48-50 the Proteinase K variants recited in Tables 2-36, among others, are substituted for IIe220Asp, with similar substantially results.

In Examples 51-52, any combination of the Proteinasevariants K

recited in Tables 2-36, among others, are substituted for IIe220Asp and Thr206Asp + IIe220Met, with substantially similar results.

Examples 53-57 Liquid Fabric Cleaning Compositions Example No.

Component 53 54 55 56 57 GIy136Ser 0.05 0.03 0.30 0.03 0.10 GIy215Pro - - - 0.01 0.20 C12- C14 alkyl sulfate, 20.00 20.00 20.00 20.00 20.00 Na 2-butyl octanoic acid 5.00 5.00 5.00 5.00 5.00 Sodium citrate 1.00 1.00 1.00 1.00 1.00 C10 alcohol ethoxylate 13.00 13.00 13.00 13.00 13.00 (3) Monethanolamine 2.50 2.50 2.50 2.50 2.50 Water/propylene glycol/ethanol balance to (100:1:1 ) 100%

In Examples 53-55 the Proteinase K variants recited in Tables 2-36, among others, are substitutedfor GIy136Ser,with substantiallysimilar results.

In Examples 56-57, any combination the Proteinasevariants of K

recited in Tables 2-36, others, are among substituted for GIy136Ser and GIy215Pro, with substantially similar results.

WO 96!28556 PCT/US96/03005 Examples 58-59 Liquid Fabric Cleaning Composition . Example No.

Component 58 59 ' 012-14 alkenyl succinic acid 3.p g.0 Citric acid monohydrate 10.0 15.0 Sodium 012-15 alkyl sulphate 8.0 8.0 Sodium sulfate of 012-15 alcohol 2 times ethoxylated - 3.0 012-15 alcohol 7 times ethoxylated - g.p 012=15 alcohol 5 times ethoxylated g.0 _ Diethylene triamine penta (methylene phosphonic acid)0.2 -Oleic acid 1.g -Ethanol 4.0 4.0 Propanediol 2.0 2.0 Pro204Asn + Thr211 Pro + GIy214G1n + GIy21 SGIu 0.2 0.2 + Thr217Ser Polyvinyl pyrrolidone 1.0 2.0 Suds suppressor 0.15 0.15 NaOH up to pH 7.5 Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Peroxidase 0.4 0.1 Waters and minors up to 100 parts In each of Examples 58 and 59 herein, the Proteinase K variants recited in Tables 2-36, among others, are substituted for Pro204Asn +

Thr211 Pro + GIy214G1n + GIy21 SGIu + Thr217Ser, with substant ially similar results.

Examples 60-62 Liquid Fabric Cleaning Composition Exa mple No.

Component 60 61 62 Citric Acid 7.10 3.00 3.00 Fatty Acid 2.00 - 2.00 Ethanol 1.93 3.20 3.20 Boric Acid 2.22 3.50 3.50 Monoethanolamine 0.71 1.09 1.09 1,2 Propanediol 7.89 8.00 8.00 NaCumene Sulfonate 1.80 3.00 3.00 NaFormate 0.08 0.08 0.08 NaOH 6.70 3.80 3.80 Silicon anti-foam agent 1.16 1.18 1.18 Leu96Met + Asn99Ser + Ser105Asp 0.0145 - -Thr211 Ser + GIy222Asp - 0.0145 -Tyr169Ser + Ser190Asp + IIe220Thr- - 0.0145 Lipase 0.200 0.200 0.200 Cellulase - 7.50 7.50 Soil release polymer 0.29 0.15 0.15 Anti-foaming agents 0.06 0.085 0.085 Brightener 36 0.095 - -Brightener 3 - 0.05 0.05 C12 alkyl benzenesulfonic acid 9.86 - -C12-15 alkyl polyethoxylate (2.5)13.80 18.00 18.00 sulfate C12 glucose amide - 5.00 5.00 C12-13 alkyl polyethoxylate (9) 2.00 2.00 2.00 Water, perfume and minors balance to 100%

c. Bar fabric cleaning compositions Bar fabric cleaning compositions t invention of the presen suitable for hand-washing soiled fabrics containeffective an amount of one or more enzyme variants of the present preferablyfrom invention, about 0.001 % to about 10%, more preferably from 01 % to ut 1 about 0. abo % by weight of the composition.

