CA2189427C

CA2189427C - Subtilisin 309 variants having decreased adsorption and increased hydrolysis

Info

Publication number: CA2189427C
Application number: CA002189427A
Authority: CA
Inventors: Philip Frederick Brode, Iii; Bobby Lee Barnett; Donn Nelton Rubingh; Chanchal Kumar Ghosh
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 1994-05-02
Filing date: 1995-04-18
Publication date: 2003-10-28
Anticipated expiration: 2015-04-18
Also published as: HU9603030D0; JPH09512434A; HUT75558A; WO1995030011A2; MX9605358A; US6599730B1; AU2294495A; WO1995030011A3; BR9507605A; CN1151187A; EP0758389A1; CA2189427A1; CZ321796A3

Abstract

The present invention relates to subtilisin (309) variants having a modified amino acid sequence of wild-type subtilisin (309) 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 different amino acids than that occurring in wild-type subtilisin (309) (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the subtilisin (309) variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild type subtilisin (309). The present invention also relates to DNA sequence encoding such subtilisin (309) variants. The present invention also relates to compositions comprising such subtilisin (309) variants for cleaning a variety of surfaces.

Description

°

W0 95/30011 21 s 9 ~

AND INCREASED HYDROLYSIS
TECHNICAL FIELD
s The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and the genes encoding such enzyme variants.
BACKGROUNC) Enzymes make up the largest class of naturally occurring proteins.
io 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) is 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.
2o 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 2s 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 andlor so 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 ss stability under quite diverse conditions.
Despite the variety of approaches described in the art, there is a continuing need for new effective variants of ~~roteases useful for cleaning a variety of surfaces.

WO 95130011 PCTlUS95104760 Objects of the Present Invention It is an object of.the present invention to provide,.subtilisin 309 enzyme variants having improved hydrolysis versus the vu~ld=type of the enzyme.
It is also an object of the present invention to provide cleaning s compositions comprising these subtilisin 309 enzyme variants.
SUMMARY
The present invention relates to subtilisin 309 variants having a modified amino acid sequence of wild-type subtilisin 309 amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a to 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 different amino acids than that occurring in wild-type subtilisin 309 (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the subtilisin 309 variant has decreased adsorption to, and is increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin 309. The present invention also relates to DNA sequences encoding such subtilisin 309 variants. The present invention also relates to compositions comprising such subtilisin 309 variants for cleaning a variety of surfaces.
2o BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A and Figure 1 B depict the amino acid sequences of subtilisin BPN' and subtilisin 309. The top line represents the amino acid sequence of subtilisin BPN' (SEQ ID N0:2), which is derived from Bacillus amyloliquefaciens; the bottom line represents the amino acid sequence of 25 subtilisin 309 (SEQ ID N0:1 ), which is derived from Bacillus lentus. The symbol * in the sequence for subtilisin 309 denotes the absence of specific amino acid residues as compared to subtilisin BPN'.
DESCRIPTION
I. Subtilisin 309 Variants 3o This invention pertains to subtilisin enzymes, in particular subtilisin 309, 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, "subtilisin 309 variants") of the present invention have decreased adsorption to and 3s increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to the mutant genes encoding for such subtilisin 309 variants.
When refering to the amino acid sequence of native subtilisin 309 PCTIUS95l04760 (having 269 amino acid residues), the amino acid sequence of subtilisin BPN' (having 275 amino acid residues) is frequently used as the standard. The use of "BPN' numbering" has become the coventional method for identifying residue positions in all subtilisins. The amino acid sequences for native s subtilisin BPN' and native subtilisin 309 are set forth in Figs 1A and 1B.
The symbol '"' in the sequence for subtilisin 309 in Figs 1A and 1 B denotes the absence of specific amino acid residues compared to native subtilisin BPN'.
However, for purposes of discussion herein, reference to amino acid positions shall be based on "true" subtilisin 309 numbering (e.g., refer to SEQ ID N0:1, to discussed below).
The subtilisin 309 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 is 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 2o increase with increasing surface concentration. This has been shown to be the case. (Erode, P.F. III and D. S. Ranch, LANGMUIR, "Subtilisin BPN':
Activity on an 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.
2s (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 3o give better pertormance. 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 3s 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 ~ 18 9 ~ 2 7 pCTlUS95104760 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 subtilisin 309, certain amino acids form exterior loops on the enzyme molecule. For purposes of s discussion, these loops shall be referred to as the first, second, third, fourth and fifth loop regions. Specifically, positions 57-64 form the first loop region;
positions 93-105 form the second loop region; positions 124-131 form the third loop region; positions 152-161 form the fourth loop region; positions 181-185 form the fifth loop region; and positions 193-214 form the sixth loop region to (position numbering analagous to positions in the amino acid sequence for wild-type subtilisin 309 (SEQ ID N0:1 )).
It 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 is this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the subtilisin 309 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 20 of the subtilisin 309 molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptideslprotein soils).
The following is a list of abbreviations used herein to describe amino acids:

Amino Acid Three-letter Abbreviation One-letter Symbol Alanine Ala A

Arginine Arg Asparagine Asn N

s Aspartic Acid Asp p Asparagine or Aspartic Acid Asp p Cysteine Cys C

Glutamine Gln Q

Glutamic Acid Glu E

io Glutamine or Glutamic Acid Glx Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu is Lysine Lys ~ K

Methionine Met M

Phenylalanine Phe F

Proline Phe F

Serine Ser S

2o Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr y Valine Val V

No amino acid at position Xaa a As used herein, "variant" means an enzymehaving an amino acid sequence which differs from that of wild-type.

As used herein, "mutant subtilisin 309 DNA sequence" means a DNA

sequence coding for a subtilisin 309 variant.

As used herein, "wild-type subtilisin refers to an enzyme 309"

3o represented by SEQ ID N0:1. The amino nce for subtilitisn acid seque 309 is further described in World Patent Publication89/06279 (1989).
See also, World Patent Publication 94!02618, published February 3, 1994 by Mulleners et al.

As used herein, the term "subtilisin 309 wild-type amino acid sequence"

3s encompasses SEQ ID N0:1 as well as SEQ having modifications ID N0:1 to the amino acid sequence other than at any of positions 57-64, 93-105, 124-131, 152-161, 181-185 and 193-214.

As used herein, "more hydrophilic amino acid" refers to any other amino 2' 189421 G
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, K.R., "Prediction of Protein Antigenic.Determinants from Amino s Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981.

Amino Acid Hydrophilicity Value Trp -3.4 to Phe -2.5 Tyr -2.3 Leu, Ile -1.8 Val -1.5 Met -1.3 15 CyS -1.0 Ala, His -0.5 Thr -0.4 Pro, Gly -0.0 Gln, Asn 0.2 2o 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 2s 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 3o 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 3s substituted with Gln, Asn, Ser, Glu or Asp.

°°
~~ WO 95130011 PCTIUS95104?60 A. Loop Region 6 Substitution Variants 1. Variants comprising at least one amino acid substitution In one embodiment of the present invention, the subtilisin 309 variant ' has a modified amino acid sequence of subtilisin . 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution s at one or more of positions 193, 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin 309.
Preferably, the positions having a substituted amino acid are 193, 194, 195, l0 196, 199, 201, 202, 203, 204, 205, 206 or 209; more preferably, 194, 195, 196, 199 or 201.
Preferably, the substituting amino acid for position 193 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid far position 194 is Asn, Asp, Gln, is Glu, Gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 195 is Asn, Asp, Gln, Glu, Gly, Ser.
Preferably, the substituting amino acid for position 196 is Asn, Asp, Gln, Glu, Pro or Ser.
2o Preferably, the substituting amino acid for position 197 is Ala, Asp, Cys, Gln, Glu, Gly, His, Met, Pro or Ser.
Preferably, the substituting amino acid for position 199 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 200 Asn, Asp, Glu or 2s Ser.
Preferably, the substituting amino acid for position 201 is Asp or Glu.
Preferably, the substituting amino acid far position 202 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
Preferably, the substituting amino acid for position 203 is Ala, Asn, Asp, 3o Gln, His, Ile, Met, Pro or Ser.
Preferably, the substituting amino acid far position 204 is Asn, Asp, Gln, Glu, Gly or Ser.
Preferably, the substituting amino acid for position 205 is Asn, Asp, Gln, Glu, Pro or Ser.
ss Preferably, the substituting amino acid for position 206 is Asp or Glu.
Preferably, the substituting amino acid for position 207 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.

s.
Preferably, the substituting amino acid for position 208 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro or Val.
Preferably, the substituting amino acid for position 209 is Asn, Asp, gln, Glu, gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 210 is Asp or Glu.
Preferably, the substituting amino acid for position 211 is Ala, Asn, Asp, Cys, Gln, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
Preferably, the substituting amino acid for position 212 is Glu.
Preferably, the substituting amino acid for position 213 is Asn, Asp, Gln, to Glu, Pro or Ser.
Preferably, the substituting amino acid for position 214 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
More preferably, the substituting amino acid for any of positions 193, 194, 195, 196, 197, 199, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 and 214 is, with reference to Table 1, is neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin 309.
More preferably still, the substituting amino acid for any of positions 193, 194, 195, 196, 197, 199, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 212, 213 and 214 is Asp or Glu; and the substituting amino acid for positions 203 and 211 is Asp.
2. Variants comprising at least two amino acid substitutions In another embodiment of the present invention, the subtilisin 309 variant has a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution at two or more of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.
3o Preferably, the positions having a substituting amino acid are 193, 194, 195, 199, 201, 202, 203, 204, 205, 206, or 209; more preferably, positions 194, 195, 196, 211 or 213.
Preferably, the substituting amino acid for position 193 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser and Thr.
Preferably, the substituting amino acid for position 194 is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 195 is Asn, Asp, Gln, Glu, Gly or Ser.

Preferably, the substituting amino acid for position 196 is Asn, Asp, Gln, Glu, Pro or Ser.
Preferably, the substituting amino acid for position 197 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr. .
s Preferably, the substituting amino acid for position 198 is Asp, Gln, Glu or Ser.
Preferably, the substituting amino acid for position 199 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 200 is Asn, Asp, Glu to or Ser.
Preferably, the substituting amino acid far position 201 is Asp or Glu.
Preferably, the substituting amino acid far position 202 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
Preferably, the substituting amino acid for position 203 is Ala, Asn, Asp, Is Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 204 is Asn, Asp, Gln, Glu, Gly or Ser.
Preferably, the substituting amino acid for position 205 is Asn, Asp, Gln, Glu, Pro or Ser.
2o Preferably, the substituting amino acid for position 206 is Asp or Glu.
Preferably, the substituting amino acid far position 207 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
Preferably, the substituting amino acid for position 208 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro or Val.
2s Preferably, the substituting amino acid for position 209 is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 210 is Asp or Glu.
Preferably, the substituting amino acid for position 211 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
3o Preferably, the substituting amino acid for position 212 is Asp, Gln, Glu or Ser. However, if position 211 is substituted with Asn, Glu or Val, then position 212 is not substituted with Asp;
p Preferably, the substituting amino acid for position 213 is Asn, Asp, Gln, Glu, Pro or Ser.
3s Preferably, the substituting amino acid for position 214 is Asn, Asp,Gln, Glu, Gly, Pro or Ser.
More preferably, the substituting amino acid for any of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214 is, with reference to Table 1, is neutral or negatively charged and equally or more more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin 309.
s More preferably still, the substituting amino acid for any of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214 is Asp and Glu.
3. Variants comprising at least three amino acid substitutions In another embodiment of the present invention, the subtilisin 309 to variant has a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution at three or more of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214;
whereby the subtilisin 309 variant has decreased adsorption to, and increased 15 hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.
Preferably, the positions having a substituting amino acid are 193, 194, 195, 199, 201, 202, 203, 204, 205, 206, or 209; more preferably, positions 194, 195, 196, 211 or 213.
Preferably, the substituting amino acid for position 193 is Ala, Asn, Asp, 2o Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 194 is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 195 is Asn, Asp, Gln, Glu, Gly or Ser.
25 Preferably, the substituting amino acid for position 196 is Asn, Asp, Gln, Glu, Pro or Ser.
Preferably, the substituting amino acid for position 197 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 198 is Asp, Gln, Glu 30 or Ser.
Preferably, the substituting amino acid for position 199 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 200 Asn, Asp, Glu or Ser.
35 Preferably, the substituting amino acid for position 201 is Asp or Glu.
Preferably, the substituting amino acid for position 202 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
Preferably, the substituting amino acid for position 203 is Ala, Asn, Asp, WO 95/30011 ' PCTIUS95104760 Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
Preferably, the substituting amino acid for position 204 is Asn, Asp, Gln, Glu, Gly or Ser.
Preferably, the substituting amino acid for position 205 is Asn, Asp, Gln, s Glu, Pro or Ser.
Preferably, the substituting amino acid for position 206 is Asp or Glu.
Preferably, the substituting amino acid for position 207 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
Preferably, the substituting amino acid for position 208 is Ala, Asn, Asp, to Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Thr or Val.
Preferably, the substituting amino acid for position 209 is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
Preferably, the substituting amino acid for position 210 is Asp or Glu.
Preferably, the substituting amino acid for position 211 is Ala, Asn, Asp, is Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
Preferably, the substituting amino acid for position 212 is Asp, Gln, Glu or Ser.
Preferably, the substituting amino acid far position 213 is Asn, Asp, Gln, Glu, Pro or Ser.
2o Preferably, the substituting amino acid for position 214 is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
More preferably, the substituting amino acid for any of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214 is, with reference to Table 1, is neutral or 2s negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in wild-type subtilisin 309.
More preferably still, the substituting amino acid for any of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 208, 30 209, 210, 211, 212, 213 or 214 is Asp or Glu.
B. Multi-Loop Re4ions Substitution Variants In another embodiment of the present invention, the subtilisin 309 variant has a modified amino acid sequence ofi subtilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a 3s substitution at one or more positions in one or more of the first, second, third, fourth, or fifth loop regions; whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin 309.

In another embodiment of the present invention, the subtilisin 309 variant further comprises one or more substitutions to the sixth loop region.
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 s is, with reference to Table 1, neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in the wild-type amino acid sequence.
1. Substitutions in the First Loop Region When a substitution occurs in the first loop region, the substitution to occurs at one or more of positions 57, 58, 59, 60, 61, 63, or 64.
When a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser.
When a substitution occurs at position 58, the substituting amino acid is Glu.
15 When a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 61, the substituting amino acid is 2o Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
2s 2. Substitutions in the Second Loop Region When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105.
When a substitution occurs at position 93, the substituting amino acid is 3o Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
35 When a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 97, the substituting amino acid is Asp or Glu.

zis~~z~
""" WO 95/30011 PCTIUS95/04760 When a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 99, the substituting amino acid is Asp or Glu.
s When a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 101, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 102, the substituting amino acid to is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 103, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 104, the substituting amino acid is Asp or Glu.
is When a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, tVlet, Pro, Ser, Thr or Val.
3. Substitutions in the Third Loop Region When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 124, 125, 126, 127, 128, 129, 130 or 131.
2o When a substitution occurs at position 124, 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 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 126, the substituting amino acid 2s is Asp or Glu.
When a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser.
When a substitution occurs at position 128, the substituting amino acid is Asp or Glu.
3o When a substitution occurs at position '129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser.
When a substitution occurs at position '130, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 131, the substituting amino acid 3s is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser, Thr.

4. Substitutions in the Fourth LoorJ Region When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 152, 153, 1 ~~4, 155, 156, 157, 158, 159, or 161.
When a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 153, the substituting amino acid s is Asp, Gln, Glu or Ser.
When a substitution occurs at position 154, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
1o When a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 158, the substituting amino acid 1s is Asp or Glu.
When a substitution occurs at position 159, 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 160, the substituting amino acid is Asp or Glu.
2o When a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.

5. Substitutions in the Fifth Loop Region When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 181, 182, 183, 184 or 185.
2s When a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 182, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 183, the substituting amino acid 3o is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val.
When a substitution occurs at position 184, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 185, the substituting amino acid 3s is Asn, Asp, Glu or Ser.

6. Substitutions in the Sixth Loop Region When a substitution occurs in the sixth loop region, in conjunction with one or more substitutions in one or more of the preceding five loop regions, the substitution occurs at one or more of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214.
When a substitution occurs at position 193, the substituting amino acid s is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 194, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 195, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser.
io When a substitution occurs at position 196, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 197, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 198, the substituting amino acid is is Asp, Gln, Glu or Ser.
When a substitution occurs at position 199, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Glu or Ser.
2o When a substitution occurs at position 201, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 202, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 203, the substituting amino acid 2s is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 2t)4, 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.
3o When a substitution occurs at position 206, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 208, the substituting amino acid ss is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Glly, His, Pro, Ser or Thr.
When a substitution occurs at position 210, the substituting amino acid ~ ~~~ z is Asp or Glu.
When a substitution occurs at position 211, 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 212, the substituting amino acid s is Asp, Gln, Glu or Ser.
When a substitution occurs at position 213, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
to C. Additional substitutions at positions other than the loop regions As indicated hereinbefore, in addition to the one or more substitutions made to the first, second, third, fourth, fifth andlor sixth loop regions of wild-type subtilisin 309, substitutions may be made at positions other than positions in such loop regions (hereafter referred to as "additional substitution"). In 15 another embodiment of the present invention, the subtilisin 309 variant has a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence wherein, in addition to being substituted at one or more positions in one or more of the above-discussed loop regions, there is substitution at position 74.
The additional substitution may occur at postion 74 alone (preferred), or in 2o combination with one or more additional substitutions.
Where the additional substitution to the subtilisin 309 variant is at position 74 alone, the substitution is preferably with Asn, Asp, Glu, Gly, His, Lys, Phe or Pro. Particularly preferred is where the substitution is with Asp or His.
25 Where there is more than one additional substitution to the subtilisin 309 variant, preferred is where the additional substitutions occur at position in combination with one or more of the following residues: 97, 99, 101, 102, 105 and 121. Preferred additional substitution combinations include the following: 74197; 74199; 741101; 741102; 741105; 741121; 74197199; 741971101;
30 74/971102; 741991101; 741991102; 7411011102; 741102/105; 7411021121;
741105/121; 74/971991101; 741971991102; 7419711011102; 74199/101 /102;
74!10111021121; 74110211051121; 74197/991101 l 102; 74197110111021121 andlor 74197199110111021121. Most preferred additional substitution combinations include the following: 74197; 741102; 741971102; 7411011102;
3s 7411021105; 7419911011102; 7419719911011102 and 741991102.
Preferably, the additional substitutions to be made at each of the identified amino acid residue positions include but are not limited to substitutions at position 74 including Asp, His, Glu, Gly, Phe, Lys, Pro and -~W O 95/30011 Asn; substitutions at position 97 including Asp, Thr, Asn, Gln, Gly and Ser;
substitutions at position 99 including Gly, Asp, Lys, Leu, Ala, Glu and Ser;
substitutions at position 101 including Gln, Thr, Asp, Glu, Tyr, Lys, Gly, Arg and Ser; substitutions at position 102 including Ser,. Tyr, Ile, Leu, M, Ala, W, s Asp, Thr, Gly and Val; substitutions at position 105 including Val, Leu, M, Tyr, Gly, Glu, Phe, Thr, Ser, Ala and Ile; and substitutions at position 121 including Asn, Thr, Ile and Ser. The specifically preferred amino acids) to be substituted at each such position are designatE~d below in Table 2. Although specific amino acids are shown in Table 2, it :>hould be understood that any to amino acid may be substituted at the identified residues. As indicated herein before, these substitutions are in addition to the one or more substitutions at one or more of the loop regions, discussed above.

Amino Acid Residue Preferred Amino Acid to be Substitutedllnserted is 74 Asp, His 97 Asp,Thr, Asn, Gly 99 Arg, Gly, Asp, Lys, Leu, Ala, Glu 101 Ala, Gln, Thr, Asp, Glu, Tyr, Lys, Gly, Arg 102 Ile, Tyr, Ser, Leu, Ala, Thr, Gly 20 105 Val, Leu, Tyr, Gly, Phe, Thr, Ser, Ala 121 Ser, Thr, Ile D. Preaaration of enzyme variants Example 1 Mutant 309 DNA Seguences 2s A phagemid (pJMA602) containing the wild type subtilisin 309 (i.e., savinase) 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 METHODS IN ENZYMOLOGY 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus, OH
30 1E9). The pUC119-pUB110 hybrid plasmid is named pJMA601. Into the BamH I restriction site of pJMA601 is cloned the polymerase chain reation-amplified 309 (savinase) gene from Bacillus lentus chromosomal DNA
(National Collections of Industrial and Marine Bacteria Bacillus lentus 10309) giving phagemid pJMA602. Phagemid pJMA602 is transformed into 3s 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 efficif~nt site-specific mutagenesis °

'~ 2189421 Is without phenotypic selection", METHODS IN ENZYMOLOGY, 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 VeCtOfS", DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed. M.J.
s 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, "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA", NUCLEIC ACIDS
RESEARCH, Vo1.10, 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 >.5 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.
20 160, pp. 15-21 ). For some of the mutants a modified pJMA602 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 at position 203 and also encoded for random substitutions at positions 57, 58, 59, 60, 61, 63, 64, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 25 103, 104, 105, 124, 125, 126, 127, 128, 129, 130, 131, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 181, 182, 183, 184, 185, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 and 214 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-3o stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing.
Example 2 Fermentation The Bacillus subtilis cells (BG2036) containing a subtilisin mutant of 35 interest are grown to mid-log phM se 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:

~T~ ~ 189427 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.
s 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 ~o 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 enzyrne is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCI
)IS 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 2o 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 25 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.
To avoid autolysis of the enzyme during storage, an equal weight of 3o 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 35 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 SephadeX G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to s 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 CaCl2 and pH adjusted to 8.6 with HCI. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C.
E. Characterization of Enzyme Variants io 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 Sachem, .Inc. (Torrence, California).
is The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3-(dimethylamino)propyl] 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 2o purge at about 70°C. The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, 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 t 7,000 pNA moleculeslpm2. The surface area will remain unchanged from the value of 50.Om21g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).
Example 5 3o Surface Hvdrolvsis Assav Using CPGaAAPF-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 3s 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-'- WO 95/30011 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 s 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 surface io 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%
is 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 2o nm on a DU-70 spectrophotometer. The Ernzyme 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 ~cM 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 LoTUST"" spreadsheet (Lotus 2s Development Corporation, Cambridge, Massac:husetts). 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 yo. The yo and so data are plotted in the standard inverse fashion to give KM and kcat~
so F. Example Subtilisin 309 Variants Subtilisin 309 variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 3-38, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number 3s second, and the substituted amino acid third.

Loop 1 - Single Mutation Variants G1n57Asn .Z2 rG1n57Asp G1n57G1u G1n57Ser Asp58Glu G1y59Asn G1y59Asp G1y59G1n G1y59G1u G1y59Pro G1y59Ser Asn60Asp Asn60Gln Asn60Glu Asn60Ser G1y61Asn Gly6lAsp G1y61G1n G1y61G1u G1y61Pro Gly6lSer G1y63Asn G1y63Asp Gly63Gln G1y63G1u G1y63Pro G1y63Ser Thr64Asn Thr64Asp Thr64Gln Thr64Glu Thr64Gly Thr69Pro Thr64Ser Loop 1 - Double Mutation Variants G1n57Ser + Asn60Glu Asp58Glu + G1y61G1n G1y59Ser + G1y63Ser Asn60Ser + G1y61Ser G1y63Asn + Thr64Asp G1y59Asn + Thr69Glu Asn60Glu + G1y63Ser Asn60Gln + G1y63G1n Asn60Asp + G1y63Ser Asp58Glu + G1y63G1n G1n57Asp + Thr64Gly G1n57G1u + G1y63G1n Asn60Glu + G1y63Asn G1y61G1n + Gly63Asn Asp58Glu + G1y59Asn Asp58G1u + Thr64Pro 21 g g ~ ~ ~ PCTIUS95104760 G1y61Asn + G1y6:3Asn Asp58Glu + G1y6:1Pro Asn60Glu + G1y6:3Gln Asn60Asp + Gly6:LGln Asp58Glu + Asn6l~Ser G1n57Asn + GlyS!aGlu Asn60Glu + Gly6:LPro G1y61Asn + Thr69Gln Asp58Glu + Thr6~~Ser Asn60Asp + Thr6~tGln G1y59G1u + G1y63Ser G1n57Asn + Gly6:Ser G1y61Ser + Thr69Asn G1n57Asp + G1y61Asn Gly59Glu + G1y63Pro Asn60Gln + G1y67.Ser G1y61Pro + G1y63Ser G1y59Asp + Gly63Asn G1y63G1n + Thr64~Asn G1n57G1u + GlyS~~Gln G1n57Asp + GlyS~~Pro G1n57G1u + G1y61Ser G1n57Asp + Gly6~'~Gln Asp58Glu + G1y61Asn G1y59Asn + Asn60Glu G1y61G1n + Thr69Glu G1n57G1u + G1y61G1n Asp58Glu + G1y63Ser G1y59G1u + G1y63Asn G1y61Ser + Thr64Asp G1y59Ser + Thr64Asp G1n57G1u + G1y63Pro G1y59Ser + Thr69Glu Asn60Gln + G1y63Pro G1n57Asn + Asn60Asp Loop 1 - Triple Mutation \/ariants G1y59Pro + G1y63G1n + Thr64Glu 4o G1n57Ser + G1y59Asn + G1y63Pro G1y59Pro + Asn60Asp + Thr69Asn G1n57G1u + Gly59Asn + G1y63Pro G1n57Asp + G1y63Pro + Thr64Asn G1n57Ser + Asn60Glu + Thr64Gly G1n57Ser + Asn60Ser + Gly63Ser Gly6lSer + G1y63Ser + Thr64Gln G1n57G1u + G1y61Pro + Thr64Gly Asp58Glu + G1y63Pro + Thr69Gly G1n57Asn + Asp58Glu + G1y61Pro Asp58Glu + G1y59Ser + Thr64Pro G1n57Asn + G1y59G1u + G1y61Ser Gly59Glu + G1y61G1n + Thr64Asn G1n57Asp + Asn60Gln + Thr69Gly Asn60Glu + Gly6lGln + Thr69Gly G1n57Asp + Gly59Pro + Gly63Pro G1n57Asn + G1y63Asn + Thr64Gly G1n57Ser + G1y59Asp + Gly6lSer Gly6lGln + G1y63Asn + Thr64Pro G1n57G1u + Gly59Pro + Thr64Ser Asn60Asp + G1y63G1n + Thr64Gly Asp58Glu + G1y63Asn + Thr64Gln Asp58Glu + Gly6lGln + G1y63G1n G1n57Asp + G1y61Pro + Thr69Gln Asp58Glu + Gly59Asn + G1y61Ser Asp58Glu + G1y59Ser + Asn60Ser G1n57Asn + Gly6lPro + Thr64Gly G1y59G1u + Gly6lPro + G1y63Pro G1y59G1u + Gly6lGln + G1y63Asn G1y59G1u + G1y61Pro + G1y63G1n G1y59G1u + G1y61Pro + Thr64Asn G1n57Asn + Asp58Glu + Thr64Gln Loop 1 - Quadruple Mutation Variants G1y59G1n + Asn60Asp + G1y61Pro + G1y63Pro G1y59Ser + Asn60Gln + Gly63Ser + Thr64Asp G1y59Pro + Asn60Ser + Gly6lSer + Gly63Gln G1n57G1u + Gly59Pro + Asn60Ser + Gly6lPro G1n57G1u + Asn60Gln + G1y63Asn + Thr64Gly G1n57Ser + Gly59Ser + Asn60Asp + G1y61Ser G1n57Ser + Asn60Glu + Gly6lPro + Thr69Pro Asn60Gln + G1y61Pro + Gly63Gln + Thr69Asp Asn60Glu + G1y61G1n + G1y63Asn + Thr64Pro G1n57G1u + Gly59Gln + G1y61Pro + Thr64Gln G1n57Asn + Asp58Glu + Gly59Ser + Asn60Gln Asp58Glu + Asn60Ser + Gly63Ser + Thr69Ser G1n57G1u + Gly59Pro + Gly63Gln + Thr64Ser Asp58Glu + Gly59Ser + Asn60Ser + G1y63Ser G1y59Asn + Asn60Asp + G1y61Ser + Thr64Gln G1n57G1u + Asp58Glu + Gly59Gln + G1y61G1n G1n57G1u + Asp58Glu + Asn60Ser + Thr69Pro G1n57Asp + Asp58Glu + G1y61Ser + G1y63Pro G1n57Asp + Asp58Glu + Asn60Gln + G1y61G1n G1n57Ser + Asp58Glu + G1y59G1u + Asn60Gln Asp58Glu + Gly59Asp + G1y61Ser + G1y63G1n Gly59Glu + Asn50Asp + Gly6lAsn + G1y63Asn G1y59Asp + Asn60Asp + Gly6lAsn + Gly63Gln G1y59G1u + Asn60Asp + Gly6lPro + Thr64Ser~

Asp58Glu + G1y59G1u + Asn60Asp + Thr64Gly Asp58Glu + G1y59G1u + Asn60Asp + Gly6lPro Asp58Glu + G1y59G1u + Asn60Glu + Gly6lAsn 2lss~az7 Loop 2 - Single Mutation Variants Val 93A1a Val 93Asn 5 Val 93Asp Val 93Cys Val 93G1n Val 93G1u Val 93G1y 10 Val 93His Val 93Met Val 93Pro Val 93Ser Val 93Thr 15 Leu 94A1a Leu 94Asn Leu 94Asp Leu 99Cys Leu 99G1n 20 Leu 99G1u Leu 99G1y Leu 94His Leu 94I1e Leu 94Met 25 Leu 94Pro Leu 94Ser Leu 94Thr Leu 94Va1 Gly 95Asn Gly 95Asp Gly 95G1n Gly 95G1u Gly 95Pro Gly 95Ser Ala 96Asn Ala 96Asp Ala 96G1n Ala 96G1u Ala 96G1y Ala 96His Ala 96Pro Ala 96Ser Ala 96Thr Ser 97Asp Ser 97G1u Gly 98Asn Gly 98Asp Gly 98G1n Gly 98G1u Gly 98Pro Gly 98Ser Ser 99Asp Ser 99G1u ~t8~d~~

Glyl00Asn Glyl00Asp G1yl00Gln G1yl00Glu G1yl00Pro G1yl00Ser SerlOlAsp SerlOlGlu Va1102A1a Va1102Asn Va1102Asp Va1102Cys Va1102G1n Va1102G1u Va1102G1y Va1102His Va1102Met Va1102Pro Va1102Ser Va1102Thr Ser103Asp Ser103G1u Ser104Asp Ser104G1u I1e105A1a I1e105Asn I1e105Asp I1e105Cys I1e105G1n I1e105G1u I1e105G1y I1e105His I1e105Leu I1e105Met I1e105Pro IlelOSSer I1e105Thr I1e105Va1 4o TABLE 8 Loop 2 - Double Mutation Variants Val 93G1n + Ser 99G1u Gly 95Ser + Gly 98G1n SerlOlAsp + I1e105A1a Leu 94Ser + Gly 95Ser Leu 94Pro + SerlOlAsp Gly 98G1n + Ser103Asp Ser 97Asp + Tyr102G1n Tyr102Cys + I1e105Met Val 93Pro + Gly 98G1n Ser 99G1u + Glyl00Pro Ser103Asp + I1e105Leu ~~894~~

Ser 97Asp + Gly 98G1n Val 93Ser + Ser 99Asp Leu 94Ser + Gly 98G1u Gly 95G1u + Tyr102Met Gly 95Asn + SerlC3Glu Gly 98Ser + Tyr102Thr-Gly 98G1n + TyrlC2Cys Leu 94I1e + IlelC~SGln Leu 99Asp + G1yl00Gln Ala 96G1y + Ser101Asp SerlOlGlu + I1e105G1n Gly 95G1n + I1e105Ser Val 93G1n + G1yl00Pro Val 93Met + Ser104Asp Gly 98Pro + Ser101Asp Val 93Pro + I1e105His Gly 95Asp + Glyl00Ser Tyr102Cys + I1e105Leu Glyl00Asn + I1e105Met Gly 98Asn + Ser101G1u Ser 99G1u + I1e105G1n Val 93Thr + Tyr102Thr Gly 98Ser + Ser109G1u Gly 95Asn + Ser109G1u Val 93Pro + Ser103G1u Gly 95Asn + Tyr102I1e Leu 94Pro + Gly 98Asp Leu 94His + Gly 98Asp Val 93Asn + Tyr102Thr Tyr102A1a + Ser103Asp Gly 98Pro + Ser103G1u Leu 94Cys + Tyr102Leu Val 93G1y + Gly 98Ser G1yl00Gln + Tyr102Ser Val 93Thr + G1yl00Asn Gly 98Asn + I1e105Pro Val 93Asp + Leu 94A1a Leu 94G1y + Ser104G1u Val 93Met + SerlOlAsp Val 93Met + Ser ~~7Glu Ala 96Pro + I1e105Asn Ser 97G1u + Gly 98pro Ala 96Thr + Ser 99G1u Val 93Asn + G1yl00Asn Gly 95G1n + Gly 98Pro Gly 95Asn + Ala ~~6His Val 93Ser + Ser103Asp Gly 98G1n + IleICI5Pro Val 93Cys + I1e105G1u ~~8g4~'~ .

Loop - Variants 2 Triple Mutation Val 93G1n + Leu 99Thr + Ser 99G1u Leu 94Met + Gly 95G1n + Ser104Asp Ser 99G1u + Tyr102Met + I1e105Thr Val 93Thr + Leu 94Thr + I1e105Cys Leu 94Asp + Ala 96Thr + Gly 98Asn Val 93Met + Gly 95G1n + Ser103Asp Val 93Cys + Ala 96Pro + Ser 99G1u Leu 94Asp + Ala 96G1y + G1yl00Gln Leu 94His + Gly 98G1n + Ser 99G1u Gly 95Pro + Ser101Asp + Tyr102G1n Leu 94Asn + Gly 95G1n + SerlOlAsp Leu 94His + Gly 98Pro + Ser104G1u Leu 94Thr + Ala 96Asn + Glyl00Pro Gly 95Ser + Gly 98Ser + Tyr102G1u Ala 96Thr + Ser 97Asp + Tyr102Thr Leu 94Asn + G1yl00Ser + SerlOlGlu Leu 94Met + Gly 98G1n + Glyl00Asn Val 93Pro + Ala 96G1u + I1e105A1a Val 93Cys + Gly 95G1u + Tyr102Leu Leu 94Cys + Gly 95Ser + Ser 97Asp Gly 95G1n + SerlOlGlu + Tyr102His Val 93G1n + Gly 95G1u + I1e105G1n Val 93G1y + Ser 99Asp + I1e105G1y Gly 95Asn + Gly 98G1u + I1e105Va1 Ser 97G1u + Gly 98Pro + Tyr102Thr Val 93G1u + Leu 99I1e + G1yl00Gln Ala 96Pro + Ser101Asp + I1e105Pro Gly 98Pro + Ser103Asp + I1e105Va1 Ala 96G1n + Ser 99Asp + Tyr102Ser Ser 97Asp + Gly 98Ser + I1e105Asn Val 93G1n + Gly 98Asn + Tyr102Leu Leu 94G1y + Tyr102Pro + Ser103G1u Val 93Thr + G1yl00Gln + Ser104G1u Val 93G1y + G1yl00Gln + Ser103G1u Ala 96Thr + Glyl00Ser + Ser103G1u G1yl00Asn + SerlOlGlu + I1e105Thr Gly 95Asp + Tyr102G1y + I1e105Met Val 93Cys + Ala 96Thr + I1e105Cys Ala 96G1y + Gly 98Asp + Ser 99Asp Gly 95Ser + Ser 97Asp + Gly 98Asp Leu 94Asn + Ser103G1u + Ser104Asp Tyr102Va1 + Ser103G1u + Ser104G1u Ser 99Asp + G1yl00Glu + I1e105Met Ser 97Asp + Gly 98Asp + Ser 99G1u Gly 95Asn + SerlOlGlu + Ser103G1u Ser101G1u + Tyr102Asn + Ser103G1u Leu 94Asp + Ser 99G1u + G1yl00Gln G1yl00Asp + SerlOlGlu + Ser104G1u Leu 94Asp + Gly 98G1u + I1e105Cys Gly 95G1u + Ser 97G1u + Tyr102Asn Tyr102Thr + Ser103G1u + I1e105G1u Gly 95Asp + Ser 99G1u + Glyl00Gln Gly 95G1u + Ser 99G1u + Tyr102Met Val 93G1n + Ser 97Asp + Ser 99G1u G1yl00Glu + Ser101Asp + Ser103Asp Val 93Asp + Ser103G1u + Ser109G1u Val 93Asp + Ser 99Asp + Glyl00Asp Ala 96G1n + G1yl00Asp + Ser104Asp Val 93Asp + SerlOlGlu + I1e105Asp Leu 94Asp + Ser 99G1u + SerlOlGlu to Loop Mutation 2 Variants -Quadruple Leu 94Asn + Ser 97G1u + Tyr102Asn +I1e105Met Leu 94Pro + Gly 95G1u + G1yl00Gln +Tyr102A1a i5 Val 93Cys + Leu 94Met + Gly 95Pro +Ala 96G1n Gly 95Pro + Ser 97G1u + Tyr102G1n +I1e105Leu Gly 95Asp + Glyl00Pro + Tyr102Va1 +I1e105Cys Ala 96Thr + G1yl00Asp + Tyrll)2Ala +I1e105Asn Val 93Asn + Leu 99Cys + Serll)lAsp +Tyr102Va1 20 Val 93Asn + Gly 95Asn + Serl()4Asp +I1e105Va1 Leu 94Ser + Gly 98Pro + Ser103G1u +I1e105Va1 Val 93Asn + Leu 94His + Gly 95Asn +I1e105Asn Ala 96Ser + Gly 98Asn + Ser ~~9Glu +Tyr102G1y Val 93Asn + Gly 98Asn + Tyr102I1e +I1e105Asp 25 Val 93Asn + Tyr102Asn + Ser103Asp +I1e105Ser Ala 96Ser + G1yl00Ser + Ser101Asp +Tyr102Pro Leu 94Pro + Ala 96Ser + Ser101Asp +Tyr102Ser Val 93Met + Ala 96Thr + Ser104Asp +I1e105G1n Val 93Met + Leu 94A1a + Gly ~a5Pro +Ser104G1u 30 Val 93Cys + Gly 95Pro + Ala ~36G1u +Tyr102Va1 Gly 95Asn + Ala 96Asn + Ser104G1u +I1e105Pro Val 93Asn + Leu 94Asn + Gly 98G1u +Ser 99G1u Leu 94His + Gly 98G1u + Ser ~~9Glu +Tyr102Ser Gly 95Pro + Gly 98G1u + Ser ~~9Asp +I1e105G1y 35 Val 93A1a + Ala 96Ser + SerlC>3Glu +Ser104G1u Leu 94G1y + Ser 97Asp + Gly 98Asp +Ser 99G1u Val 93Cys + Leu 94Asp + Gly ~~5Asn +G1yl00Glu Val 93Ser + Ser101G1u + Ser103Asp +Ser104Asp Ala 96Ser + SerlOlGlu + Tyr102G1n +Ser103Asp 40 Val 93Thr + G1yl00Asn + SerlOlGlu +Ser103Asp Val 93Asn + Leu 99G1u + Gly 95G1u +Ser 99G1u Leu 94Asp + Ala 96G1y + Ser 99G1u +I1e105Cys Leu 94A1a + Ser101G1u + Ser104Asp +I1e105Ser Ala 96His + Ser101G1u + SerlC4Glu +I1e105A1a 45 Val 93G1y + G1yl00Pro + SerlClGlu +Ser109Asp Gly 95G1n -+Ser101G1u + SerlC4Asp +I1e105Met Gly 98G1n + Ser101G1u + Ser104G1u +I1e105Asn Val 93G1y + SerlOlAsp + Tyr102G1u +I1e105Asp Ser1 01Asp + Tyr102Va1 + Ser104G1u +I1e105G1u 50 Val 93Cys + Gly 95Asp + Gly 98Asp +G1yl00Pro Gly 95Asp + Ser 97G1u + Tyr102A1a +I1e105Cys Val 93Thr + Gly 95G1u + Ala 96Asn +Ser 99Asp 21$g ~2'~
Val 93Cys + Gly 95Asp + Ser 99G1u + I1e105Va1 Val 93Cys + Ser101Asp + Ser103G1u + I1e105Asp Ala 96His + Ser 97G1u + Ser 99Asp + I1e105Asn Ala 96Asn + Ser 97G1u + Ser 99G1u + Tyr102Pro 5 Val 93Cys + Ser 97G1u + Ser 99Asp + I1e105His Val 93Ser + Ser 97G1u + Gly 98G1h + Ser 99G1u Gly 95Asn + Ser 97Asp + Ser 99G1u + G1yl00Glu Val 93Met + G1yl00Asp + Tyr102Asp + Ser103Asp Leu 99G1y + SerlOlAsp + Tyr102A1a + I1e105G1u 10 Ser 97G1u + Gly 98Asp + Glyl00Asp + Tyr102G1y Leu 94Ser + Ser 99G1u + Glyl00Glu + Ser104G1u Val 93Asn + Ser 99G1u + G1yl00Glu + Ser104Asp Val 93A1a + Ser 99G1u + G1yl00Asp + Ser104Asp Leu 99Cys + Gly 95Ser + Ser 99Asp + Ser101G1u 15 Gly 95Ser + Ala 96Thr + Ser 99G1u + SerlOlAsp Leu 94Cys + Ala 96Ser + Ser 99Asp + Ser101Asp Ser 99Asp + Ser101G1u + Tyr102Asn + I1e105Va1 Val 93G1u + Gly 95Pro + Ser101G1u + Ser103Asp Val 93A1a + Gly 95Asp + Ser 99Asp + Ser101G1u 20 Ser 99G1u + SerlOlGlu + Tyr102Pro + Ser104G1u Loop 3 - Single Mutation Variants Leu129A1a 5 Leu124Asn Leu124Asp Leu124Cys Leu124G1n Leu129G1u 30 Leu124G1y Leu124His Leu124I1e Leu124Met Leu124Pro Leu124Ser Leu124Thr Leu124Va1 G1y125Asn G1y125Asp G1y125G1n G1y125G1u G1y125Pro G1y125Ser Ser126Asp Ser126G1u Pro127Asn Pro127Asp Pro127G1n Pro127G1u Pro127G1y Pro127Ser Ser128Asp "'~ WO 95/30011 PCT/US95104760 zis9~z~