The bar fabric cleaning composi tion embodiment the present of invention is illustrated by the following examples.

Examples 63-66 Bar Fabric Cleaning Compositions - Example No.

Component 63 64 65 66 Leu96Ala + Asp97Glu + Ser101Asp 0.3 - 0.1 0.02 + IIe107Asp .

Ser193Asp - - 0.4 0.03 C12-C16 alkyl sulfate, Na 20.0 20.0 20.0 20.00 C12-C14 N-methyl glucamide 5.0 5.0 5.0 5.00 C11-C13 alkyl benzene sulfonate, Na 10.0 10.0 10.0 10.00 Sodium carbonate 25.0 25.0 25.0 25.00 Sodium pyrophosphate 7.0 7.0 7.0 7.00 Sodium tripolyphosphate 7.0 7.0 7.0 7.00 Zeolite A (0.1-.10~) 5.0 5.0 5.0 5.00 Carboxymethylcellulose 0.2 0.2 0.2 0.20 Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20 Coconut monethanolamide 5.0 5.0 5.0 5.00 Brightener, perfume 0.2 0.2 0.2 0.20 CaS04 1.0 1.0 1.0 1.00 MgS04 1.0 1.0 1.0 1.00 Water 4.0 4.0 4.0 4.00 Filler* balance %
to *Can be selected from convenient materials such as CaC03, talc, clay, silicates, and the like.
In Examples 63-64 the Proteinase K variants recited in Tables 2-36, among others, are substituted for Leu 96A1a + Asp 97GIu + Ser101Asp +
IIe107Asp, with substantially similar results.
In Examples 65-66, any combination of the Proteinase K variants recited in Tables 2-36, among others, are substituted for Leu96Ala +
Asp97Glu + Ser101Asp + IIe107Asp and Ser193Asp, with substantially similar results.

Examples 67-70 Bar Fabric Cleaning Compositions Example No.
Component 67 68 69 70 Arg64Asp + GIy68Gln 0.3 - 0.1 0.02 Tyr137G1n - 0.3 0.4 0.03 C12-C16 alkyl sulfate, Na 20.0 20.0 20.0 20.00 C12-C14 N-methyl glucamide 5.0 5.0 5.0 5.00 C11-C13 alkyl benzene sulfonate, Na 10.0 10.0 10.0 10.00 Sodium carbonate 25.0 25.0 25.0 25.00 Sodium pyrophosphate 7.0 7.0 7.0 7.00 Sodium tripolyphosphate 7.0 7.0 7.0 7.00 Zeolite A (0.1-.10u) 5.0 5.0 5.0 5.00 Carboxymethylcellulose 0.2 0.2 0.2 0.20 Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20 Coconut monethanolamide 5.0 5.0 5.0 5.00 Brightener, perfume 0.2 0.2 0.2 0.20 CaS04 1.0 1.0 1.0 1.00 MgS04 1.0 1.0 1.0 1.00 Water 4.0 4.0 4.0 4.00 Filler" balance to 100%
*Can be selected from convenient materials such as CaC03, talc, clay, silicates, and the like.
In Example 67, the Proteinase K variants recited in Tables 2-36, among others, are substituted for Arg64Asp + GIy68Gln, with substantially similar results.
In Example 68, the Proteinase K variants recited in Tables 2-36, among others, are substituted for Tyr137G1n, with substantially similar results.
In Examples 69-70, any combination of the Proteinase K variants recited in Tables 2-36, among others, are substituted for Arg64Asp +
GIy68Glnand Tyr137G1n, with substantially similar results. ' B. Additional Cleaning Compositions In addition to the hard surtace cleaning; dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated into a variety of other cleaning compositions where hydrolysis of an insoluble substrate is desired. Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions.
1 Oral cleanin4 compositions In another embodiment of the present invention, a pharmaceutically-acceptable amount of one or more enzyme variants of the present invention are included in compositions useful for removing proteinaceous stains from teeth or dentures. As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Preferably, the oral cleaning compositions comprise from about 0.0001 % to about 20% of one or more enzyme variants of the present invention, more preferably from about 0.001 °~ to about 10%, more preferably still from about 0.01 % to about 5%, by weight of the composition, and a pharmaceutically-acceptable carrier. As used herein, "pharmaceutically-acceptable" means that drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio.
Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99.99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99°~, by weight of the composition.
The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art. A wide variety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in U.S. Patent 5,096,700, Seibel, issued March 17, 1992; U.S. Patent 5,028,414, Sampathkumar, issued July 2, 1991; and U.S. Patent 5,028,415, Benedict, Bush and Sunberg, issued July 2, 1991 , The oral cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 71-74 Dentifrice Composition Example No.