Ser128G1u Pro129Asn Pro129Asp Pro129G1n Pro129G1u Pro129G1y Pro129Ser Ser130Asp Ser130G1u A1a131Asn A1a131Rsp A1a131G1n A1a131G1u A1a131G1y IS A1a131His A1a131Pro A1a131Ser A1a131Thr 2o TABLE 12 Loop 3 - Double Mutation Variants Leu124G1n + Ser128G1u G1y125Ser + Serl:?6Glu G1y125Ser + Ser128G1u 25 Pro127G1y + Ser130G1u Leu124Thr + Serla?6Glu Ser126G1u + Prola?9Ser G1y125G1n + Serla?8Glu Pro127Ser + Ser130G1u 30 G1y125G1u + Prop?7Asn Pro129G1n + Ser1:30G1u G1y125Asp + A1a131His Pro127G1y + Alal.'3lGlu Leu124His + Ser126Asp 35 Pro127G1y + Pro129Rsp Ser126G1u + Alal'.3lGln Leu124Asp + A1a131Pro G1y125Pro + Pro127G1u G1y125Ser + Ser126Asp 40 Leu124G1n + A1a131Rsn G1y125Rsp + Pro129Ser Pro129Asn + A1a131Asp Pro127Asn + Serl~'.8Asp Pro129G1y + A1a131G1u 45 Pro127Ser + Pro129Asp Pro127Ser + Ser11.8G1u Leu124G1y + Ser130G1u G1y125Asn + Ser130Asp Leu124G1u + Pro127Asn 50 Ser130G1u + A1a131G1y Ser130Asp + A1a131Thr Leu129G1n + Ser128Asp WO 95/30011 218 9 ~ 2'~ PCTIUS95104760 Leu124Ser + A1a131Pro Pro129Ser + Ser130G1u G1y125G1n + Pro127Asp Leu124Cys + A1a131Thr G1y125G1n + Pro129G1n Leu124Met + A1a131G1n G1y125G1n + A1a131Asp Ser128G1u + A1a131Asn Ser130G1u + A1a131Pro G1y125Pro + A1a131Ser Pro127Ser + Pro129Asn Ser126G1u + Pro127G1n Pro127Ser + Ser130Asp Pro127G1y + Ser128Asp G1y125G1u + Pro127Ser Leu124Cys + Pro129Asn G1y125G1n + Pro129G1u G1y125Asn + A1a131Asp G1y125Ser + Pro129G1n Ser126Asp + Pro129G1n Leu124Va1 + Pro129Asn Leu124His + Ser126G1u Pro127G1u + A1a131G1y Leu124Thr + G1y125Pro 5 Leu124His + Ser130G1u G1y125Asn + Ser126Asp Pro129Asn + A1a131Pro Pro127G1n + Pro129Asn Leu124G1y + Pro129G1u Loop 3 - Triple Mutation Variants G1y125G1n + Ser126G1u + A1a131His Ser126Asp + Pro127Ser + A1a131His Leu124Va1 + Ser130Asp + A1a131Asn Leu124G1n + Ser126G1u + A1a131Ser G1y125Ser + Pro129G1u + A1a131G1y Leu124Ser + Pro127Asp + A1a131G1n Pro127Ser + Ser128Asp + A1a131His Leu124Thr + Pro127G1u + A1a131Ser Leu124G1n + Ser126G1u + A1a131Asn G1y125Asn + Pro127G1u + A1a131G1n G1y125G1n + Ser126Asp + Pro127G1n Pro127Asn + Ser130Asp + A1a131Thr G1y125Asn + Pro127G1u + Pro129Asn Leu129Thr + Ser128Asp + A1a131G1n G1y125Ser + Pro129G1n + A1a131G1u Pro127G1n + Pro129Asn + Ser130G1u Leu124I1e + G1y125G1n + Ser130G1u Pro129Asn + Ser130G1u + A1a131G1y Leu129Ser + G1y125Ser + Pro129G1n Leu124Asn + G1y125G1n + Ser128Asp ~IBg!~~7 "'~"'° WO 95/30011 PCT/US95104760 Leu124G1n + Pro127Ser + Ser130Asp G1y125Ser + Pro127Ser + A1a131Asp G1y125Ser + Ser128G1u + Pro129G1n Pro127Asn + Ser128Asp + A1a131G1y Leu124Thr + G1y125Ser + A1a131Asn G1y125G1n + Pro129Ser + Ser130Asp G1y125Pro + Ser126G1u + Pro129Asn Pro129G1y + Ser130Asp + A1a131Pro G1y125Asn + Ser126Asp + Pro129G1y to G1y125Asp + Ser126Asp + A1a131Asn G1y125G1u + Ser126G1u + A1a131Ser G1y125G1u + Ser126Asp + A1a131His G1y125G1n + Ser128G1u + Pro129G1u Ser128G1u + Pro129G1u + A1a131G1y Leu124Cys + Ser128G1u + Pro129Asp Prol2_7Gly + Ser128G1u + Pro129G1u Pro127G1y + Ser128Asp + Pro129Asp Leu124Asn + Ser128G1u + Pro129Asp Ser126G1u + Pro127Asp + A1a131Asn Ser126G1u + Pro127G1u + Pro129G1y Leu124His + Ser126Asp + Pro127G1u Ser126Asp + Pro127G1u + Pro129G1y Pro127Asp + Ser128Asp + Pro129Asn Pro127Asp + Ser128G1u + A1a131His Pro127G1u + Ser128Asp + A1a131Pro Leu124I1e + Pro127G1u + Ser128G1u Pro129Asp + Ser130Asp + A1a131G1y Leu124Pro + Pro129G1u + Ser130G1u Ser126Asp + Pro127G1u + Ser128Asp Ser126G1u + Pro127Asp + Ser128Asp Pro129G1u + Ser130Asp + A1a131G1u Leu124Asp + G1y125Asp + Ser126Asp Leu124Ser + Ser126Asp + Ser128Asp Ser126G1u + Ser128Asp + Pro129Asn G1y125Asn + Ser126G1u + Ser128G1u Ser126G1u + Ser128G1u + A1a131Thr Leu124Asn + Ser126G1u + Ser128G1u Ser126G1u + Ser128G1u + Pro129G1u G1y125G1u + Ser126G1u + Ser128Asp Ser128Asp + Pro129Ser + Ser130G1u Loop 3 - Quadruple Mutation Variants G1y125G1n + Ser126G1u + Prol2'7Ser + A1a131Ser G1y125Asn + Pro127Ser + Ser130Asp + A1a131Asn Leu124A1a + Pro127G1n + Ser12f3G1u + A1a131Pro G1y125Pro + Pro127Ser + Pro129Ser + Ser130G1u G1y125Asn + Pro129Asn + Ser130Asp + A1a131G1n G1y125Asn + Pro129Asn + Ser130G1u + A1a131Pro Leu124Ser + Ser126G1u + Prol2~~Gly + A1a131G1n G1y125Asn + Ser126G1u + Prol2~~Gly + A1a131Ser Leu124I1e + Ser128Asp + Prol2~aGln + A1a131Ser W095130011 21g942~

Leu124His + G1y125Ser Pro129G1y A1a131Asn + +

Leu124A1a + G1y125G1u Pro127G1n Pro129G1n + +

Leu124Ser + G1y125G1u Ser126Asp Pro129Ser + +

G1y125G1u + Ser126G1u Pro127G1n A1a131His + +

Leu124Met + G1y125Asp Ser126Asp Pro127Asn + +

Leu124G1n + Ser128G1u Pro129Asp A1a131G1n + +

Leu124Va1 + Ser128G1u Pro129G1u A1a131Asn + +

Pro127Ser + Ser128Asp Pro129Asp A1a131Thr + +

Leu124I1e + Ser128G1u Pro129Asp A1a131Ser + +

l0 G1y125Ser + Ser126G1u Pro127G1u A1a131G1n + +

Leu124His + G1y125G1n Ser130G1u A1a131G1u + +

Leu124Thr + G1y125G1n Ser130Asp A1a131Asp + +

Leu124His + Pro127G1u Ser128G1u A1a131His + +

G1y125Ser + Pro127Asp Ser128Asp Pro129G1n + +

Leu124Asn + Ser126G1u Pro127Asp Ser128Asp + +

Pro127G1n + Ser128G1u Pro129Asp Ser130Asp + +

G1y125Pro + Ser128Asp Pro129G1u Ser130G1u + +

Leu124Asn + G1y125Asp Ser126Asp Pro127Asp + +

Leu124Asp + G1y125Asp Ser126Asp Pro129G1n + +

Leu124Asp + G1y125G1u Ser126Asp Pro129G1y + +

G1y125G1n + Ser126G1u Ser128G1u A1a131Thr + +

Leu124Asn + Ser126G1u Pro127G1n Ser128Asp + +

Leu129Met + G1y125Pro Ser126Asp Ser128G1u + +

Leu124Asn + G1y125Ser Ser126Asp Ser128Asp + +

5 Leu124Va1 + Ser126G1u Ser128G1u Pro129Asp + +

Leu124Cys + Ser126Asp Ser128G1u Pro129G1u + +

Ser126Asp + Pro127Asp Pro129G1u A1a131Ser + +

G1y125Asn + Ser126G1u Pro127G1u Pro129G1u + +

Leu129Ser + Ser126Asp Pro127Asp Pro129Asp + +

G1y125Asp + Ser126G1u Ser128Asp Pro129Ser + +

Leu124Va1 + G1y125Asp Ser126G1u Ser128G1u + +

G1y125G1u + Ser126G1u Ser128Asp Pro129Asn + +

G1y125G1u + Ser126Asp Pro127Ser Ser128Asp + +

Leu124Met + Ser128G1u Ser130G1u A1a131Ser + +

Leu124Cys + Ser128G1u Pro129Ser Ser130Asp + +

G1y125G1n + Ser128G1u Ser130G1u A1a131His + +

Leu124Met + Pro127G1n Ser128G1u Ser130G1u + +

Leu124G1y + Ser126Asp Pro129G1u Ser130Asp + +

G1y125Asn + Ser126G1u Pro129G1u Ser130G1u + +

Leu124G1y + G1y125Asp Pro127Asp Pro129G1y + +

Ser126Asp + Ser128Asp Ser130G1u A1a131G1n + +

G1y125Pro + Ser126Asp Ser128G1u Ser130Asp + +

Ser126Asp + Pro127G1y Ser128G1u Ser130G1u + +

Ser126G1u + Pro127G1y Ser128Asp Ser130G1u + +

Ser126Asp + Ser128Asp Pro129Ser Ser130Asp + +

Ser126G1u + Ser128Asp Ser130Asp A1a131His + +

Ser126G1u + Ser128G1u Ser130G1u A1a131Ser + +

G1y125Pro + Ser126G1u Ser128G1u Ser130Asp + +

Leu124Va1 + Ser126Asp Ser128G1u Ser130Asp + +

G1y125G1n + Ser126G1u Ser128Asp + + Ser130G1u Ser126G1u + Ser128Asp + Pro129G1y + Ser130G1u "'WO 95130011 PCT/US95/04760 Loop 4 - Single Mutation Variants G1y152Asn G1y152Asp G1y152G1n G1y152G1u G1y152Pro G1y152Ser Asn153Asp 10 Asn153G1n Asn153G1u Asn153Ser Ser154Asp Ser154G1u 15 G1y155Asn G1y155Asp G1y155G1n G1y155G1u G1y155Pro 20 G1y155Ser A1a156Asn A1a156Asp A1a156G1n A1a156G1u A1a156G1y A1a156His A1a156Pro A1a156Ser A1a156Thr 30 G1y157Asn G1y157Asp G1y157G1n G1y157G1u G1y157Pro 3S G1y157Ser Ser158Asp Ser158G1u I1e159A1a I1e159Asn 4o I1e159Asp I1e159Cys I1e159G1n I1e159G1u I1e159G1y I1e159His I1e159Leu I1e159Met I1e159Pro I1e159Ser I1e159Thr I1e159Va1 Ser160Asp Ser160G1u Tyr161A1a Tyr161Asn Tyr161Asp Tyr161Cys Tyr161G1n Tyr161G1u Tyr161G1y Tyr161His Tyr161I1e Tyr161Leu Tyr161Met Tyr161Pro Tyr161Ser Tyr161Thr Tyr161Va1 Loop 4 - Double Mutation Variants Ser159G1u + G1y155Asn Ser154Asp + G1y157G1n Ser154G1u + I1e159His G1y152G1n + G1y157Rsp Ser154G1u + G1y155Ser Asn153Ser + Ser154G1u A1a156Asn + Ser160G1u A1a156Thr + Ser158G1u Asn153G1u + G1y157Ser G1y155Asn + A1a156Asp I1e159G1y + Tyr161G1y G1y155Ser + Tyr161G1u Ser159Asp + G1y155Pro G1y155Asp + A1a156Ser G1y152G1u + Rsn153Ser Ser158G1u + I1e159Asn Asn153Asp + A1a156His Ser154G1u + Tyr161Cys I1e159Leu + Tyr161Asn G1y157Asp + I1e159G1y Ser158Asp + I1e159G1y G1y152Pro + G1y155G1u G1y155G1n + Ser160Asp G1y152Asp + Tyr161Met Ser154G1u + G1y155G1n G1y155G1u + Tyr161A1a A1a156Ser + Ser160Asp A1a156G1u + Tyr161Leu G1y152Pro + G1y157Asn Ser158G1u + I1e159Ser G1y157Asp + Tyr161Thr Ser158Asp + I1e159Cys G1y155Pro + Tyr161A1a G1y152Asp + Tyr161A1a ~"~' WO 95/30011 PCTlUS95104760 A1a156His + I1e159Asn Ser154Asp + Alal~~6His G1y157Pro + Serl_'s8Asp Asn153Ser + Ilel_'~9Cys Ser159Asp + Ala1_'~6Ser Ser154G1u + TyrlEilAla I1e159Leu + TyrlEilAla G1y152G1u + G1y155G1n A1a156Pro + Glyl_'i7Glu to Asn153Asp + TyrlEilHis A1a156G1n + Ilel_'i9Asn G1y152Pro + Serl_'i4Asp G1y155G1n + Alal_'~6Asp A1a156Ser + Ilel'_~9Gly Asn153Asp + Glyl_'i7Gln I1e159Leu + SerlEiOAsp G1y155G1u + I1e159G1n Ser160Asp + TyrlFilThr Ser158Asp + Ilel'_i9Gln Ser154G1u + Glyl_'i7Asn A1a156G1y + I1e159His A1a156Ser + Serl'_iBAsp Asn153G1n + Glyl_'iSGlu G1y155Asn + Serl_'iBGlu Ser158G1u + Ilel_'i9Pro Ser158Asp + Ile1_'i9Leu Loop 4 - Triple Mutation Variants A1a156Asn + G1y157Asn + Tyr161Cys G1y152G1n + G1y155G1u 1-Tyr161Asn G1y152G1n + Asn153G1n + Ser154G1u G1y155Ser + Ser158G1u + Tyr161G1n G1y152G1u + Asn153Ser ~-A1a156Ser G1y152G1u + I1e159Pro 1-Tyr161Asn G1y152Asp + G1y157G1n ~-Tyr161Met G1y155Asn + I1e159Thr -~-Tyr161His Asn153Ser + G1y155Ser ~-Ser158G1u G1y152Ser + G1y155Pro ~-Ser158G1u G1y152Pro + G1y157G1n ~-Ser160Asp G1y152G1n + Ser154Asp -~-Tyr161Met A1a156Pro + I1e159Asn ~-Tyr161I1e I1e159Asn + Ser160G1u a-Tyr161Ser G1y152Asp + I1e159A1a ~-Tyr161Cys Asn153G1n + I1e159A1a ~-Ser160G1u Asn153Asp + G1y155Pro ~-Tyr161Asn G1y155Asn + A1a156Thr ~-Ser158Asp G1y152G1n + Ser154Asp a-Tyr161G1n G1y157Ser + Ser158Asp ~-Tyr161Pro Ser154G1u + G1y155Ser ~-I1e159Cys G1y155G1n + A1a156Asn ~- Ser158Asp G1y155Asn + G1y157Asn + I1e159A1a W095/30011 ~~ PCT/US95104760 G1y157Asn + Ser158G1u + I1e159His Asn153Ser + I1e159Leu + Ser160G1u A1a156G1n + G1y157Asp + Tyr161Asn G1y155Asn + A1a156His + Tyr161Va1 Asn153G1n + A1a156Asp + I1e159G1y A1a156His + Ser160G1u + Tyr161His A1a156G1n + Ser160Asp + Tyr161A1a G1y152Asp + G1y157G1n + I1e159G1y Asn153G1u + A1a156Pro + I1e159A1a l0 G1y152G1n + G1y155Asn + A1a156Thr Asn153G1n + I1e159Met + Ser160G1u G1y152Asp + A1a156Pro + Tyr161Cys G1y152Ser + Ser154G1u + Tyr161Va1 G1y155Asp + G1y157Ser + Tyr161Asn Asn153G1u + Ser159Asp + G1y155Ser Asn153G1u + Ser154Asp + A1a156Pro G1y157Asn + Ser160Asp + Tyr161Asp G1y152G1u + Asn153Asp + Tyr161Met G1y152Asp + Asn153Asp + I1e159Leu A1a156Asp + G1y157G1u + Tyr161Thr Asn153Ser + Ser154G1u + G1y155Asp Ser154Asp + G1y155Asp + A1a156Pro Asn153Ser + Ser159Asp + G1y155G1u Ser154G1u + G1y155Asp + Tyr161A1a G1y157Asp + Ser158G1u + Tyr161Met G1y155G1n + G1y157Asp + Ser158G1u A1a156His + G1y157G1u + Ser158Asp G1y155Asp + A1a156G1u + G1y157Asp Ser154Asp + A1a156G1u + Tyr161Cys Ser154Asp + A1a156G1u + I1e159Pro Ser154Asp + A1a156G1u + I1e159Asn G1y152Pro + A1a156G1u + Ser158G1u G1y155Asn + A1a156G1u + Ser158G1u Asn153G1u + G1y155Asp + I1e159Pro G1y152G1u + I1e159Cys + Ser160Asp G1y152G1u + Ser154G1u + G1y157G1n G1y152G1u + Ser154G1u + G1y155Asn 4o Loop 4 - Quadruple Mutation Variants A1a156Pro + I1e159Pro + Ser160G1u + Tyr161Thr Asn153G1n + G1y157Asn + I1e159G1n + Ser160Asp Asn153G1n + A1a156G1y + G1y157G1u + I1e159Asn G1y152Asn + I1e159G1n + Ser160Asp + Tyr161Met G1y152Pro + Ser159G1u + G1y157Pro + I1e159A1a A1a156Ser + G1y157Ser + I1e159G1n + Tyr161G1u Asn153Ser + G1y157Ser + I1e159A1a + Ser160Asp G1y155G1n + Ser158Asp + I1e159G1y + Tyr161Met G1y152Ser + Ser154Asp + A1a156G1n + I1e159Cys SO A1a156His + G1y157Pro + Ser158G1u + Tyr161Met A1a156Pro + G1y157Asn + I1e159Met + Ser160Asp Ser154G1u + G1y155Asn + I1e159Met + Tyr161A1a G1y152Ser + A1a156His + Glyl.'>7Pro+ I1e159G1y Asn153Ser + I1e159Leu + SerlEiOGlu+ Tyr161Thr G1y152Asn + Ser154Asp + Alal'.>6Ser+ G1y157Asn Ser154Asp + G1y155G1n + I1e159Leu + Tyr161Thr Asn153Ser + Ser154Asp + G1y155Asn + I1e159Met G1y152Asn + Asn153G1n + G1y155Asn + I1e159Va1 Asn153G1u + Ser159G1u + G1y157Asn + I1e159Leu Asn153G1u + Ser154Asp + G1y155Pro + A1a156Ser Ser154Asp + G1y155Asp + A1a156G1y + G1y157Asn G1y152Ser + G1y157Asp + Ser158Asp + I1e159Ser G1y155Pro + G1y157Asp + Ser158Asp + I1e159Pro A1a156G1u + G1y157G1u + Ser158G1u + Tyr161I1e Asn153G1u + Ser154Asp + G1y155Asp + A1a156Pro Ser154G1u + A1a156G1u + G1y157Ser + I1e159Ser A1a156G1u + G1y157Asn + Serl.'~8Asp+ I1e159Asn G1y152Asn + Asn153Asp + G1y155G1u + Tyr161G1n G1y152Pro + Asn153Asp + Glyl.'~SGlu+ G1y157G1n G1y152Asp + A1a156Pro + I1e159Va1 + Ser160Asp G1y152Asp + Asn153G1n + Ilel.'~9Ala+ Ser160Asp G1y152G1u + Ser154G1u + I1e159G1n + Tyr161Cys G1y152G1u + Ser154Asp + Glyl!~7Pro+ Tyr161Leu G1y155Asp + A1a156Asp + Glyl.'~7Pro+ Ser158Asp G1y155G1u + A1a156Asp + Glyl.'~7Gln+ Ser158Asp G1y152Asp + Asn153G1u + Ilel.'~9Thr+ Ser160G1u G1y152G1u + Asn153Asp + Ilel.'~9Leu+ Ser160G1u A1a156Thr + Ser158Asp + Ser160G1u + Tyr161Cys Ser158G1u + I1e159Met + Ser160Asp + Tyr161Pro Ser158G1u + I1e159Cys + Ser160G1u + Tyr161G1y G1y155Pro + Ser158G1u + I1e159G1n + Ser160Asp G1y157Pro + Ser158Asp + Ser160G1u + Tyr161Met G1y152G1u + Ser154G1u + G1y157Asn + Ser160G1u G1y152G1u + Ser154Asp + G1y157G1n + Ser160Asp G1y152G1u + Ser154Asp + G1y157Ser + Ser160G1u G1y152G1u + A1a156G1y + Ser158Asp + Ser160G1u G1y152Asp + Ser158Asp + I1e159Leu + Ser160G1u G1y152Asp + G1y155G1n + Ser158Asp + Ser160G1u A1a156G1u + Ser158G1u + Ser1~50Asp+ Tyr161G1y G1y157Asp + Ser158G1u + I1e159His + Ser160G1u G1y152Asn + Ser159G1u + Glyl.'~5Asp+ Ser158Asp Asn153G1u + Ser154Asp + Glyl:~7Pro+ Ser160G1u Asn153Asp + Ser154Asp + Ilel.'~9Ser+ Ser160G1u Asn153Asp + Ser154Asp + Serl~oOAsp+ Tyr161I1e G1y152Asn + Ser154G1u + Glyl~7Glu + Ser158Asp G1y152G1u + A1a156Asp + Ser1:58G1u+ I1e159Va1 Asn153Asp + Ser154G1u + A1a1:56Ser+ G1y157Asp Asn153G1u + Ser154G1u + G1y1.55Ser+ G1y157Asp Ser154G1u + G1y155Asp + G1y1.57Pro+ Ser160Asp G1y152Asp + Asn153G1u + Ser158G1u + I1e159A1a so TABLE 19 Loop 5 - Single Mutation Variants A1a181Asn 218942' A1a181Asp A1a181G1n A1a181G1u A1a181G1y s A1a181His A1a181Pro A1a181Ser A1a181Thr Ser182Asp 10 Ser182G1u Phe183A1a Phe183Asn Phe183Asp Phe183Cys 15 Phe183G1n Phe183G1u Phe183G1y Phe183His Phe183I1e 20 Phe183Leu Phe183Met Phe183Pro Phe183Ser Phe183Thr 2s Phe183Tyr Phe183Va1 Ser184Asp Ser184G1u G1n185Asn 30 G1n185Asp G1n185G1u G1n185Ser 3s Loop 5 - Double Mutation Variants A1a181Asp + Phe183G1n Ser182Asp + G1n185Asn Phe183Met + G1n185G1u Ser182G1u + G1n185Asn 40 A1a181Pro + Ser182G1u A1a181Asn + G1n185G1u Ser182G1u + Phe183Leu A1a181Pro + G1n185Asp Phe183Ser + G1n185G1u 4s A1a181G1n + Ser182G1u Phe183I1e + G1n185G1u A1a181Ser + G1n185Asp A1a181G1n + Phe183Ser A1a181Thr + Phe183Asn A1a181G1y + G1n185Asp A1a181His + Ser182G1u Phe183G1n + G1n185Ser '"~' WO 95130011 PCTlUS95104760 Phe183Pro + G1n185Ser A1a181Asn + Ser18.2Asp A1a181Ser + G1n185G1u A1a181Asn + Phe183Asn s A1a181His + Phe183Asp A1a181Asn + Ser182G1u Ser182Asp + Phe183His A1a181Asp + Phe183I1e Phe183Leu + G1n185Asp Ser182G1u + Phe183Ser A1a181His + G1n185Asp Phe183Va1 + G1n185Asp Ser182Asp + Phe183Thr Phe183G1y + G1n185Asp is A1a181Thr + G1n185G1u A1a181His + G1n185G1u Ser182Asp + Phe183Va1 A1a181Asp + Phe183Ser Ser182Asp + Phe183Cys A1a181Pro + Phe183Asp A1a181G1y + Phe183A1a A1a181Pro + Phe183Asn Phe183Pro + G1n185Asp Ser182G1u + Phe183Tyr 2s Phe183Cys + G1n185Ser A1a181Thr + Ser182G1u A1a181Pro + Phe183A1a Ser182G1u + Phe183Thr A1a181Pro + Phe183Pro Ser182G1u + G1n185Ser A1a181G1n + Ser182Asp Phe183G1y + G1n185G1u A1a181G1n + G1n185Asp Phe183Ser + G1n185Asp 3s Ser182Asp + Phe183Met Phe183A1a + G1n185Asn A1a181Asp + Phe183His Phe183Va1 + G1n185G1u A1a181Ser + Ser182G1u A1a181Asn + Phe183Pro Phe183Tyr + G1n185G1u A1a181G1u + Phe183Cys Ser182G1u + Phe183I1e 4s TABLE 21 Loop 5 - Triple Mutation Wariants A1a181G1u + Phe183Va1 + G1n185Ser A1a181Pro + Ser182Asp + G1n185Asn A1a181Thr + Phe183His + G1n185Asp Ser182G1u + Phe183A1a + G1n185Asn A1a181Thr + Ser182Asp + G1n185Asn A1a181G1n + Ser182Asp + Phe183A1a ~18942'~

Ser182Asp + Phe183Asn + G1n185Asn A1a181G1n + Phe183G1n + G1n185G1u Ser182Asp + Phe183Met + G1n185Asn A1a181Pro + Ser182G1u + Phe183G1y A1a181His + Phe183A1a + G1n185Ser A1a181Ser + Ser182G1u + G1n185Asn A1a181G1y + Phe183I1e + G1n185G1u A1a181Asn + Phe183Tyr + G1n185Asn Ser182Asp + Phe183Leu + G1n185Asn l0 Ser182Asp + Phe183Va1 + G1n185Asn A1a181G1u + Phe183Leu + G1n185Ser A1a181G1u + Phe183Tyr + G1n185Ser A1a181G1n + Ser182Asp + Phe183G1y A1a181Thr + Phe183I1e + G1n185Ser A1a181G1n + Ser182G1u + G1n185Ser A1a181G1n + Ser182G1u + Phe183His A1a181Asp + Phe183A1a + G1n185Asn A1a181Thr + Ser182Asp + Phe183Thr A1a181Ser + Phe183Pro + G1n185G1u Ser182G1u + Phe183A1a + G1n185Ser A1a181Asn + Phe183Cys + G1n185Ser A1a181Pro + Phe183Thr + G1n185Asp A1a181Thr + Phe183Va1 + G1n185Asn A1a181G1n + Ser182Asp + G1n185Ser A1a181Asn + Ser182Asp + G1n185Ser Ser182Asp + Phe183Thr + G1n185Ser A1a181Ser + Phe183Asp + G1n185Asn Ser182Asp + Phe183Thr + G1n185Asn A1a181G1y + Phe183Asn + G1n185Ser A1a181Asp + Phe183Met + G1n185Ser A1a181Asp + Phe183Pro + G1n185Asn A1a181G1y + Ser182Asp + Phe183G1y A1a181G1n + Ser182G1u + G1n185Asn A1a181Thr + Ser182Asp + G1n185Ser A1a181Asn + Phe183Asn + G1n185Asp A1a181Thr + Phe183Asp + G1n185Ser A1a181G1n + Phe183Ser + G1n185Ser Ser182Asp + Phe183G1y + G1n185Ser A1a181G1y + Ser182G1u + Phe183Leu A1a181Asn + Ser182G1u + G1n185Asn A1a181G1u + Ser182G1u + Phe183Ser A1a181G1u + Ser182G1u + Phe183G1y A1a181G1u + Ser182G1u + Phe183Leu A1a181G1u + Ser182G1u + Phe183Va1 A1a181G1u + Ser182Asp + Phe183Met A1a181Asp + Ser182Asp + Phe183Met A1a181Asp + Ser182Asp + Phe183Cys A1a181G1u + Ser182Asp + G1n185Asn A1a181Asp + Ser182Asp + G1n185Asn A1a181G1u + Ser182G1u + Phe183Asn A1a181Asp + Ser182G1u + Phe183Ser A1a181G1u + Ser182Asp + G1n185Ser A1a181G1u + Ser182G1u + Phe183Thr A1a181G1u + Ser182Asp + Phe183Leu WO 95/30011 ~~ ~ PCT/US95/04760 Loop 5 - Quadruple Mutation Variants A1a181Ser + Ser182Asp + PhelE33Al~+ G1n185Ser A1a181Ser + Ser182G1u + PhelE33Pro+ G1n185Ser A1a181G1y + Ser182Asp + Phe183G1y + G1n185Asn A1a181G1y + Ser182G1u + Phe183Pro + G1n185Ser A1a181Thr + Ser182G1u + Phe183Thr + G1n185Ser A1a181G1y + Ser182G1u + Phe183Thr + G1n185Ser A1a181His + Ser182G1u + Phe183His + G1n185Asn A1a181G1y + Ser182G1u + Phe183Ser + G1n185Ser A1a181G1n + Ser182G1u + PhelE33His+ G1n185Asn A1a181Asn + Ser182G1u + PhelE33Cys+ G1n185Ser A1a181Pro + Ser182G1u + Phel~~3Met+ G1n185Ser A1a181G1n + Ser182Asp + Phel~i3Gln+ G1n185Asn A1a181Thr + Ser182Asp + Phe183His + G1n185Asn A1a181G1y + Ser182G1u + Phe183Leu + G1n185Ser A1a181Asn + Ser182Asp + Phe183I1e + G1n185Ser A1a181G1y + Ser182Asp + Phe183A1a + G1n185Ser A1a181Asn + Ser182Asp + Phe183Ser + G1n185Asn A1a181Asn + Ser182G1u + Phe18~3G1y+ G1n185Ser A1a181Asn + Ser182G1u + Phe183Met + G1n185Asn A1a181His + Ser182G1u + PhelE~3Asn+ G1n185Asn A1a181G1y + Ser182G1u + Phe183Tyr + G1n185Asn A1a181Asn + Ser182Asp + PhelE'3Asn+ G1n185Asn A1a181G1u + Ser182G1u + PhelB'3Asn+ G1n185Asn A1a181Asp + Ser182G1u + Phe183I1e + G1n185Asn A1a181G1u + Ser182Asp + Phe183G1n + G1n185Asn A1a181Asp + Ser182Asp + Phe183His + G1n185Asn A1a181G1u + Ser182Asp + Phe183Ser + G1n185Asn A1a181G1u + Ser182Asp + Phe183Leu + G1n185Asn A1a181G1u + Ser182Asp + Phe183Leu + G1n185Ser A1a181Asp + Ser182G1u + Phe183Thr + G1n185Ser A1a181Asp + Ser182Asp + Phe183G1n + G1n185Asn A1a181Asp + Ser182Asp + Phe183A1a + G1n185Asn A1a181Asp + Ser182G1u + Phe183A1a + G1n185Asn A1a181G1u + Ser182Asp + Phe183Met + G1n185Ser A1a181G1u + Ser182G1u + Phe183Thr + G1n185Asn A1a181Asp + Ser182Asp + Phe183G1y + G1n185Ser A1a181G1u + Ser182Asp + Phe183A1a + G1n185Asn A1a181His + Ser182G1u + Phe183G1u + G1n185Asn A1a181Ser + Ser182Asp + Phe183G1u + G1n185Ser A1a181Ser + Ser182Asp + Phe183Asp + G1n185Ser A1a181Pro + Ser182G1u + Phe183Asp + G1n185Ser A1a181Asp + Ser182Asp + Phe183Asp + G1n185Asn A1a181G1u + Ser182Asp + Phe183G1u + G1n185Ser A1a181Ser + Ser182Asp + Phe183G1u + G1n185G1u A1a181G1y + Ser182G1u + Phe183G1u + G1n185G1u A1a181G1n + Ser182G1u + Phe183Asp + G1n185Asp A1a181Ser + Ser182Asp + Phe183Asp + G1n185Asp A1a181Thr + Ser182Asp + Phe183Asp + G1n185Asp A1a181Pro + Ser182Asp + Phe183G1u + G1n185Asp WO 95!30011 ~ PCT/US95/04760 A1a181Asn + Ser182G1u + Phe183Asp + G1n185Asp A1a181Thr + Ser182Asp + Phe183Asp + G1n185G1u A1a181Pro + Ser182G1u + Phe183G1u + G1n185G1u A1a181Asn + Ser182Asp + Phe183Asp + G1n185G1u A1a181Thr + Ser182G1u + Phe183Asp + G1n185Asp A1a181His + Ser182G1u + Phe183G1u + G1n185G1u A1a181G1u + Ser182G1u + Phe183Ser + G1n185G1u A1a181G1u + Ser182G1u + Phe183Va1 + G1n185G1u A1a181Asp + Ser182G1u + Phe183Pro + G1n185G1u A1a181G1u + Ser182G1u + Phe183Thr + G1n185Asp A1a181Asp + Ser182Asp + Phe183Cys + G1n185G1u Loop 6 - Single Mutation Variants Va1193A1a Va1193Asn Va1193Asp Va1193Cys Va1193G1n Va1193G1u Va1193G1y Va1193His Va1193Met Va1193Pro Va1193Ser Va1193Thr A1a194Asn A1a194Asp A1a194G1n A1a199G1u A1a194G1y A1a194His A1a194Pro A1a194Ser A1a194Thr Pro195Asn Pro195Asp Pro195G1n Pro195G1u Pro195G1y Pro195Ser G1y196Asn G1y196Asp G1y196G1n G1y196G1u G1y196Pro G1y196Ser Va1197A1a Va1197Asp Va1197Cys Va1197G1n Va1197G1u ~~8~4~'~

Va1197G1y Va1197His Va1197Met Va1197Pro Va1197Ser Va1199A1a Va1199Asn Va1199Asp Va1199Cys 10 Va1199G1n Va1199G1u Va1199G1y Va1199His Va1199Met 15 Va1199Pro Va1199Ser Va1199Thr G1n200Asn G1n200Ser 20 Ser201Asp Ser201G1u Thr202Asn Thr202Asp Thr202G1n 25 Thr202G1u Thr202G1y Thr202Pro Thr202Ser Tyr203A1a 30 Tyr203Asn Tyr203Asp Tyr203G1n Tyr203His Tyr203I1e 35 Tyr203Met Tyr203Pro Tyr203Ser Pro204Asn Pro204Asp 40 Pro204G1n Pro209G1u Pro209G1y Pro209Ser G1y205Asn 45 G1y205Asp G1y205G1n G1y205G1u G1y205Pro G1y205Ser SO Ser206Asp Ser206G1u Thr207Asn Thr207G1n Thr207G1u z~~94z~

Thr207G1y Thr207Pro Thr207Ser Tyr208A1a Tyr208Asn Tyr208Asp Tyr208Cys Tyr208G1n Tyr208G1u Tyr208G1y Tyr208His Tyr208I1e Tyr208Leu Tyr208Met Tyr208Pro Tyr208Va1 A1a209Asn A1a209Asp A1a209G1n A1a209G1u A1a209G1y A1a209His A1a209Pro A1a209Ser A1a209Thr Ser210Asp Ser210G1u Leu211A1a Leu211Asn Leu211Asp Leu211Cys Leu211G1n Leu211G1y Leu211His Leu211I1e Leu211Met Leu211Pro Leu211Ser Leu211Thr Leu211Va1 Asn212G1u G1y213Asn G1y213Asp G1y213G1n G1y213G1u G1y213Pro G1y213Ser Thr214Asn Thr214Asp Thr214G1n Thr214G1u Thr214G1y Thr214Pro Thr214Ser w° WO 95/30011 PCTIUS95/04760 Loop 6 - Double Mutation Variants G1y196Asn + A1a209Asp Va1199Ser + Tyr208Asn Pro195Asn + Thr207Asp Va1197A1a + Asn27_2Asp Asn198G1n + Thr207Ser Va1199G1y + Tyr208I1e G1y196Asn + A1a209Pro Va1199G1y + G1n200Ser Pro195Ser + G1y205G1n Va1199Ser + Leu2l.lAsp G1y196G1n + Va1197Asn Thr202Asn + Thr21.4G1u A1a194Thr + Thr2C)2Pro Va1199Asn + Ser2l.OGlu Thr202Ser + Asn21.2G1n Ser210Asp + Thr21.4Asn Pro195G1y + Asn21.2Asp Asn198G1n + Tyr208G1n Va1197Asn + Thr21.4G1u Tyr208Asp + Leu21.1G1n Thr202Ser + Ser2l.OGlu Va1197Cys + Ser2lOGlu G1n200G1u + Tyr203G1y A1a209Thr + Leu21.1G1u Va1197Ser + G1n200G1u Thr202G1y + Ser210G1u G1n200Ser + G1y213Pro G1y196Ser + Thr207G1y Pro204G1n + Ser210G1u Va1199Cys + Pro209G1y G1y213Pro + Thr219Pro G1y196Asn + Ser210G1u Thr207G1n + Ser210G1u Va1199Met + G1n200Asp Thr202Ser + Tyr2C3Ile Pro195Ser + Asn212Ser Va1197G1u + Tyr2C8Gln Asn198G1u + Leu211Cys G1y196G1n + Ser206G1u A1a194Ser + Thr214Ser Va1197Ser + Tyr203A1a Ser210Asp + G1y213Pro Tyr203His + G1y213Asp Va1197Ser + Va1199Ser A1a209Pro + Ser210Asp Thr207Asp + Thr214Ser SO Thr207G1y + A1a209His Pro195Asn + Asn198Asp Va1197Pro + Ser206Asp as G1n200Ser + Asn212Asp Va1197Thr + Asn212Asp G1n200Asn + Thr214G1u A1a209Asp + Thr214Ser A1a209Asn + Asn212Ser A1a194Ser + Asn212G1u Va1197G1u + G1y213Asn Tyr203Cys + Asn212Asp Pro195G1n + Va1197Cys Asn198Ser + Tyr208A1a G1y205G1n + A1a209G1u G1y205Ser + Leu211Ser G1y205G1n + Tyr208A1a A1a194Ser + Asn198Asp A1a194Thr + Pro195Ser Va1199Thr + Pro209G1u Thr202G1n + Ser210Asp Ser206G1u + Leu211His Asn198G1u + Thr202Asn Ser210Asp + G1y213Ser G1y196Ser + Thr202G1n Pro204G1n + G1y213Asp Asn198G1u + Pro204G1y Thr202G1y + Tyr208Met Pro195G1y + G1y213G1u Pro195Ser + Pro204Asp Pro195Ser + Tyr203Thr A1a194Asn + Tyr203Pro A1a194Pro + G1n200Asp Pro209Asp + G1y205Asn G1y196Pro + Asn198G1n Thr207Ser + Leu211G1y Pro195Ser + Leu211I1e Pro209G1u + Leu211G1y Va1199Ser + Tyr208A1a G1y196Ser + Ser210Asp A1a194Thr + Leu211G1y Thr207Ser + Ser210G1u G1y196Pro + Thr207G1u Pro204G1u + Leu211Met Pro195Asn + Asn198Ser Tyr203Met + Thr207G1n Pro204G1y + A1a209G1u Va1197G1u + Asn212Ser Pro195Ser + A1a209Pro G1y196Ser + Leu211G1y G1y205Pro + Leu211Met Va1197G1n + Pro204Ser Va1199Asn + Ser206Asp Va1197Asp + Thr202Asn G1y196G1n + Thr219G1y Pro204Asn + Asn212G1n Va1199Asn + G1n200Asn Pro195G1y + Leu211G1y ~"~ WO 95/30011 PCT/US95/04760 Tyr203Cys +Pro204Ser G1n200Asp +Leu2:11Cys Asn198G1n +Ser2:LOGlu A1a199G1y +G1n200G1u G1y196G1n +Pro21)4Glu Va1197Pro +Thr202Asn A1a199G1y +Va1199A1a A1a194Thr +Leu2:llAsn Asn198G1n +G1y205Asp Tyr203Asn +Leu2:llHis Asn212Asp +Gly2:L3Gln Asn198G1n +Ser206Asp Va1199Thr +G1n200G1u Thr202Ser +Asn2:L2Asp A1a194G1y +Ser2:lOGlu Ser206G1u +A1a209Asn A1a209Asp +Asn2:L2Ser Va1199G1y +Asn2:L2Ser A1a194Pro +Gly2:l3Asp G1n200Asp +Pro204G1y Va1197Thr +Tyr203A1a Asn198G1n +Leu211Pro Pro195G1n +Leu2:.lGln G1y196G1n +Thr2:.4G1u Pro195Asn +Ser2J.OGlu Thr207Asn +Leu2J.lAsp Ser210G1u +G1y213Pro G1y205Pro +Thr207Asp G1n200G1u +Thr202G1n Thr202Ser +Gly2J.3Asp Pro204G1n +Thr2J.4Gln G1y196Ser +G1n200Asp Va1197Ser +G1n200Asp Thr202Pro +Thr207Asp Tyr203Thr +Tyr208His Pro195G1n +Thr202Asn Va1197Asn +Asn2J.2Asp Va1197Met +Thr202G1n Va1199Met +Thr202Pro A1a209G1u +Leu2J.lMet G1y196Asn +Va1199His A1a194G1y +Ser206G1u G1y196Asn +Pro204Asn G1y196Asn +G1n200G1u Va1197Ser +Ser2J.0Asp Pro195G1n +Pro204G1n A1a194His +Asn198G1n Va1197Cys +Asn2J.2Gln Asn198G1u +A1a209Ser A1a194G1n +Vall~a9Pro Asn198Asp +Va11~j9Gln Asn198Asp +Thr27.4Pro Pro204Asn +Tyr208Asp Tyr203Ser +Tyr208Leu 218 9 .4 2 7 PCTIUS95J04760 G1y196G1n + A1a209Asp Ser206G1u + Asn212G1n Thr207Ser + A1a209His Va1197Thr + Ser210Asp 5 Tyr208G1n + Ser210Asp Asn198Ser + Ser210Ghu G1y196Ser + Thr214Asp A1a194G1n + Thr202Asn Pro195G1y + Thr214Asn 10 Leu211Thr + Thr214Asp Pro195Ser + Asn198G1u G1y196Ser + G1y205Ser A1a199His + Thr202Asn Thr207G1y + Tyr208Met 15 A1a194Ser + Va1197His Loop 6 - Triple Mutation Variants A1a194Thr + Ser206G1u + Tyr208Cys 20 G1y196Asn + Tyr203Pro + A1a209G1y Pro195G1y + Asn198Ser + Thr207G1u Pro195G1y + G1y205G1n + G1y213Ser Tyr203Leu + Pro209G1n + G1y205G1n G1y196G1n + Va1197Asn + Ser206Asp 25 Leu211Ser + G1y213Ser + Thr219G1n Pro195Asn + Pro209G1u + G1y205G1n Asn198Asp + Thr202G1y + G1y213G1n Tyr203A1a + Leu211His + Thr214G1n A1a194Thr + Va1197Ser + Va1199G1y 30 A1a194G1n + Thr207Asp + Thr214Ser G1y196Asn + Pro204G1n + A1a209Asn Thr202Pro + Tyr208Asp + Leu211Thr Va1197G1y + A1a209G1n + Asn212G1n G1y196Ser + G1y205Asp + Leu211I1e 35 A1a199Ser + Va1197Pro + Asn198Asp G1y196Asn + Asn198G1n + Leu211Ser A1a194Thr + A1a209His + Thr214Asp A1a194Pro + Pro195Ser + Asn212Asp Thr202Ser + Tyr203G1n + Ser210Asp 40 Thr202Ser + Tyr203Leu + Thr214G1u A1a194Ser + Pro195G1y + Leu211Va1 A1a194Pro + Thr207G1u + Leu211A1a G1y196Asn + Ser206G1u + Leu211G1y G1y196Ser + G1y205Ser + Ser210Asp 45 A1a194Thr + Va1197G1y + Leu211Ser Pro204G1u + A1a209His + Thr214Pro G1y196Ser + G1y205Asp + Leu211His Va1197Cys + Thr202G1y + Thr207G1n Asn198Ser + Pro204G1y + G1y213Asp 50 A1a194G1y + A1a209Pro + Ser210Asp G1y196Pro + Tyr203Va1 + Tyr208Cys Va1199A1a + Thr207Asn + Tyr208Va1 "' WO 95130011 PCT/US95104760 ~~~'g4~~