Component 71 72 73 74 GIy160Pro 2.000 3.500 1.500 2.000 Sorbitol (70% aqueous solution) 35.000 35.000 35.000 35.000 PEG-6' 1.000 1. 000 1.000 1.000 Silica dental abrasive" 20.000 20.000 20.000 20.000 Sodium fluoride 0.243 0.243 0.243 0.243 Titanium dioxide 0.500 0.500 0.500 0.500 Sodium saccharin 0.286 0.286 0.286 0.286 Sodium alkyl sulfate (27.9% 4.000 4.000 4.000 4.000 aqueous solution) Flavor 1. 040 1.040 1. 040 1.040 Carboxyvinyl Polymer"~' . 0.300 0.300 0.300 0.300 Carrageenan"" 0.800 0.800 0.800 0.800 Water balance to 100%

'PEG-6 = Polyethylene glycol having a molecular weight of 600.

'"'Precipitated silica identified as Zeodent 119 offered by J.M. Huber .
""Carbopo offered by B.F. Goodrich Chemical Company.

""iota Carrageenan offered by Hercules Chemical Company In Examples 71-74 the Proteinase K variants ables recited in T 2-36.

among others, are substituted for GIy160Pro, with substantially similar results.

Examples 75-78 Mouthwash Composition E xample No.

Component 75 76 77 78 AIa166G1n + Ser216Asp 3.00 7.50 1.00 5.00 SDA 40 Alcohol 8.00 8.00 8.00 8.00 Flavor 0.08 0.08 0.08 0.08 Emulsifier 0.08 0.08 0.08 0.08 Sodium Fluoride 0.05 0.05 0.05 0.05 Glycerin 10.00 10.0010.00 10.00 Sweetener 0.02 0.02 0.02 0.02 Benzoic acid 0.05 0.05 0.05 0.05 Sodium hydroxide 0.20 0.20 0.20 0.20 Dye 0.04 0.04 0.04 0.04 Water balance to 100%

In Examples 75-78 , the Proteinase recited in Tables K variants 2-36, among others, are substituted for + Ser216Asp, with AIa166G1n substantially similar results.

Examples 79-82 Lozenge Composition Example No.

Component 79 80 81 82 GIy70Ser + IIe107G1y + Leu133Met0.01 0.03 0.10 0.02 + Phe192His + Asn194Asp Sorbitol 17.50 17.50 17.50 17.50 Mannitol 17.50 17.50 17.50 17.50 Starch 13.60 13.60 13.60 13.60 Sweetener 1.20 1.20 1.20 1.20 Flavor 11.70 11.70 11.70 11.70 Color 0.10 0.10 0.10 0.10 Corn Syrup balance to 100%

In Examples 79-82, the Proteinase bles K variants recited in Ta 2-36, among others, are substitutedGIy70Ser IIe107G1y 133Met for + + Leu +

Phe192His + Asn194Asp, with tantiallyar results.
subs simil 14.1 Examples 83-86 Chewing Gum Composition Example No.
Component 83 84 85 86 GIy214Asn 0.03 0.02 0.10 0.05 Sorbitol crystals 38.44 38.40 38.40 38.40 Paloja-T gum base' 20.00 20.00 20.00 20.00 Sorbitol (70% aqueous solution) 22.00 22.00 22.00 22.00 Mannitol 10.00 10.00 10.00 10.00 Glycerine 7.56 7.56 7.56 7.56 Flavor 1.00 1.00 1.00 1.00 'Supplied by L.A. Dreyfus Company.
In Examples 83-86, the Proteinase K variants recited in Tables 2-36, among others, are substituted for GIy214Asn, with substantially similar results.
2. Denture cleanin4 compositions In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more enzyme variants of the present invention. Such denture cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.0001 % to about 50% of one or more of the enzyme variants, more preferably from about 0.001 % to about 35%, more preferably still from about 0.01 % to about 20%, by weight of the composition, and a denture cleansing carrier. Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see for example U.S. Patent 5,055,305, Young), and are generally appropriate for incorporation of one or more of the enzyme variants for removing proteinaceous stains from dentures.
The denture cleaning composition embodiment of the present invention is illustrated by the following examples.