Tyr203Cys +Ser210Asp + Leu211Pro A1a194Ser +Va1197Ser + G1n200Asp A1a194Asn +Pro195G1y + Leu211I1e Va1199Ser +Leu211Asn + G1y213Asp A1a194Asn +Ser206G1u + Thr214G1y A1a194G1y +Pro195G1y + V~1197Asp Pro195G1n +A1a209Ser + Ser210Asp Va1197Thr +Asn198G1n + Tyr203Leu Va1197A1a +Thr202G1n + Ser206G1u G1y196Pro +Pro204G1y + Asn212G1u Thr202G1y +Pro204G1u + Tyr208Ser G1n200Ser +Thr202Pro + Pro204Ser G1n200G1u +Tyr203Thr + Pro204Asn Asn198Asp +Va1199G1n + Pro204Asn A1a194Pro +Pro195G1n + Va1199G1y Pro195Asn +Pro204Asn + Ser206G1u Thr202Asn +Leu211Thr + Asn212G1n G1y196G1n +Thr202Pro + G1y213Asp Asn198G1u +G1n200Asn + Leu211G1y Va1199G1y +Thr202G1n + G1y213G1u Tyr203Ser +Thr207Pro + Ser210Asp Pro195G1y +G1y196G1n + Va1197G1u Pro195G1y +Thr207G1y + Tyr208Ser Thr202G1y +Leu211G1u + G1y213G1n Va1197G1y +Thr202Pro + Asn212G1u G1y196G1n +G1y205G1n + Ser206G1u Pro195G1y +Va1199Thr + Thr214G1u G1y196Ser +G1n200Ser + Leu211Asp G1y196Asn +Tyr208G1u + Thr219Pro Tyr203Thr +Pro204Asn + Ser206G1u Va1199Thr +Thr202Asn + Tyr208G1y Thr202Pro +Tyr203Thr + Pro204G1u G1y196G1n +Asn212Asp + Thr214G1n Pro195G1y +Thr202G1y + Asn212Ser Pro195G1y +Tyr208G1n + Ser210Asp A1a194Pro +Ser206G1u + Leu211Ser A1a194His +Pro204Asn + G1y213G1n G1y196Ser +Tyr208Met + Ser210Asp Va1197G1u +Va1199Ser + Tyr203Asn Thr202G1y +A1a209Asn + Asn212Asp Va1197Ser +Ser210Asp + G1y213Pro A1a194Ser +Tyr208I1e + Asn212G1u A1a194G1y +Asn198G1n + A1a209His A1a199Ser +Thr207Pro + G1y213G1u Va1199Thr +Leu211Asp + Asn212Ser Va1199Pro +Thr202Ser + Leu211Asn Ser206G1u +Tyr208Cys + Thr219Asn A1a209Ser +Leu211I1e + Asn212G1u Asn198G1u +Tyr208A1a + A1a209Ser A1a194Pro +Asn198Asp + G1y213G1n A1a199Asn +Pro195Ser + Leu211G1u G1y196Pro +Pro204G1y + G1y205Pro Tyr203I1e +Tyr208Thr + A1a209G1u G1y196G1n +Thr202G1n + Asn212Asp A1a194Asn + Va1197Pro + Thr202Pro A1a194G1n + Va1199G1y + A1a209Asp A1a194Ser + Pro195G1y + Asn212G1n Pro195G1n + Tyr203Pro + Ser210Asp Va1197Thr + Thr202G1n + Tyr208Asn A1a194Thr + G1y196Ser + Leu211Asn A1a194G1y + Thr202Ser + Pro204Asp A1a199G1y + G1y205G1u + G1y213Ser Va1199Pro + Thr207Pro + Tyr208Met 1p A1a194Pro + Asn198G1u + A1a209His Tyr208Leu + Ser210G1u + Thr214Pro G1y196Asn + Va1199A1a + G1y205Asp G1y196Pro + G1y205G1u + Tyr208Ser Pro195Asn + G1y196G1n + Va1197Cys Asn198Ser + G1n200G1u + Leu211Asn Thr202Ser + Tyr203Cys + Thr214G1n A1a194Ser + G1y196G1n + Thr207Asp G1y196Ser + G1y205G1u + Tyr208I1e Tyr203Met + G1y205Asn + G1y213Pro Pro204G1n + G1y205Asp + A1a209His G1y205Asn + Leu211Asp + Thr214Ser Tyr203Met + A1a209His + Leu211Met Va1199Asn + Thr202Ser + G1y213G1u Va1197Thr + Pro209Asp + Asn212G1n A1a194G1y + Ser210G1u + Leu211Pro Va1197His + A1a209G1y + Ser210Asp G1n200G1u + Thr202G1n + Tyr208His A1a199Ser + Ser206Asp + Leu211A1a Pro195Ser + Asn198Asp + G1y205Pro Tyr203Pro + Thr207Asp + Thr214Pro Pro195Ser + Va1199His + Leu211Pro Tyr203Met + Thr207G1u + A1a209His Va1197Cys + G1n200Asn + Ser206G1u Va1197G1u + Thr202G1n + Tyr208His Pro195G1n + Va1197Cys + Pro204G1y Va1199Pro + G1y205Ser + G1y213Asp A1a199Thr + Thr207Ser + A1a209G1u Va1197Met + G1y205Pro + Tyr208G1y G1y196Ser + G1y205G1u + A1a209Thr Pro195Ser + Va1199Thr + G1y205Ser A1a194Ser + Asn198Asp + G1y213Ser Asn198G1u + Va1199Ser + Thr207Pro A1a194Thr + G1y196Pro + Ser210G1u G1y196Asn + Thr202Pro + Leu211Met Tyr203Met + Asn212Asp + Thr214Ser Tyr203G1y + A1a209Ser + Ser210Asp A1a194Ser + Thr207Ser + Leu211Pro Tyr208Cys + G1y213Asn + Thr214Pro Va1197Met + Pro204G1u + G1y213Ser G1n200Ser + Tyr203A1a + Pro204Asp Pro195G1y + Ser206Asp + Leu211I1e Tyr203G1y + Tyr208Asp + Leu211Asn Va1197Thr + Ser206G1u + Thr214Pro Tyr203A1a + Pro204Asn + G1y213G1u ~"' WO 95/30011 PCTlUS95/04760 Tyr208I1e + A1a209G1y + Leu211G1n Asn198Ser + Pro209G1y + Asn212G1u A1a194G1n + Va1197Cys + Pro204Asn A1a209Thr + Leu211Ser + G1y213Pro G1y196Ser + Thr202Pro + Asn212Asp Va1197His + Tyr203G1n + Tyr208Cys Va1199Ser + Leu211Thr + G1y213Pro Pro195Asn + Va1197Asn + Thr202Pro Va1199Pro + Pro204G1y + G1y205Asn Pro195G1y + Asn198Asp + Thr214G1y G1n200G1u + Tyr203Ser + Asn212G1n Pro195G1y + Va1199G1n + Thr219Pro Thr202Ser + Pro209Ser + G1y213G1u Pro195G1y + Thr202Pro + G1y205G1u Thr202G1n + Tyr203G1y + A1a209Asn Pro195Asn + G1y196G1n + Asn212Rsp Thr202G1y + Tyr208I1e + Leu211Va1 Asn198G1u + Tyr203Cys + Pro204G1y A1a199Ser + Thr207Ser + Ser210Asp G1n200Asp + Thr202Pro + Tyr203A1a Va1199Cys + A1a209G1n + Ser210Asp A1a194Asn + Va1199Cys + Leu211Pro G1n200Ser + Ser210G1u + Leu211Asn Tyr203G1n + Thr207G1u + Tyr208Ser A1a194Thr + Thr202Asn + Thr207G1y Pro195G1y + G1y196G1n + Ser210Asp G1n200Ser + Pro204G1y + Ser206G1u A1a194Asn + G1y196G1n + Asn198G1u Va1197Thr + Pro204G1n + G1y205G1u Va1199A1a + G1n200Asp + G1y213Asn Va1197Met + Tyr203Va1 + G1y213Pro A1a194G1n + Tyr203Va1 + Thr214G1u Va1199Cys + A1a209His + Leu211Met Thr202G1n + Tyr203I1e + A1a209G1n A1a194G1y + Ser206G1u + Leu211G1y G1y196Pro + Tyr208G1y + Ser210G1u Va1197G1n + Thr202G1y + G1y205Asp Va1197Pro + G1y205Asp + Thr219Pro 4o TABLE 26 Loop 6 - Quadruple Mutation Variants Pro195Rsn + Va1197A1a + Thr207Ser + Asn212Asp Asn198G1n + Va1199G1y + Tyr208I1e + Asn212Asp Tyr203His + Leu211Pro + Asn212G1n + G1y213G1n A1a194His + Asn198Ser + Thr202Asn + A1a209Asp G1y196G1n + Asn198Ser + Thr202Ser + Pro209G1u Pro195G1n + Asn198G1n + G1y205G1u + A1a209Pro Va1199His + Pro204Asp + G1y205Pro + Leu211G1y G1y196G1n + G1y205Asn + Ser206Asp + Thr214Ser G1n200G1u + Thr202Pro + Leu211Asn + G1y213Asn G1y196G1n + Pro204Asn + Asn212G1n + Thr214G1y Pro195G1y + Va1199Asn + G1n200Asn + Leu211G1y WO 95130011 21 g 9 4 2 ~ PCT/US95I04760 Pro195G1n + Pro204Ser + G1y205G1u + Tyr208I1e Va1199A1a + Tyr203G1n + Thr207G1y + Ser210G1u A1a194His + Va1199Ser + Pro204Asn + G1y205G1u G1y196Pro + Asn198Asp + G1n200Asn + A1a209Pro Pro195G1n + G1n200Asp + Tyr203Asn + Leu211Cys Pro195Ser + Thr202Pro + Asn212Asp + Thr214Ser A1a194G1y + Va1197G1u + Tyr203Leu + Leu211Cys Asn198G1n + Thr202Pro + Tyr208Met + Thr214G1u G1y196Ser + Va1199Ser + G1y205G1n + Ser210G1u Tyr203A1a + G1y205G1n + Ser206Asp + Leu211G1n G1y196Ser + Va1199His + G1n200Asp + Thr207Ser Pro195G1n + G1y196G1n + G1n200G1u + Tyr203Ser G1y196Ser + G1n200G1u + G1y205G1n + G1y213Asn Pro195Ser + G1y196Asn + Ser206G1u + Thr214G1y G1y196Asn + Va1199Asn + Ser206G1u + Tyr208Pro Va1197G1n + G1y205Ser + Ser210G1u + Thr214Ser Pro195Asn + G1n200G1u + G1y205Asn + Leu211Pro Tyr203Pro + Tyr208His + Leu211Va1 + Thr219Pro A1a194His + Va1199Ser + Thr202G1y + Tyr208Asp Pro195Asn + Va1199Asn + Ser206Asp + G1y213Ser A1a199Ser + Pro195Asn + Va1199A1a + G1y205G1u A1a199Thr + G1y196G1n + Va1199G1y + Tyr203I1e Va1199A1a + A1a209Asp + Leu211Va1 + Asn212G1n Va1197G1y + Asn198Ser + Tyr203Leu + Ser210Asp G1n200Ser + Thr207G1y + Tyr208Thr + Ser210Asp Pro204G1y + G1y205Asp + Thr207Asn + Tyr208Leu A1a194G1n + Thr202Ser + A1a209Ser + Asn212G1u Va1197Met + Thr202G1y + Pro204G1y + Thr214G1u Va1199A1a + Pro209G1n + G1y205Asp + Tyr208G1y Asn198G1u + Tyr208Cys + A1a209G1n + Leu211Va1 G1n200Ser + Pro204Ser + A1a209Asn + Leu211G1u Pro195Ser + Va1197Thr + G1y205G1n + Thr219Pro Pro195Asn + Thr202Asn + G1y205Asp + Tyr208Ser Pro195G1y + Tyr203Pro + Leu211A1a + Asn212G1u Thr202G1y + Pro209Asn + G1y205Asn + G1y213G1n Va1197G1y + G1n200G1u + Tyr208A1a + Asn212Ser A1a199Ser + Pro195Ser + Tyr203A1a + A1a209Asp Pro195G1n + Va1197Thr + Va1199Pro + A1a209His G1y196Asn + Ser206Asp + Leu211His + Thr214Ser Va1197Asp + Tyr203His + Tyr208Cys + Thr219G1y A1a194G1n + Pro195Asn + A1a209His + Ser210Asp Va1199Ser + G1n200Ser + Tyr203Asn + Ser206Asp A1a194Asn + Pro195G1n + Ser206G1u + G1y213Ser Pro195Ser + Tyr203Met + Ser206Asp + G1y213G1n A1a199G1y + Va1199Thr + Tyr208His + G1y213Pro A1a194Pro + Va1197Cys + G1n200Asp + Pro204G1y Thr202G1n + Thr207G1u + Tyr208Cys + Leu211Pro G1y196G1n + Asn198G1u + Tyr203G1n + G1y205Pro Thr202Asn + Tyr203Pro + G1y205Ser + G1y213G1u Pro195Asn + Asn198Ser + A1a209G1y + Leu211Thr Asn198Asp + G1n200Ser + Tyr203Pro + G1y205Asn Pro195G1n + Va1199G1y + Tyr203Asn + Thr207G1u A1a194G1n + Thr202G1n + Tyr203His + Tyr208His A1a199Ser + Pro209Asp + Thr207Pro + Tyr208Thr Asn198G1n + Va1199G1n + G1W?OOAsn + A1a209G1n A1a194G1y + Asn198Ser + Pro?04Asp + A1a209His G1y196G1n + Va1197Thr + Sera?06G1u+ A1a209Pro G1n200Ser + Thr202Pro + Tyr~?03Thr+ Thr207G1.y S A1a194Thr + Va1197G1y + G1n200Asn + Thr207Asp Va1197Pro + Va1199Asn + G1n200G1u + Leu211I1.e G1y196Pro + Pro204Ser + A1a209Ser + Leu211G1n Pro195G1y + G1y196G1n + Asn7.98G1n+ Tyr208G1u G1n200Ser + Tyr203Met + G1y205G1u + Tyr208Asn 10 A1a194G1n + Pro195G1y + A1a209G1y + Ser210G1u G1y196Asn + Va1197Asn + Ser210Asp + Asn212G1n G1y196G1n + Thr202Ser + G1y205Ser + Ser210G1u G1n200Asn + G1y205Pro + Leu211Cys + Thr214Pro A1a194Ser + G1y196G1n + Thr207Ser + Ser210Asp 15 Va1197G1n + Thr202Pro + Tyr203A1a + Ser206Asp Glyl96Gln + Asn198G1u + Asn212Ser + G1y213Asn Pro195Asn + Va1197G1n + G1y205G1n + Leu211G1u Thr207G1u + Tyr208Leu + Leu211Met + G1y213G1n Pro195Ser + Va1197Pro + Pro209G1u + Leu211Cys 20 Pro195G1y + G1y196Ser + Va11.99G1n+ Thr214Asp Va1197Asn + G1y205Asn + Thr207Ser + Ser210Asp A1a194His + Tyr203His + Gly~:05Pro+ Ser206G1u Pro195Asn + Asn198G1n + Va1199G1n + Leu211G1n Va1197Pro + Thr202G1y + Pro204G1y + Thr207Ser 25 Thr202Pro + Tyr203Leu + Leu211A1a + Thr214Asp Pro195Asn + Va1199His + Tyr203Met + Thr207G1u Va1199Cys + Pro204G1y + Thr207Ser + Thr214Asp A1a194Ser + Pro195G1n + Tyr203Thr + Tyr208Pro Va1199Cys + Tyr203Cys + Ser206Asp + Tyr208A1a 30 Va1197Thr + Thr202G1n + Leu211G1u + Asn212Ser G1y196Ser + Va11~9Pro + A1a209G1n + Leu211Asn A1a194G1y + A1a209G1n + Asn212Asp + Thr214G1n A1a194G1n + Thr207Asn + A1a209Pro + Leu211His G1y196Ser + Va1197Ser + Thr202G1n + Leu211G1n 35 A1a194Thr + Va1197Ser + Thr202Pro + Thr207Pro G1y196Pro + Thr202Pro + G1y205G1u + G1y213Pro A1a194Pro + Pro195G1y + Va1197Asn + Va1199Cys Thr202G1y + Thr207Ser + Tyr208Pro + Leu211Pro Thr202Asn + Pro204Asp + Thr207Ser + G1y213Asn 40 G1y196G1n + Va1197Cys + Tyr203Met + A1a209Asn G1y196G1n + Ser206G1u + Thr207Ser + G1y213Pro Pro195Ser + Va1199Cys + Ser210G1u + Asn212Ser A1a194Ser + Ser206Asp + Asn212Ser + Thr214G1n G1y196G1n + G1n200Ser + Thr202G1n + A1a209Ser 45 G1y196G1n + Tyr203Thr + Thr207G1n + Asn212G1u A1a194Pro + Thr202G1n + Tyr208Cys + Leu211Pro G1n200Asp + Tyr203Cys + Leu211G1n + Asn212G1n G1y196Asn + Tyr203Met + Leu211Asn + G1y213Asn Pro195G1n + G1y196Asn + G1n200Ser + Thr202G1n 50 Thr202Pro + Tyr203G1y + Asn212Ser + Thr214G1y G1n200Asp + Tyr203Thr + Pro204G1n + G1y205Ser G1y196Ser + Thr202Pro + Pro204Asn + A1a209Ser G1y196Ser + G1n200Ser + Pro204G1y + Ser210Asp A1a194G1n + G1n200Asn + Thr202Ser + G1y205G1n WO 95/30011 ~ PCT/US95104760 SG
Pro195G1y + G1n200Asn + Thr207Asp + A1a209His G1n200Asn + Thr202Ser + Tyr203A1a + Thr214G1y G1n200G1u + A1a209Pro + Leu211I1e + G1y213Asn A1a199Asn + Thr202Ser + Tyr203Met + Ser206G1u Va1197Asn + Asn198Ser + Thr207G1n + Thr214G1u Va1197Met + G1n200Ser + Pro209G1u + A1a209Pro G1y196Asn + Va1199Asn + Leu211G1u + Asn212G1n Pro195G1n + Thr202G1n + G1y205Asp + Tyr208Asn Thr202Pro + Ser206Asp + Leu211I1e + G1y213Ser G1y196Ser + G1n200G1u + Thr202G1n + Tyr208Met Asn198G1u + Tyr203Thr + Tyr208Asn + G1y213G1n Asn198Ser + Ser210G1u + Leu211His + Thr214Pro G1y196Ser + Va1197G1y + Pro204G1u + Thr214Ser Tyr203Met + Ser206G1u + Tyr208ser + A1a209His G1y196G1n + G1n200Ser + Leu211Cys + G1y213Pro A1a194G1y + Va1199Asn + Thr202Asn + G1y213Pro Asn198Ser + Thr202Asn + Tyr203G1y + Ser210G1u Va1199G1n + G1n200Ser + Tyr203His + Thr214Asp Thr202G1n + Ser205Asp + Thr207Ser + Thr219G1y A1a199Thr + Tyr203G1n + Thr207Asp + Leu211I1e A1a209Ser + Ser210Asp + Asn212G1n + G1y213Ser Va1197Ser + Tyr203G1y + Ser206G1u + Asn212G1n A1a194Ser + Asn198Ser + G1y205G1n + Ser210Asp A1a194G1n + Pro195Ser + Va1197G1n + Pro204Asn A1a194G1y + Asn198Ser + A1a209G1u + Thr214Asn Va1199Met + Ser206Asp + G1y213Asn + Thr214Ser Thr202G1n + G1y205Ser + Leu211Asp + Asn212G1u Pro195Ser + A1a209G1y + G1y213Asp + Thr214Asp Thr202G1n + Thr207Pro + G1y213G1u + Thr214Asp A1a194Ser + Thr207Pro + A1a209G1u + Ser210G1u G1n200Asn + Thr202G1n + A1a209G1u + Ser210G1u G1n200Ser + A1a209Asp + Ser210Asp + Asn212G1n G1y205Asn + A1a209Asp + Ser210G1u + Asn212G1n Pro204G1y + Leu211G1n + Asn212G1u + G1y213Asp Tyr203G1y + Pro204Asp + G1y205G1u + Leu211A1a Thr202Ser + Pro204G1u + G1y205Asp + Thr207G1n Thr202G1y + Pro204Asp + G1y205G1u + G1y213Asn Va1199G1y + Ser210Asp + Leu211Asp + Asn212G1n G1y196Pro + Thr202Ser + G1y205Asp + Ser206G1u Va1197Thr + G1y205Asp + Ser206G1u + G1y213G1n Pro204Asn + G1y205G1u + Ser206G1u + Asn212Ser A1a194His + G1y205Asp + Ser206Asp + Leu211His Pro195Asn + Ser206G1u + Thr207Asp + Asn212Ser Tyr203Met + Ser206G1u + Thr207G1u + Leu211G1y G1y196G1n + Pro204Asn + Ser206Asp + Thr207G1u A1a194Thr + Va1197Cys + Ser206Asp + Thr207Asp A1a194Pro + Tyr208Asp + A1a209Asp + G1y213G1n G1n200Asp + Tyr208Thr + A1a209Ser + Ser210G1u Va1199Asn + G1n200Asp + Thr202G1n + Ser210Asp A1a194Ser + G1n200G1u + Thr202G1y + Ser210G1u Va1199Cys + G1n200Asp + A1a209G1n + Ser210Asp A1a194G1n + Va1197Asp + Tyr208G1n + Asn212Asp Pro195G1y + Asn198G1u + Ser210Asp + Leu211G1n A1a194Pro + Pro195Asn + Asn198Asp + Ser210Asp ~18942~