WO 96!28556 PCT/US96/03005 Examples 87-90 Two-layer Effervescent Denture Cleansing Tablet Example No.
Component 87 88 gg gp Acidic Laver Tyr137Leu + Tyr169Cys 1.0 1.5 0.01 0.05 + Phe192Asn + Ser207Asp + Arg218Glu + IIe220Leu Tartaric acid 24.0 24.0 24.00 24.00 Sodium carbonate 4.0 4.0 4.00 4.00 Sulphamic acid 10.0 10.0 10.00 10.00 PEG 20,000 4.0 4.0 4.00 4.00 Sodium bicarbonate 24.5 24.5 24.50 24.50 Potassium persulfate 15.0 15.0 15.00 15.00 Sodium acid pyrophosphate 7.0 7.0 7.00 7.00 Pyrogenic silica 2.0 2.0 2.00 2.00 TAED" 7.0 7.0 7.00 7.00 Ricinoleylsulfosuccinate 0.5 0.5 0.50 0.50 Flavor 1.0 1.0 1.00 1.00 Alkaline Layer Sodium perborate monohydrate 32.0 32.0 32.00 32.00 Sodium bicarbonate 19.0 19.0 19.00 19.00 EDTA 3.0 3.0 3.00 3.00 Sodium tripolyphosphate 12.0 12.0 12.00 12.00 PEG 20,000 2.0 2.0 2.00 2.00 Potassium persulfate 26.0 26.0 26.00 26.00 Sodium carbonate 2.0 2.0 2.00 2.00 Pyrogenic silica 2.0 2.0 2.00 2.00 Dye/flavor 2.0 2.0 2.00 2.00 "Tetraacetylethylene diamine In Examples 87-90, the Proteinase K var iants recited in Tables 2-36, among others, are substituted for Tyr137Leu Tyr169Cys e192Asn + + Ph +

Ser207Asp + Arg218Glu + IIe220Le u, with similarsults.
substantially re 3. Contact Lens Cleaning Comp ositions In another embodiment of the present invention, ntact co lens cleaning compositions comprise one or more zyme riants en va of the present invention. Such contact lens cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.01 to about 50% of one or more of the enzyme variants, more preferably from about 0.01 % to about 20%, more preferably still from about 1 % to about 5%, by weight of the composition, and a contact lens cleaning carrier.
Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art (see for example U.S. Patent 4,863,627, Davies, Meaken and Rees, issued September 5, 1989; U.S.
Patent Re. 32,672, Huth, Lam and Kirai, reissued May 24, 1988; U.S. Patent 4,609,493, Schafer, issued September 2, 1986; U.S. Patent, 4,690,793, Ogunbiyi and Smith, issued September 1, 1987; U.S. Patent 4,614,549, Ogunbiyi, Riedhammer and Smith, issued September 30, 1986; and U.S.
Patent 4,285,738, Ogata, issued August 25, 1981), and are generally appropriate for incorporation of one or more enzyme variants of the present invention for removing proteinaceous stains from contact lens.
The contact lens cleaning composition embodiment of the present invention is illustrated by the following examples.

Examples 91-94 Enzymatic Contact Lens Cleaning Solution Example No.

Component 91 92 93 94 GIy102G1n 0.01 0.5 0.1 2.0 Glucose 50.00 50.0 50.0 50.0 Nonionic surfactant (polyoxyethlene-2.0 2.0 2.0 2.00 polyoxypropylene copolymer) Anionic surfactant (polyoxyethylene-1.0 1.0 1.0 1.00 alkylphenylether sodium sulfricester) Sodium chloride 1.00 1.0 1.0 1.0 Borax 0.30 0.3 0.3 0.3 Water balance to 100~