WO 95/30011 pCTIUS95/04760 Pro195G1n + Asn198G1u + Va1199Ser + Ser210Asp Va1197His + Asn198G1u + Thr207Pro + Ser210Asp G1y196Pro + Va1197G1y + Asn198Asp + Ser210G1u Asn198G1u + A1a209G1n + Leu211G1u + Thr214Pro G1n200G1u + Tyr203Asn + Thr207G1n + A1a209G1u Pro195Ser + G1y196Ser + G1n200G1u + A1a209Asp Pro195Ser + G1n200Asp + A1a209G1u + Leu211A1a to Loop 6 Variants - Quintuple Mutation Va1197A1a + Asn198G1n + Thr207Ser + Tyr208I1e + Asn212Asp A1a194G1y + G1y196Ser + Va1197G1y + G1n200G1u + Leu211G1y G1n200Asp + Thr202Ser + Tyr203Ser + Pro204Ser + Asn212Ser Pro195Ser + Va1199A1a + G1y205Asn + Ser206G1u + G1y213Ser Va1199His + Pro204Asp + G1y205Pro + Leu211G1y + Thr214Ser A1a194G1y + Thr207Pro + Ser210G1u + Leu211Cys + G1y213G1n Pro195Asn + G1y196Pro + Asn198Ser + Thr202G1y + G1y213Asn G1y196Ser + Va1197G1n + Pro204Ser + G1y205Asp + Leu211G1y Pro195Ser + G1y196G1n + Pro204Asn + G1y213Asp + Thr214G1y 2o Pro195G1y + Va1199Asn + G1n200Asn + Ser210G1u + Leu211G1y Va1197Ser + Va1199G1y + Tyr203Thr + G1y205Ser + Asn212G1u Pro195Asn + Asn198Asp + Thr202Ser + A1a209His + Asn212G1n G1y196Asn + G1y205Pro + Thr207Pro + Asn212Asp + G1y213G1n Va1197G1y + Asn198Asp + Va1199His + G1y205G1n + Tyr208His Tyr203Va1 + Thr207G1y + A1a209Ser + Asn212G1u + G1y213G1n A1a194Thr + Pro195G1y + G1y196Ser + Tyr203Asn + Asn212Ser Pro195G1n + Thr202G1y + Tyr203Leu + Pro204G1y + Ser210Asp Pro195Asn + Va1199A1a + Thr202Asn + Tyr208Pro + Ser210Asp A1a194Pro + Pro195G1y + Asn198G1u + Pro204Asn + Tyr208Pro A1a194Ser + Va1197Thr + Asn198Ser + Thr202Pro + Asn212Ser G1y196Ser + Va1199Pro + Thr207Ser + Tyr208Thr + Asn212G1n G1y205G1u + Tyr208G1y + Leu211I1e + Asn212Ser + Thr214G1y G1n200Ser + Thr202G1y + Thr207Asp + Leu211Ser + Thr214G1n Pro195Asn + Thr207Asp + Tyr208Asn + Asn212Ser + G1y213Asn Pro195Asn + Tyr203Ser + Pro209G1u + Leu211Va1 + Asn212G1n G1n200Asp + G1y205Ser + Tyr208Met + Leu211Asn + Asn212Ser A1a199Ser + Va1199Pro + G1y205G1u + Tyr208Met + G1y213Ser Asn198Ser + G1n200G1u + Thr202G1y + A1a209G1n + Leu211Asn G1y196Pro + Asn198G1u + Thr202Ser + A1a209Thr + Thr214G1y A1a194G1n + Pro195G1y + G1y196Asn + Tyr203I1e + Thr214Pro Pro195G1n + Va1197Cys + Thr202G1n + Tyr203Cys + Tyr208His Va1199Pro + Pro209G1y + G1y205Ser + Thr207Ser + G1y213Asp Tyr203G1y + Pro204Ser + G1y205Asn + A1a209Thr + Ser210Asp A1a194Ser + Thr207Ser + Tyr208Cys + Leu211Pro + Thr214Pro Pro195G1y + Va1199His + G1y205G1u + A1a209Asn + Leu211Pro Pro204Asn + Tyr208I1e + A1a209G1y + Leu211G1n + G1y213G1u A1a194G1n + Va1197Cys + Asn198Ser + Pro204G1y + Asn212G1u Asn198G1u + Pro204Asn + A1a209Thr + Leu211Ser + G1y213Pro G1y196Ser + Va1199Ser + Pro204Asp + Thr207G1y + Tyr208I1e G1n200G1u + Thr202Ser + Tyr203A1a + Pro204G1y + Tyr208Met Asn198G1n + Thr202Pro + G1y205Asn + Thr207G1u + Thr214Pro G1y196Ser + Va1199Pro + A1a209G1n + Ser210Asp + Leu211Asn WO 95130011 21 g 9 ~ 2'~ PCT/US95104760 ss A1a194G1y +Thr207Asn +A1a209G1n + Asn212Asp +Thr214G1n Pro195G1y +Asn198G1n +Va1199Thr + Tyr208His +Leu211I1e A1a194Pro +Thr202G1y +Thr207Ser + Tyr208Pro +Leu211Pro G1y196Ser +Va1197A1a +G1n200Asn + Thr202Pro +Ser210G1u Va1197G1n +Thr202Asn +Tyr203Asn + G1y205Asp +Tyr208A1a G1y196Pro +Tyr203I1e +Pro204Asn + Leu211A1a +Thr214G1n A1a194Asn +G1n200Asn +Pro204G1n + A1a209Thr +G1y213Ser A1a194His +Pro195G1y +G1n200Ser + Leu211Asn +Asn212Ser A1a194Ser +G1y196Ser +G1n200Asn + Ser206Asp +Thr207G1y G1y196Asn +Tyr203Asn +Tyr208A1a + A1a209Pro +G1y213G1n A1a194Thr +Va1199Asn +Tyr203Ser + Tyr208His +Asn212Asp Va1197G1u +Va1199A1a +Tyr208G1n + A1a209G1y +Leu211Va1 G1n200Asn +Thr202Ser +Tyr203A1a + Thr207G1n +Thr214G1y Va1197Asn +Asn198Ser +Thr202Pro + Thr207G1n +Thr214G1u Pro195G1n +Asn198G1u +G1n200Ser + Tyr203Cys +G1y213G1n G1y196G1n +Asn198Ser +Thr202Asn + Tyr203Ser +G1y205Pro A1a194Thr +Thr207Pro +Tyr208I1e + A1a209Pro +Thr214Asn Asn198Ser +Va1199Pro +Thr202Asn + Tyr203G1y +Asn212G1u A1a199Asn +Thr202Asn +Ser210Asp + Leu211Va1 +Asn212Ser Va1197Ser +Va1199His +Thr202Asn + Ser210Asp +Leu211Cys G1y196Pro +Va1197Cys +Va1199Met + Tyr203Ser +Leu211Asn A1a194G1y +G1y196Pro +Va1199His + Thr202Pro +Ser210G1u G1y196Ser +Va1197His +Asn198G1u + Thr202Pro +Asn212Ser A1a194His +G1y196Asn +Va1197G1n + Thr202Ser +G1y205G1u Pro195G1y +G1y196Pro +Asn198Ser + G1n200Ser +Thr202Pro Va1197Asn +Asn198Asp +G1n200Asn + Thr202G1n +Tyr208A1a Pro195Asn +Thr202Ser +Pro204Asp + Tyr208A1a +A1a209Ser Tyr203His +Pro204Asn +G1y205Asp + Thr207G1n +Leu211G1y A1a194Asn +Pro195G1n +G1y196G1n + Pro204Asn +G1y213G1u Va1197Thr +Va1199His +Ser206G1u + G1y213Asn +Thr214Asn G1y196G1n +Va1197Asn +Tyr203Thr + Pro204Ser +Thr214Asp G1y196Asn +Va1199G1y +G1n200Ser + Tyr203Asn +G1y213G1u A1a194Thr +G1n200Asn +Thr202Ser + G1y205G1n +G1y213Asp Pro195Ser +Asn198Ser +Va1199G1y + Tyr203Va1 +Thr207G1u A1a199Asn +Pro195G1y +Thr202G1y + Tyr203G1y +Leu211Asp Pro195Ser +Va1197Ser +Tyr203Cys + G1y205G1u +A1a209Thr G1y196G1n +Va1197G1y +G1y205Ser + Tyr208Leu +Thr214Asp A1a194G1n +G1y196Asn +Tyr203Pro + Pro209Ser +G1y213Asp G1y196G1n +Thr202G1y +Tyr203Cys + Pro209G1n +Ser206G1u Va1197Pro +Va1199Met +Tyr203Va1 + Pro204G1y +Thr214Asp G1y196Pro +G1n200Asn +Tyr203Cys + A1a209Thr +Thr214Asn Asn198G1n +Pro204Asn +Thr207G1n + G1y213Pro +Thr214Asp Pro195G1y +Va1197Ser +Va1199A1a + Thr207Asp +Leu211G1y Pro195G1n +Asn198G1n +Ser206Asp + Thr207Ser +A1a209Ser A1a194Ser +Tyr203Leu +A1a209Asn + Ser210Asp +Thr214Ser Va1199His +Thr202Ser +G1y205Asn + Leu211Pro +Asn212G1n G1y196Ser +Asn198Ser +Thr207Asn + Tyr208G1n +Ser210G1u Pro195Asn +Asn198G1n +Thr207Ser + G1y213Asn +Thr214G1y G1y196G1n +G1n200Ser +Pro204Asn + G1y205G1n +G1y213Asp Pro204Asn +Ser206G1u +Tyr208Leu + A1a209G1y +Asn212G1n Va1197Thr +Va1199G1y +G1n200Asp + Thr202Pro +Thr207G1y Pro195G1n +Va1199Ser +Thr202Asn + G1y205Asp +A1a209G1n G1y196G1n +Tyr203Thr +Tyr208Cys + Leu211A1a +Thr214G1u Asn198G1n +G1n200Ser +Pro204G1n + Asn212Ser +G1y213Pro 2I89~~~
~""""' WO 95130011 PCT/US95/04760 ~9 Va1197Pro + Va1199Asn + Thr202G1n + Tyr203Asn + G1y213Ser A1a194Ser + G1n200Ser + Thr202G1n + Thr207G1n + A1a209G1u Thr202G1y + Tyr203Thr + Tyr208Pro + G1y213Asp + Thr219G1u Va1197G1n + Pro204G1y + G1y205Ser + A1a209G1u + Ser210Asp A1a194Ser + Va1199A1a + Tyr208A1a + A1a209G1u + Ser210G1u Va1199A1a + G1n200Ser + G1y205Pro + Asn212G1u + G1y213Asp A1a194Pro + Thr202Ser + Pro209G1u + G1y205Asp + A1a209Pro Pro209G1u + G1y205G1u + Thr207G1y + Tyr208Ser + Leu211I1e Pro195G1n + Va1197Thr + Pro209G1u + G1y205Asp + Leu211Va1 l0 Va1199His + G1y205Asp + Ser206G1u + A1a209G1n + Asn212G1n Tyr203A1a + G1y205G1u + Ser206Asp + Tyr208I1e + Asn212G1n Pro195Asn + G1y205G1u + Ser206Asp + Tyr208Ser + A1a209Ser Va1197His + Pro204G1y + G1y205G1u + Ser206Asp + Tyr208Cys Thr202Asn + Pro204Ser + G1y205Asp + Ser206Asp + Tyr208Thr A1a194Asn + Thr202Ser + Tyr203G1y + Ser206G1u + Thr207G1u Pro195Ser + G1y195G1n + Va1199Thr + Ser206G1u + Thr207Asp G1y196Pro + Ser206Asp + Thr207G1u + Tyr208Thr + Leu211His A1a194His + G1y196G1n + Pro204Asn + Tyr208Asp + A1a209Asp Va1199A1a + Tyr203Pro + Tyr208Asp + A1a209Asp + Asn212G1n Va1199A1a + Thr202Ser + Thr207G1u + Tyr208Asp + Thr214Ser G1y196Asn + Thr202G1n + Pro204Ser + Thr207Asp + Tyr208Asp Pro195G1n + G1n200G1u + Thr202Pro + Tyr203Pro + Ser210Asp G1y196Pro + Va1197Thr + Asn198G1n + G1n200Asp + Ser210Asp G1y196G1n + Asn198G1u + Thr207Pro + Tyr208Ser + Ser210G1u A1a194Pro + Asn198G1u + Thr207Pro + A1a209His + Ser210G1u Asn198G1u + Va1199G1n + Pro204Ser + Tyr208Asn + Ser210Asp Pro195G1y + Asn198G1u + Thr207G1y + Tyr208Cys + Ser210Asp Asn198Asp + Va1199Thr + Leu211Asp + Asn212Ser + Thr214G1y G1y196Pro + Asn198G1n + G1n200G1u + Tyr203Asn + A1a209G1u G1y196Pro + Tyr203Thr + Pro209Asp + Ser206Asp + A1a209G1n Pro195Asn + G1y196Pro + Asn198Asp + A1a209Pro + Asn212G1u Pro195Asn + G1y196G1n + G1y205Asp + Thr207G1u + Leu211Thr Thr202Asn + Pro204Asp + Thr207Asp + Tyr208Met + Thr214Pro Tyr203G1y + Pro209Asp + Thr207Asp + Asn212G1n + Thr214G1y Pro195Ser + Tyr203His + Pro204G1u + Thr207Asp + G1y213G1n Asn198G1u + G1n200G1u + Tyr203Cys + Pro209G1y + Asn212G1n Va1197G1u + Va1199G1y + Thr202G1y + Tyr203Ser + G1y213G1u Pro204G1y + Thr207Asn + A1a209His + Ser210Asp + Asn212Asp Pro195G1y + G1y196G1n + Thr202G1n + Ser210Asp + Asn212Asp Thr202Asn + Tyr203Asn + G1y205Pro + Ser210Asp + Asn212Asp Thr202G1n + Tyr203G1n + Ser210Asp + Asn212Asp + G1y213G1n G1y196Ser + Ser210Asp + Leu211G1y + Asn212Asp + Thr214G1n G1y196Pro + A1a209Ser + Ser210G1u + Leu211Asn + Asn212Asp G1y205Ser + A1a209Pro + Ser210G1u + Asn212Asp + Thr214Pro Pro195G1y + Tyr203G1y + Ser210Asp + Asn212Asp + Thr214Ser Pro195G1n + Va1197Asn + Va1199Cys + Tyr208G1u + Ser210Asp Va1197Ser + Tyr203G1y + Pro204G1u + Thr207Ser + Tyr208Asp Pro195Ser + G1y196Asn + Va1197Asp + Va1199G1y + Ser210G1u G1y196Pro + Va1:197Asp+ Thr202Pro + Ser210G1u + Thr214Pro Va1197G1u + Va1199His + Tyr208Thr + Leu211G1n + Thr219G1u Asn198Asp + Va1199Asn + Thr207Asn + A1a209His + G1y213Asp Va1197His + Asn198G1u + Tyr203G1n + Tyr208Cys + G1y213Asp Pro195Asn + Va1197Asn + Asn198G1u + Thr202Pro + G1y213Asp Pro195Ser + Asn198G1u + Tyr203Ser + G1y205Ser + A1a209Asp GO
Pro195G1y + G1y196Asn + Asn198Asp + Va1199Met + A1a209G1u Pro195G1n + Asn198G1u + Thr202Asn + Pro209G1y + A1a209Asp Va1197G1u + G1n200G1u + Thr202G1y + Tyr208A1a + G1y213G1n Va1197Asp + G1n200Asp + Thr207Ser + Tyr208I1e + A1a209Ser Va1197Asp + G1n200G1u + A1a209Ser + Leu211G1y + Asn212Ser Va1197G1u + G1n200Asp + Thr202Asn + G1y205G1n + Asn212G1n A1a194Pro + G1y196Pro + Va1197Met + G1n200G1u + Asn212Asp G1y196G1n + G1n200G1u + Thr202Pro + Tyr203I1e + Asn212G1u A1a194His + G1n200Asp + Asn212G1u + G1y213Pro + Thr214Asn Va1199Pro + G1n200G1u + Pro204G1y + Asn212G1u + G1y213Ser Asn198Ser + G1n200Asp + G1y205Pro + Thr207Ser + Asn212G1u A1a194Asn + G1n200Asp + G1y205Asn + Tyr208Asn + Asn212Asp Pro195Asn + G1n200Ser + Thr202Pro + Pro204Asp + A1a209G1u Pro195Ser + Thr202Asn + Tyr203Cys + Pro209G1u + A1a209Asp A1a194His + Asn198Ser + Thr202Asn + Pro204G1u + A1a209Asp A1a194His + Pro204Asn + Ser206G1u + A1a209G1u + Thr214Ser Pro195G1y + Va1197Cys + Ser206G1u + A1a209G1u + Asn212Ser Thr202G1y + Ser206G1u + Thr207Ser + Tyr208Met + A1a209G1u A1a194G1y + G1n200G1u + Thr207Asp + Leu211Ser + Thr219Pro Pro195Asn + Va1199Ser + G1n200G1u + G1y205Asn + Thr207Asp A1a194G1n + Asn198G1n + G1y205Pro + Ser210Asp + G1y213Asp G1y196Ser + Thr207G1y + Ser210G1u + Leu211Thr + G1y213Asp Asn198G1n + Pro204G1n + Thr207Asp + Tyr208Thr + Ser210G1u A1a194Ser + G1y196G1n + Thr207G1u + Ser210G1u + Leu211Thr Thr207G1u + Ser210G1u + Asn212Ser + G1y213Ser + Thr214Ser Va1197Cys + Thr207G1u + A1a209His + Ser210G1u + Asn212Ser Asn198G1u + Va1199Met + Tyr208A1a + G1y213G1n + Thr214Asp A1a194G1n + Asn198G1u + A1a209Pro + Leu211His + Thr214Asp Asn198Asp + Thr207Pro + Leu211G1y + Asn212Ser + Thr214Asp A1a194Ser + G1y196Asn + Asn198Asp + Pro204G1n + Thr214G1u Loop 6 - Sextuple Mutation Variants A1a194G1n + Va1197Ser + Thr202Ser + Tyr203Ser + A1a209Ser + Asn212Asp Pro195Ser + Va1199Cys + G1y205Ser + Leu211Thr + Asn212G1u + G1y213Pro Va1197Thr + Asn198Ser + Va1199Pro + G1n200Ser + Thr202Ser + Leu211Asn Thr202G1y + Pro204Ser + G1y205Asn + Tyr208Asp + G1y213G1n + Thr214Pro Va1197G1y + G1n200G1u + Thr202G1y + Tyr208A1a + Leu211Va1 + Asn212Ser A1a194Pro + G1y196Ser + Va1199Cys + Tyr203A1a + Pro204Ser + A1a209Asp Pro195Asn + Va1199Pro + Thr202Asn + Tyr203G1y + Thr207Asp + G1y213Asn A1a199G1n + Thr202Ser + Tyr203I1e + Pro2~9Asn + G1y205Asp + Tyr208Ser A1a194G1n + Va1197His + A1a209Ser + Ser210Asp + Leu211G1y + Asn212G1n Thr202G1y + G1y205Pro + Ser210Asp + Leu211A1a + G1y213Ser "~ WO 95130011 PCTIUS95104760 + Thr214G1n A1a194His + G1n200Ser + Thr202G1y 7-Pro204Ser + Tyr208His + G1y213G1u A1a194Asn + Pro195G1n + G1y205Asn ~-Tyr208Ser + Leu211Va1 + Thr214Pro G1y196Pro + Asn198Ser + G1n200Ser ~-Thr202Asn + Tyr208I1e + Thr214G1u Pro195Asn + G1y205Pro + Thr207G1n ~-Ser210G1u + Leu211Asn + G1y213Pro toA1a199Ser + Va1197G1y + Asn198Asp ~-Tyr203Cys + G1y205G1n + Leu211Met A1a194Ser + Va1197Asp + Va1199G1n ~-Tyr203Cys + G1y205Ser + G1y213G1n A1a194Pro + Thr202G1y + Thr207Ser -~-Tyr208Met + Leu211Pro 15+ G1y213G1u A1a194Thr + Pro195Asn + G1y196G1n ~-G1n200Ser + Thr202Asn + Pro204G1u A1a194G1n + G1y196Asn + Va1197Ser -+-Asn198Ser + Va1199Thr + Ser210G1u 20G1y196Ser + Va1199G1y + Tyr203I1e ~-Pro204Asn + Leu211Asp + Thr214G1n A1a194Asn + G1n200Asn + Pro204G1n + A1a209Thr + Leu211Asn + G1y213Ser A1a199His + Pro195G1y + G1y196Asn ~-G1n200Ser + Tyr203Ser 25+ Asn212Ser A1a194Pro + Va1197Met + Va1199Asn -+-Thr202Ser + Ser206Asp + Thr207Asn Pro195G1n + Thr202Pro + G1y205Asn ~-A1a209Pro + Asn212Ser + Thr214Asn 30Pro195Asn + Asn198G1u + Va1199His -+-Pro204Asn + Thr207G1y + Leu211Met G1y196Ser + Pro204Asn + Thr207Pro ~-Asn212Asp + G1y213Ser + Thr219Pro A1a194Ser + Pro195Ser + Va1197Cys ~-Pro204Asn + Thr207G1u 35+ G1y213G1n A1a194His + Pro195G1y + Va1197G1y -+-Va1199G1n + Thr207G1y + Asn212G1u A1a194G1n + Pro195Ser + Va1197Ser ~-Tyr203Met + Leu211G1u + G1y213Asn 40A1a194Asn + G1y196Asn + Pro204Asp ~-Thr207Pro + Tyr208Ser + Thr214Ser Pro195Asn + Thr202G1y + G1y205Asp -+-Thr207G1n + Tyr208Thr + A1a209Asn A1a194His + Pro195G1y + Va1197His -+~G1y205G1n + A1a209G1u 45+ Thr214Asn A1a194G1n + Asn198G1n + Tyr203Met -+-A1a209G1n + Ser210G1u + Leu211G1y A1a194Pro + G1y196Ser + G1n200Ser + Thr202Pro + Thr207Asp + A1a209G1y 50Pro195G1n + G1y196Ser + Asn198G1n ~~G1y205Ser + Ser210Asp + Leu211Va1 Asn198G1n + Va1199Met + G1n200G1u + Thr202G1y + G1y205Asn + Tyr208A1a A1a194Pro + Va1199A1a + Pro204Asn + Thr207G1n + Leu211G1u WO 95/30011 ~ ~ PCT/US95104760 + G1y213Ser G1y196Pro + Va1197Thr + Va1199His + G1y205Asn + Ser206Asp + Thr207Asn Pro195Asn + G1y196Ser + Thr202Asn + Tyr203G1y + Leu211Asn + Asn212G1u A1a194Ser + Pro195G1n + Va1197Cys + Ser206G1u + Tyr208His + Asn212G1n Pro195G1n + G1y196Pro + Va1197Thr + Ser210Asp + G1y213Ser + Thr214Pro A1a194G1y + Pro195Ser + Thr202G1y + Pro204G1u + Tyr208Thr + G1y213Pro A1a194Asn + Pro195Asn + G1y196Asn + G1y205Pro + A1a209Asn + Ser210Asp Pro195Ser + Va1199G1n + Tyr208Asn + A1a209G1n + Leu211G1y + G1y213G1n Asn198Asp + Va1199A1a + Thr202Asn + A1a209Pro + Asn212G1n + Thr214G1y Pro195Asn + G1y196G1n + Va1199Pro + Ser206G1u + Tyr208A1a + Leu211Va1 A1a194G1n + Pro195Ser + Asn198Asp + Va1199Ser + Thr202Pro + A1a209G1n Pro195G1y + Va1199Met + G1y205G1u + Thr207Asn + A1a209Pro + G1y213Pro Pro195Asn + G1y196G1n + G1n200Asp + Thr207Asn + A1a209G1y 2,5+ Asn212Ser G1y196G1n + Va1197His + Thr207Asn + Ser210Asp + Leu211A1a + G1y213G1n A1a194G1y + Va1197Thr + Tyr203Met + G1y205G1u + A1a209G1y + Thr214G1y G1n200G1u + Thr202Asn + Tyr203G1y + Thr207Asn + Asn212G1n + G1y213Pro G1y196Asn + Thr202Ser + Pro204G1u + Leu211Pro + G1y213Ser + Thr214Asn Pro195G1y + Va1197Asn + Va1199G1n + G1n200Asn + Thr207G1y + Asn212Ser Pro195G1n + G1y196Pro + Va1197His + Tyr203G1y + Ser206Asp + Thr214Ser G1y196G1n + Tyr203His + Thr207Asn + Leu211Asn + G1y213Pro + Thr214Asp A1a194Asn + Asn198Ser + G1n200Asn + Thr202Ser + Tyr203I1e + A1a209Thr A1a199His + Va1197Cys + G1n200Ser + Tyr203G1y + Thr207G1y + A1a209His Asn198G1n + Va1199Thr + G1n200Asp + Pro204Asn + Asn212ser + G1y213G1n G1y196Pro + Tyr203G1n + Pro204Ser + Thr207Asn + Tyr208Met + Leu211Asp G1y196Ser + Va1197Met + Asn198Ser + Thr202Asn + Thr207Asp + Asn212G1n SO Pro195G1y + G1y196Pro + Thr202Asn + Tyr203I1e + G1y213Asp + Thr214Asn Pro195G1y + G1y196Pro + G1n200G1u + Pro204Ser + Tyr208l1e + G1y213Ser Va1199A1a + Thr202G1n + G1y205Ser + A1a209Asn + Ser210G1u ~'189~,~~