In Examples 91-94, the Proteinase K variants recited in Tables 2-36, among others, are substituted for with substantially GIy102G1n, similar results.
While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BRODE, PHILIP F
BARNETT, BOBBY L
RUBINGH, DONN N
(ii) TITLE OF INVENTION: PROTEINASE K VARIANTS HAVING DECREASED
ADSORPTION AND INCREASED HYDROLYSIS
(iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: THE PROCTER & GAN~LE COMPANY -(B) STREET: 11810 EAST MIAMI RIVER ROAD
(C) CITY. ROSS
(D) STATE. OH
(E) COUNTRY: US
(F) ZIP: 45061 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE. Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME : HAKE , RICHARD A
(B) REGISTRATION NUMBER: 37,343 (C) REFERENCE/DOCKET NUMBER: 5605 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (513)-627-0087 (B) TELEFAX: (513)-627-0260 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 279 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY. linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
Ala Ala Gln Thr Asn Ala Pro Tzp Gly Leu Ala Arg Ile Ser Ser Thr Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu -Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Tyr Ser Ser Arg Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu ' 85 90 95 Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr I1e Ile Ala Gly Met Asp Phe Val Ala Ser Asp Lys Asn Asn Arg Asn Cys Pro Lys Gly Val Val Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser Val Cys Thr Val Gly Ala Ser Asp Arg Tyr Asp Arg Arg Ser Ser Phe Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Gly Pro Gly Thr Ser Ile Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu Gly Lys Thr Thr Ala Ala Ser Ala Cys Arg Tyr Ile Ala Asp Thr Ala Asn Lys Gly Asp Leu Ser Asn Ile Pro Phe Gly Thr Val Asn Leu Leu Ala Tyr Asn Asn Tyr Gln Ala

Claims

Claims:

1. A Proteinase K variant having a modified amino acid sequence identified as SEQ ID NO. 1 of Proteinase K wild-type amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region;
wherein the modified amino acid sequence comprises a substitution at one or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 64, 65, 66, 68, 70 or 71; wherein a. when a substitution occurs at position 64, the substituting amino acid is Asp or Glu;
b. when a substitution occurs at position 65, the substituting amino acid is Glu;
c. when a substitution occurs at position 66, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 68, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
e. when a substitution occurs at position 70, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and f. when a substitution occurs at position 71, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 95, 96, 97, 98, 100, 102, 103, 104, 106 or 107; wherein a. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;
b. when a substitution occurs at position 96, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;

c. when a substitution occurs at position 97, the substituting amino acid is Glu;
d. when a substitution occurs at position 98, the substituting amino acid is Glu;
e. when a substitution occurs at position 100, the substituting amino acid is Asn, Gln, Pro or Ser;
f. when a substitution occurs at position 102, the substituting amino acid is Asn, Gln, Glu, Pro or Ser;
g. when a substitution occurs at position 103, the substituting amino acid is Asn or Ser;
h. when a substitution occurs at position 104, the substituting amino acid is Asp, His, Ile, Leu, Met, Pro or Val;
i. when a substitution occurs at position 106, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; and j. when a substitution occurs at position 107, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Leu, Met, Pro, Ser, Thr or Val;
C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 133, 134, 136, 137, 138, 139 or 140; wherein a. when a substitution occurs at position 133, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Met, Pro, Ser, Thr or Val;
b. when a substitution occurs at position 134, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
c. when a substitution occurs at position 136, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; with the proviso that when position 136 is substituted, the variant is not a single mutation variant wherein the substituting amino acid is Gln;
d. when a substitution occurs at position 137, the substituting amino acid is Asp, His, Ile, Leu, Met or Pro;

e. when a substitution occurs at position 138, the substituting amino acid is Asp or Glu;
f. when a substitution occurs at position 139, the substituting amino acid is Asp or Glu; and g. when a substitution occurs at position 140, the substituting amino acid is Asp or Glu; with the proviso that when position 140 is substituted, the variant is not a single mutation variant wherein the substituting amino acid is Asp;
D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 161, 162, 163, 164, 165, 166, 167, 168, 169 or 170; wherein a. when a substitution occurs at position 161, the substituting amino acid is Gln;
b. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser;
c. when a substitution occurs at position 163, the substituting amino acid is Asp, Gln, Glu or Ser;
d. when a substitution occurs at position 164, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
e. when a substitution occurs at position 165, the substituting amino acid is Glu;
f. when a substitution occurs at position 166, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
g. when a substitution occurs at position 167, the substituting amino acid is Asp or Glu;
h. when a substitution occurs at position 168, the substituting amino acid is Asp, Gln, Glu or Ser;
i. when a substitution occurs at position 169, the substituting amino acid is Asp, His, Ile, Leu, Met or Pro; and j. when a substitution occurs at position 170, the substituting amino acid is Asp or Glu;

E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 190, 192, 193 or 194; wherein a. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu;
b when a substitution occurs at position 192, the substituting amino acid is Asn, Cys, Gln, His, Ile, Met, Pro, Thr, Tyr or Val;
with the proviso that when position 192 is substituted, the variant is not a single mutation variant wherein the substituting amino acid is Val:
c. when a substitution occurs at position 193, the substituting amino acid is Asp or Glu; and d. when a substitution occurs at position 194, the substituting amino acid is Asp, Gln, Glu or Ser; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 203, 204, 205, 206, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 or 223;
wherein a. when a substitution occurs at position 203, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
b. when a substitution occurs at position 204, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;
c. when a substitution occurs at position 205, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 206, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; with the proviso that when the variant is substituted at position 206.
the variant is a single mutation variant wherein the substituting amino acid not selected from the group consisting of Asp and Glu;
e. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val;

f. when a substitution occurs at position 209, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
g. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu;
h. when a substitution occurs at position 211, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
i. when a substitution occurs at position 212, the substituting amino acid is Asp or Glu;
j. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val;
k. when a substitution occurs at position 214, the substituting amino acid is Asn, Gln, Pro or Ser; with the proviso that when the variant is substituted at position 214, the variant is not a single mutation variant wherein the substituting amino acid is Ser:
l. when a substitution occurs at position 215, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
m. when a substitution occurs at position 216, the substituting amino acid is Asp or Glu;
n. when a substitution occurs at position 217, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
o. when a substitution occurs at position 218, the substituting amino acid is Asp or Glu; with the proviso that when position 218 is substituted. the variant is not a single mutation variant wherein the substituting amino acid is Asp:
p. when a substitution occurs at position 219, the substituting amino acid is Asp or Glu;
q. when a substitution occurs at position 220, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val;
r. when a substitution occurs at position 221, the substituting amino acid is Asp or Glu;

s. when a substitution occurs at position 222, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and t. when a substitution occurs at position 223, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
whereby the Proteinase K variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Proteinase K.

2. The Proteinase K variant of Claim 1, wherein one or more substitutions occur in the first loop region.

3. The Proteinase K variant of Claim 1, wherein one or more substitutions occur in the second loop region.

4. The Proteinase K variant of Claim 1, wherein one or more substitutions occur in the third loop region.

5. The Proteinase K variant of Claim 1, wherein one or more substitutions occur in the fourth loop region.

6. The Proteinase K variant of Claim 1, wherein one or more substitutions occur in the fifth loop region.

7. The Proteinase K variant of Claim 1, wherein one or more substitutions occur in the sixth loop region.

8. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 1 and a cleaning composition carrier.

9. The cleaning composition of Claim 8, wherein the cleaning composition is a hard surface cleaning composition.

10. The cleaning composition of Claim 8, wherein the cleaning composition is a fabric cleaning composition.

11. The fabric cleaning composition of Claim 10, wherein the composition is in the form of a liquid.

12. The fabric cleaning composition of Claim 11, wherein the composition comprises at least about 5% surfactant and at least about 5% builder, by weight of the composition.

13. The fabric cleaning composition of Claim 12 further comprising cleaning composition materials selected from the group consisting of solvents, buffers, enzymes, soil release agents, clay soil removal agents, dispersing agents, brighteners, suds supressors, fabric softeners, suds boosters, enzyme stabilizers, bleaching agents, dyes, perfumes, and mixtures thereof.

14. The fabric cleaning composition of Claim 12 further comprising at least one bleaching agent.

15. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 2 and a cleaning composition carrier.

16. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 3 and a cleaning composition carrier.

17. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 4 and a cleaning composition carrier.

18. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 5 and a cleaning composition carrier.

19. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 6 and a cleaning composition carrier.

20. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, wherein the cleaning composition comprises the Proteinase K variant of Claim 7 and a cleaning composition carrier.

21. A DNA sequence encoding the Proteinase K variant of Claim 1.