+ Leu211Cys A1a194His + Pro195Ser + Va1199Thr + G1n200G1u + Tyr203Pro + Tyr208Cys G1y196G1n + Pro204G1y + Ser206Asp + Thr207Pro + A1a209G1n + G1y213Ser Pro195Asn + Thr202Asn + Pro204Asn + A1a209G1n + Asn212Asp + G1y213G1n G1y196Ser + Asn198G1n + Tyr203His + Asn212Ser + G1y213Asp + Thr214G1y 10A1a194G1n + Va1197Pro + Va1199G1n + G1n200Asn + Tyr208G1u + Thr219Ser Va1199Cys + G1n200Asn + Pro209G1y + Ser206Asp + Thr207Ser + Asn212Ser A1a194G1n + G1y196G1n + Va1199His + Thr202Pro + Tyr203Thr 15+ G1y205Ser Asn198G1n + Va1199G1y + Thr202Ser + Ser210Asp + Asn212Ser + G1y213G1n Va1199Thr + G1n200Asn + Pro204Ser + Tyr208Va1 + Asn212G1n + Thr214Asp 20A1a194His + G1y196G1n + Va1199G1n + Thr202G1y + Pro204Asp + Leu211Cys A1a194Ser + Va1197G1y + Pro204G1n + A1a209Pro + Ser210G1u + Thr214G1y A1a194G1n + G1y196Pro + Tyr203G1n + Thr207Pro + Tyr208Pro 25+ A1a209Asn G1y196Ser + Thr202Asn + Tyr208I1e + A1a209G1n + Leu211A1a + G1y213G1u Pro195Asn + G1y196Pro + Va1197G1u + G1n200Asn + Tyr203Ser + Tyr208Ser 30G1y196Ser + Va1199His + G1y205Asp + Tyr208Va1 + A1a209G1y + Thr214Pro Va1197Ser + Va1199Met + G1n200Asn + Thr202Ser + Tyr208Cys + G1y213G1n A1a194Ser + G1y196G1n + Va1199Pro + Leu211Va1 + G1y213G1u 35+ Thr214G1y A1a194G1n + Asn198G1n + Va1199Cys + Thr207G1y + Leu211Asp + Asn212G1u A1a194Thr + Asn198G1n + Thr202Asn + Pro204Ser + G1y213G1u + Thr214Asp 40A1a194Thr + Pro195Ser + Va1199A1a + A1a209G1u + Ser210G1u + Leu211Cys Tyr203Leu + G1y205Ser + A1a209G1u + Ser210G1u + Asn212Ser + G1y213Asn Va1197G1u + Asn198G1u + Tyr203Pro + Thr207G1y + A1a209Ser 45+ Asn212Ser Va1197Asp + Asn198Asp + Va1199His + Thr202Ser + Pro204G1n + A1a209Asn G1y196Asn + Va1197G1u + Asn198Asp + G1n200Ser + Pro204Asn + Thr207G1y SOPro195G1n + Thr202Ser + Tyr203Ser + Thr207G1n + Asn212Asp + G1y213G1u G1y196G1n + Tyr203His + G1y205Ser + Leu211Asn + Asn212Asp + G1y213G1u Asn198Ser + G1y205Ser + Tyr208I1e + Asn212G1u + G1y213G1u + Thr219Pro A1a194Ser + G1y196G1n + Pro204G1u + G1y205Asp + Tyr208Asn + Thr214Asn A1a194Ser + Pro195G1y + Va1199Cys + Pro204Asp + G1y205G1u + Asn212G1n A1a194Thr + Va1199Pro + G1y205Asn + A1a209G1n + Ser210G1u + Leu211Asp Thr202G1n + Thr207G1y + Ser210Asp + Leu211G1u + G1y213G1n + Thr214Ser Thr202G1n + G1y205Asp + Ser206G1u + Tyr208Met + G1y213G1n + Thr214Asn A1a194G1y + G1y205G1u + Ser206G1u + A1a209G1y + Leu211G1n + Thr214G1y G1n200Ser + Thr202Ser + Tyr203G1y + G1y205Asp + Ser206G1u + Thr214G1n Va1197Asn + Thr202Ser + G1y205G1u + Ser206G1u + A1a209His + Thr219Ser A1a194G1n + Thr202Ser + Tyr203G1y + Ser206Asp + Thr207G1u + G1y213Ser G1y196Pro + Va1199G1n + Tyr203Met + Ser206G1u + Thr207Asp + A1a209Thr Pro195G1y + G1y196Pro + Thr202Ser + Pro204G1n + Tyr208Asp + A1a209G1u Pro195G1n + G1y196Pro + Va1197Pro + G1n200Asp + G1y205Ser + Ser210Asp G1y196Ser + Asn198G1u + G1y205Pro + A1a209His + Ser210G1u + Asn212G1n A1a194Thr + Asn198Asp + Thr202Ser + Tyr203Cys + A1a209Pro + Ser210G1u G1y196Pro + Asn198G1u + G1y205Ser + Ser210G1u + Leu211Asn + Thr214Asn A1a194Pro + G1y196Ser + Asn198G1u + G1y205Ser + Thr207Pro + Ser210G1u G1y196Ser + Va1197His + Asn198Asp + G1n200Ser + Thr207G1n + Ser210Asp Asn198G1u + Va1199Met + G1y205Pro + A1a209His + Ser210G1u + Thr214Pro Va1197Ser + Asn198G1u + Pro204Ser + Thr207Ser + A1a209Pro + Leu211Asp Pro195Ser + G1n200Asp + Thr202Asn + Tyr203Ser + A1a209Asp + Asn212Ser G1n200Asp + Pro204Ser + G1y205Pro + Tyr208His + A1a209Asp + Leu211Asn Pro204G1u + Ser206Asp + Tyr208I1e + Leu211G1y + Asn212G1n + Thr214Asn G1y196Ser + Asn198Ser + Va1199A1a + Pro204Asp + Ser206G1u + Tyr208Ser Pro195Asn + Pro209Asp + Ser206Asp + Tyr208Ser + Leu211Ser + Asn212G1n A1a194G1y + Asn198G1u + G1n200Ser + Thr207G1y + A1a209Asn + Asn212G1u Asn198G1u + G1y205G1n + A1a209Pro + Leu211Cys + Asn212Asp + G1y213Pro A1a194Thr + Va1197Thr + Asn198G1n + Pro204G1y + G1y205Asp WO 95/30011 ~ ~ PCTIUS95/04760 GS
+ Thr207Asp A1a194Asn + G1y196Pro + Pro204Asp + Thr207Asp + Tyr208Cys + G1y213Asn A1a194Thr + Pro195G1n + G1n200Ser ~+Pro204G1u + Thr207Asp + G1y213Pro Va1197Pro + Asn198G1u + G1n200Asp -~Leu211A1a + Asn212Ser + G1y213Pro Asn198Asp + G1n200G1u + Tyr203Va1 -~Leu211Thr + Asn212Ser + G1y213Pro l0 Pro195G1n + Asn198Asp + Va1199G1n -~G1n200G1u + Pro204Asn + A1a209Pro A1a194G1y + G1y196Asn + Asn198Asp -~G1n200Asp + Thr207G1y + Asn212Ser G1n200Asp + Thr202Asn + Tyr203A1a -~Thr207G1n + Leu211G1u + Thr214Pro Va1197Asp + Va1199His + Tyr203Va1 -~Tyr208His + Leu211Asp + G1y213Pro Pro195Asn + Va1197G1n + Va1199G1n - Thr207Asp + A1a209G1u + G1y213Ser Asn198G1n + G1n200Asn + Tyr203A1a ,-Thr207Asp + Tyr208Va1 + A1a209Asp Va1197A1a + Asn198G1n + Pro204Ser ~-Tyr208Thr + Ser210G1u + Asn212G1u G1y196Asn + G1n200Asn + Pro209G1n ~-Ser210Asp + Asn212G1u + Thr214Pro Pro195G1n + Va1199Pro + Ser210Asp 1-Asn212G1u + G1y213Asn + Thr214G1y Pro195G1y + G1n200Ser + Thr207Pro ~-Tyr208G1n + Ser210G1u + Asn212G1u 3o Pro195G1y + Va1199Cys + Thr202Asn ~-Tyr208His + Ser210G1u + Asn212Asp Va1199His + Thr202G1y + Pro204G1n -~-A1a209Thr + Ser210Asp + Asn212Asp Asn198G1n + Thr202Pro + A1a209Ser ~-Ser210G1u + Asn212G1u + G1y213Pro A1a199Ser + Va1199G1n + Tyr208G1y ~-Ser210Asp + Leu211G1n + Asn212Asp A1a194Asn + Va1199G1y + G1y205Ser i-A1a209His + Ser210G1u + Asn212G1u 4o A1a199Ser + Thr202Asn + Pro209G1y -+~Tyr208G1u + Ser210G1u + G1y213G1n Pro195G1n + Va1199Asn + Thr207G1y -+~Tyr208Asp + Ser210Asp + Leu211I1e Pro195G1n + G1n200Asn + Tyr203G1n ~~Tyr208G1u + Ser210G1u + Asn212G1n A1a194His + Tyr203Asn + Tyr208Asp ~ Ser210Asp + Leu211His + Thr214Ser G1y196Asn + Va1197G1n + Thr202Asn + Tyr208G1u + A1a209Asn + Ser210G1u A1a194Asn + G1n200Ser + Tyr203Met + Ser206G1u + Tyr208G1u + Leu211Va1 Asn198Ser + Pro209G1y + Ser206G1u ~ Tyr208Asp + Asn212G1n + Thr214G1y A1a194Pro + G1n200Asn + Thr202Pro + Tyr203Met + Asn212Asp PCTlUS95/04760 GG
+ Thr214Asp Pro195Ser + Asn198G1u + G1n200Ser +Thr202Asn + G1y205Pro + G1y213Asp Pro195Ser + Asn198G1u + Va1199A1a +A1a209G1u + Leu211Asn + Thr214G1y Pro195G1y + Asn198G1u + G1n200Asn +Thr202Pro + Tyr203I1e + A1a209G1u Va1197Ser + Asn198Asp + Tyr203Pro +Tyr208Met + A1a209Asp + Leu211G1n Pro195Ser + Asn198Asp + G1n200Ser +Tyr208Met + A1a209Asp + Leu211Met G1y196Asn + Va1197Asp + Va1199G1n +G1n200G1u + G1y205Asn + G1y213Pro G1n200G1u + Thr202G1n + Tyr203Met +A1a209Asn + Leu211A1a + Asn212Asp A1a194Pro + G1y196Pro + G1n200Asp +Thr202Asn + Tyr208Ser + Asn212G1u Va1197G1y + Va1199Ser + G1n200G1u +Tyr203G1y + Asn212G1u + G1y213Pro Pro204Asp + Thr207G1n + Tyr208Leu +A1a209Asp + Asn212Ser + Thr214Asn A1a194Ser + Pro195G1n + Thr202Ser +Pro204Asp + A1a209Asp + Thr214Asn Pro195Asn + Thr2025er + Tyr203Leu +Tyr208Asp + A1a209His + Leu211Asp A1a194Asn + G1y196Pro + Ser206G1u +Tyr208His + A1a209Asp + Thr214G1y Pro204G1n + G1y205Asn + Ser206G1u +Thr207G1y + A1a209Asp + Leu211Cys Va1197G1n + Thr202G1n + Pro204G1y +Ser206Asp + A1a209Asp + Thr214Ser A1a194G1y + Va1197G1n + Asn198G1n +Tyr203Va1 + Ser206Asp + A1a209Asp G1y196Ser + Va1197Asn + G1n200Asp +Thr202Pro + Thr207Asp + Asn212G1n A1a194G1y + G1y196Ser + Ser210Asp +Leu211Ser + Asn212G1n + G1y213G1u Va1197Thr + Tyr203His + Thr207G1n +Tyr208Pro + Ser210Asp + G1y213Asp Asn198Ser + Va1199Cys + Tyr203Va1 +G1y205Ser + Ser210Asp + G1y213Asp Va1197His + Va1199Asn + Pro204G1n +Ser210Asp + Asn212Ser + G1y213Asp G1y196Ser + Va1197A1a + G1n200Asn +Thr202Pro + Ser210G1u + G1y213G1u Asn198G1u + Tyr203G1y + G1y205G1n +Thr207Pro + Tyr208Va1 + Thr214G1u A1a194G1y + G1y196Asn + Asn198Asp +Tyr203Leu + Tyr208His + Thr214G1u Pro195G1y + Asn198G1u + Va1199Cys +A1a209G1n + Asn212G1n + Thr214Asp Asn198Asp + Va1199Asn + Thr202Asn +Pro204G1n + Asn212G1n + Thr214Asp Va1199Pro + G1n200Asp + Tyr203Asn +Leu211Pro + G1y213G1u "~"' WO 95130011 ~ PCT/iJS95/047G0 + Thr214Asn Pro195Ser + G1y196Pro + Va1199A1a + G1n200Asp + Asn212G1n + G1y213Asp A1a194Thr + Pro1.95G1y + G1y196Pro + G1n200G1u + G1y205Asn + G1y213G1u Va1199His + G1n200G1u + Pro209Ser + Leu211His + G1y213G1u + Thr214G1n Asn198Asp + G1n200Ser + G1y205Ser + Tyr208Asp + Leu211A1a + Asn212Ser l0 A1a194Ser + Asn198Asp + Thr202Ser + Tyr208Asp + A1a209Pro + G1y213G1n G1n200Asn + Tyr203Pro + Pro204G1u + G1y205Pro + Ser210Asp + Leu211Pro Va1197Ser + Tyr203Pro + Pro209Asp + A1a209G1y + Ser210Asp + Thr214Asn G1y196Asn + Pro209G1u + Ser210G1u -+ Leu211Pro + Asn212Ser + Thr214G1y 2o Loop 6 - Heptuple Mutation Variants Pro195Ser + Asn198Ser + Va1199Met ~~ Ser206G1u + Thr207Ser + G1y213Asn + Thr214G1n A1a194G1n + Pro195Asn + Va1197Ser ~+- Thr202Ser + Tyr203Ser + A1a209Ser + Asn212Asp Va1197Asp + G1n200Asn + Thr202Pro -~ G1y205G1n + Thr207G1y + Tyr208Met + Leu211Va1 A1a194Ser + Pro195G1y + Asn198Ser -~ Thr202Pro + Ser206G1u + Tyr208A1a + Thr219G1n A1a194Pro + Pro195Asn + Thr202G1y -~ Tyr203Thr + Ser206G1u + Leu211Ser + Thr214Pro G1y196G1n + Va1197Thr + G1n200Asn -~ Tyr203Cys + G1y205Ser + Ser210G1u + Leu211Va1 A1a194His + Va1199Ser + Thr202G1y -~ Thr207Asn + Leu211Met + G1y213G1u + Thr214Pro Va1199G1y + Tyr203Cys + Pro204G1y -~ Thr207Asn + A1a209His + Asn212Asp + G1y213Asn A1a194G1n + G1y196Pro + Tyr203Ser -~ Pro204Ser + Tyr208Ser + Leu211G1u + Thr214Pro G1y196G1n + G1y205Asn + Thr207Asp - Tyr208Met + Asn212G1n + G1y213Ser + Thr214Pro G1y196G1n + Va1199Cys + Ser206G1u - Thr207Pro + A1a209Pro + Leu211I1e + G1y213Ser Thr202Pro + Tyr203Va1 + Pro204Asn ,- Ser206Asp + Asn212Ser + G1y213Ser + Thr214G1y A1a194Thr + Pro195Asn + G1y196Pro ~- G1n200Asn + Pro204Ser + Asn212G1u + G1y213G1n Asn198G1n + G1n200Ser + Pro209G1n -~- G1y205Asn + Thr207G1u + Asn212Ser + G1y213Ser A1a194Ser + G1y196Ser + Asn198Ser ~- Tyr203G1y + G1y205Pro SO + Ser206G1u + G1y213Pro A1a194Thr + G1y196G1n + Va1197Pro ~- Thr202G1n + Pro204Ser + A1a209G1y + Asn212G1u WO 95130011 218 9 4 2 7 PCTlUS95/04760 ~s Pro195G1n + G1y196Pro + Asn198G1n + Pro209Ser + G1y205G1u + Tyr208G1n + G1y213Asn Va1199Thr + Tyr203G1n + Pro204G1n + G1y205Asn + Thr207G1u + Leu211Thr + Thr214G1n Va1197Thr + Asn198G1u + Thr202G1y + G1y205Pro + Thr207Pro + Tyr208Met + G1y213G1n Pro195G1y + G1y196Ser + Thr202G1y + Tyr203G1n + Pro204G1n + Tyr208Pro + Thr214G1n Pro195Asn + Asn198Asp + G1n200Ser + Pro204G1y + G1y205G1n + Tyr208G1y + A1a209Asn Pro195Asn + Va1199His + Pro204Asn + A1a209Pro + Leu211Thr + Asn212Ser + Thr214Pro Asn198G1n + Va1199Thr + G1n200Asp + Pro204Asn + Leu211Va1 + Asn212Ser + G1y213G1n Pro195Ser + G1y196Ser + Va1197Met + Va1199G1y + G1y205Pro + Ser210G1u + Asn212G1n A1a194Thr + Pro195G1n + Asn198G1n + Thr202Asn + Thr207Asp + Tyr208Thr + G1y213Ser Pro195G1n + G1y196Asn + G1y205G1u + Thr207Pro + Tyr208Asn + A1a209His + Asn212Ser Asn198G1n + Thr202Pro + Thr207Pro + Leu211Pro + Asn212G1u + G1y213Asn + Thr214Ser A1a194Pro + Va1197Met + Pro204Ser + Thr207G1n + Tyr208Asn + A1a209G1n + Thr219G1y A1a194Asn + Va1197Cys + Pro204Asn + Tyr208Thr + A1a209Pro + Ser210G1u + Leu211Ser G1y196Asn + Va1197G1y + Tyr203Pro + G1y205Asp + Leu211A1a + G1y213Asn + Thr214Ser A1a194Thr + Pro195Asn + Tyr203Pro + G1y205Ser + Ser210G1u + Leu211A1a + G1y213Pro A1a199His + G1y196Asn + Va1197Met + Asn198Ser + Ser206Asp + Leu211Asn + Asn212Ser A1a194Thr + G1y196Ser + Va1199Ser + Thr202Asn + A1a209Pro + Leu211Pro + G1y213G1n G1y196G1n + Va1199His + Thr202Pro + Tyr203Thr + Pro204Asp + G1y205Ser + A1a209Asn Va1197Cys + Va1199G1y + Thr202G1n + Tyr203His + G1y205G1u + Tyr208I1e + Thr214G1n A1a194G1y + Va1197Cys + Asn198Asp + G1n200Ser + Tyr203Leu + A1a209G1n + Asn212G1n A1a194G1y + Tyr203Cys + G1y205G1n + Ser206G1u + Tyr208Met + Leu211Cys + Thr214G1y G1y196Ser + Va1199G1n + Thr202Ser + Tyr203His + Pro204G1n + Tyr208Leu + G1y213Pro Asn198G1n + Thr202Asn + Tyr203A1a + Pro204G1u + Tyr208G1y + A1a209G1n + Thr214G1n A1a194Asn + Pro195G1n + Pro204G1y + Ser206G1u + Thr207Ser + Tyr208A1a + Asn212Ser A1a194Pro + Pro195G1n + Va1197Ser + Asn198Ser + G1n200G1u + Thr202Ser + G1y205Ser Pro195Ser + G1y196Ser + Va1199G1y + Thr202G1y + Pro209G1u + Leu211A1a + Thr214Asn A1a194Thr + G1y196Pro + Asn198Ser + Pro209Asn + Tyr208G1y + Ser210Asp + Thr214Asn ~' WO 95/30011 PCTIUS95104760 A1a194Pro + Va1199Asn + Thr202G1n + Pro204G1n + G1y205Asp + Thr207Ser + A1a209Pro A1a194Pro + Pro195G1y + Va1197Cys + Va1199G1n + Tyr203Cys + Tyr208His + G1y213G1u Va1197Asp + Va1199Ser + G1y205Pro + Thr207G1y + Leu211Thr + Asn212G1n + G1y213Ser A1a194Pro + Pro195Ser + G1n200Asn + Thr202Pro + Tyr208His + G1y213Asp + Thr214G1y A1a194Thr + Va1197Asn + Va1199Pro + G1n200Asp + Thr202Ser + Thr207Asn + Tyr208Leu Pro195Ser + G1y196G1n + Va1197A1a + Thr202G1n + Tyr203G1y + Tyr208Cys + G1y213Asn A1a194G1n + Va1199G1n + Thr202G1y + Pro204Asn + G1y205G1u + A1a209His + Leu211His Pro195Asn + Va1199Thr + Tyr203G1y + G1y205G1n + Ser206Asp + Asn212Ser + G1y213Pro A1a194G1n + Pro195Ser + G1y196G1n + Thr202G1n + A1a209His + Asn212Ser + Thr214G1u G1y196Pro + Va1197Cys + Tyr203Pro + G1y205Ser + Ser206Asp + Leu211I1e + Thr214G1y G1y196Asn + Va1197G1y + Va1199His + Thr202Ser + A1a209His + Asn212G1u + Thr219G1n G1y196G1n + G1n200Ser + Thr202G1n + G1y205G1n + A1a209Ser + G1y213Asp + Thr214G1u Va1199G1y + G1n200Asn + Thr202G1n + G1y205Ser + A1a209G1u + Ser210Asp + Leu211Asn Va1199Pro + G1n200Asn + Tyr203Va1 + A1a209Asp + Ser210Asp + Leu211Ser + G1y213Pro A1a199G1y + Tyr203Va1 + G1y205Asn + Thr207G1y + A1a209G1u + Ser210Asp + Leu211Pro Pro195G1n + Thr202G1y + Tyr203Va1 + A1a209G1u + Ser210G1u + G1y213Ser + Thr214G1n Pro195Asn + Va1197Ser + Tyr208Thr + A1a209G1u + Ser210Asp + Asn212Ser + Thr214G1n A1a194Asn + G1y196Ser + Va1199G1y + Thr202Pro + Tyr203Leu + Asn212G1u + G1y213Asp Va1197Cys + Asn198Ser + G1n200Ser + Thr202Asn + Thr207G1n + Asn212Asp + G1y213Asp G1y196Pro + Thr202Asn + Pro204Asp + G1y205G1u + A1a209His + Leu211His + G1y213Ser Pro195G1y + G1y196G1n + Tyr203Thr + Thr207Asn + Ser210Asp + Leu211Asp + Asn212Ser A1a194G1y + G1y196Pro + Thr202G1n + Tyr203Va1 + G1y205Ser + Ser210Asp + Leu211Asp A1a194His + Va1199Cys + Tyr203A1a + Tyr208G1y + Ser210Asp + Leu211Asp + G1y213Pro A1a194His + G1y196Ser + Va1197Cys + Va1199Ser + G1y205G1u + Ser206G1u + Tyr208Ser A1a194His + G1y196Ser + Va1197Thr + Va1199Cys + G1y205Asp SO + Ser206G1u + Thr214G1n Thr202Asn + Tyr203Thr + G1y205G1u + Ser206Asp + A1a209G1n + Leu211Thr + Thr214G1y A1a194Pro + Va1197Met + Asn198Ser + Va1199Asn + Thr202Ser + Ser206Asp + Thr207G1u W O 95/30011 21 g 9 4 2 ?
Pro195Asn + Va1199Met + Thr202G1y + Ser206G1u + Thr207Asp + Tyr208Thr + Asn212Ser Pro195G1y + G1y196Asn + Va1199G1n + Tyr203His + Ser206G1u + Thr207Asp + Asn212G1n 5 Pro195Asn + Va1199A1a + G1y205Pro + Ser206G1u + Thr207Asp + Tyr208G1y + Asn212Ser Pro195Asn + G1y196Ser + Va1197A1a + Pro209Ser + Ser206G1u + Thr207Asp + Asn212G1n Pro195Asn + Va1197Thr + Ser206Asp + Thr207Asp + Leu211Thr 10 + G1y213Ser + Thr219Ser A1a194Thr + Va1197Cys + Asn198Ser + Thr207Asp + Tyr208Asp + Asn212Ser + G1y213Asn A1a194Thr + G1n200Asp + Thr202G1n + A1a209His + Ser210Asp + Leu211G1y + Thr214G1y 15 Pro195G1n + G1n200Asp + Tyr203A1a + G1y205Asn + Tyr208Ser + Ser210Asp + G1y213Asn A1a194Thr + Va1199Pro + G1n200G1u + Thr207G1y + Ser210G1u + Leu211Ser + G1y213Asn Va1197Ser + G1n200G1u + Tyr203Leu + Pro204Asn + A1a209G1y 20 + Ser210Asp + Leu211Asn G1y196G1n + Asn198G1n + Va1199Pro + G1n200Asp + Thr202G1y + Pro204G1y + Ser210Asp Asn198G1n + Va1199Cys + G1n200G1u + G1y205G1n + Ser210Asp + G1y213Ser + Thr214G1y 25 Asn198G1n + G1n200G1u + G1y205Pro + Thr207Ser + A1a209Asn + Ser210G1u + Thr214Asn G1n200G1u + Thr202Asn + Thr207Ser + Tyr208Pro + Ser210G1u + G1y213G1n + Thr214G1y A1a194Pro + Pro195Ser + G1y196Asn + Va1197Asp + Va1199Thr 30 + Pro204G1y + Asn212G1u G1y196Asn + Asn198G1u + Tyr203G1y + Pro209G1n + Ser210Asp + Leu211Va1 + Asn212Ser G1y196Pro + Va1197G1y + Asn198Asp + Thr202Pro + Tyr203Va1 + Thr207Asn + Ser210G1u 35 Pro195Asn + G1y196Pro + Asn198Asp + Thr202Ser + Pro204G1n + Ser210Asp + G1y213G1n Pro195Ser + Va1197Pro + Asn198G1u + Va1199Met + Thr207Ser + Ser210Asp + Thr214G1n A1a194Ser + G1y196Asn + Va1197Met + Asn198Asp + Va1199Thr 40 + Tyr208Met + Leu211G1u Asn198Asp + Va1199G1n + Pro204Ser + Leu211Asp + Asn212Ser + G1y213Pro + Thr214Asn Pro195G1n + Asn198Asp + Thr202Pro + G1y205G1n + Tyr208Met + Leu211G1u + G1y213Asn 45 G1y196Pro + G1n200Asp + Thr202Ser + Tyr203Thr + Tyr208His + A1a209G1u + Leu211I1e A1a194Thr + G1n200Asn + Thr202Pro + Pro204Asp + Ser206G1u + Thr207Asn + A1a209Ser G1y196Pro + Pro204Asp + G1y205Ser + Ser206Asp + Tyr208G1y 50 + G1y213G1n + Thr214G1y Pro195G1n + Va1197G1n + G1n200Ser + Pro204G1u + Ser206Asp + A1a209G1y + Thr214G1n A1a194Thr + Pro195G1y + Va1197Pro + Pro204Asp + Ser206G1u + Leu211Va1 + G1y213Ser '~~8'9~~?
'° WO 95/30011 PCT/US95/04760 7l Pro195Asn + G1y196Ser + Pro204G1u + Ser206Asp + Thr207G1y + Tyr208Va1 + A1a209Thr Pro195Asn + G1y196Pro + Va1197G1n + Asn198Asp + Tyr203G1n + Tyr208Asn + Asn212Asp G1y196G1n + Va1197Thr + Asn198Asp + Va1199G1y + Thr202Pro + Tyr208Asn + Asn212G1u Pro195Asn + Asn198Asp + Va1199Asn + Thr202G1n + A1a209Pro + Asn212Asp + G1y213G1n A1a194G1y + Va1197Met + Asn198Asp + G1n200G1u + Thr202Asn + Pro204Asn + Tyr208Asn Pro195Ser + Va1197G1n + Asn198G1u + Va1199Cys + G1n200Asp + G1y205Ser + A1a209Pro Pro195G1n + G1y196Pro + Asn198G1u + Va1199Ser + G1n200Asp + G1y205Ser + Thr214Asn A1a194G1n + Pro195Ser + Asn198G1u + G1n200Asp + Thr207G1n + Tyr208Leu + Asn212G1n A1a194Pro + Pro195Asn + G1n200G1u + Pro209G1n + Thr207G1n + Tyr208Asp + G1y213G1n Va1199Thr + Gln'?OOAsp + Tyr208G1u + A1a209Thr + Leu211Ser + Asn212Ser + G1y213Asn G1y196Pro + Va1197G1u + Asn198G1n + Va1199Met + Tyr208Met + G1y213Asp + Thr214Asn Pro195G1y + Va1197G1u + Va1199Thr + G1n200Ser + G1y205Asn + A1a209G1y + Leu211G1u Va1197Met + Va1199Cys + G1y205Pro + Tyr208I1e + Leu211Asp + Asn212Ser + G1y213Asp Pro195G1n + Asn198Ser + Va1199G1n + Pro204Ser + Ser210Asp + Leu211Cys + Asn212G1u Pro195Ser + G1y196Pro + Asn198G1n + Pro209G1n + A1a209Thr + Ser210G1u + Asn212G1u A1a194G1y + Asn198G1n + Va1199G1n + Thr207Ser + Ser210Asp + Leu211G1y + Asn212Asp A1a194G1n + Va1197G1y + Thr202Ser + Pro204Ser + Ser210G1u + Asn212G1u + G1y213Ser A1a194Asn + Asn198Ser + Thr202Pro + Ser210G1u + Leu211Cys + Asn212Asp + G1y213Ser Pro195G1y + ValI97Thr + Thr202Asn + G1y205Ser + Ser210Asp + Asn212G1u + G1y213Ser Pro195G1n + G1y205G1n + A1a209G1n + Ser210Asp + Leu211Asn + Asn212G1u + Thr214G1n Va1199His + Pro209Asn + Tyr208I1e + A1a209Thr + Ser210G1u + Leu211G1n + Asn212Asp Va1197Asn + Asn198G1n + Tyr203Pro + Pro204Asn + Ser210G1u + Asn212G1u + Thr214G1n A1a194Pro + G1y196Ser + G1n200Ser + G1y205G1n + Tyr208G1y + Ser210G1u + Asn212G1u Pro195Ser + Va1197G1y + Asn198G1n + Va1199G1n + G1n200Ser + Ser210G1u + Asn212Asp Asn198G1n + G1n200Asn + Thr207G1y + Tyr208G1u + Ser210G1u + Asn212Ser + Thr219Asn Pro195G1y + Thr202Ser + Tyr208G1u + A1a209Asn + Ser210G1u + Asn212G1n + Thr214Asn Va1199Thr + Thr202Ser + G1y205Pro + Ser206G1u + Tyr208G1u + Leu211His + Asn212Ser 2~1g~4~~
A1a194G1n + G1y196Pro + Asn198G1n + Tyr203I1e + Ser206Asp + Tyr208Asp + G1y213G1n A1a194Ser + Pro195Ser + Thr202Ser + G1y205G1n + Ser206Asp + Tyr208Asp + Asn212G1n Pro195G1y + Va1197His + Tyr203G1n + Thr207Pro + Asn212Asp + G1y213Ser + Thr214Asp Pro195G1y + Va1197Met + Thr202Asn + Tyr208Met + Leu211Thr + Asn212G1u + Thr214Asp Va1197Ser + Asn198G1n + Thr202Ser + Pro204G1u + Thr207Ser + Tyr208Asp + A1a209Ser A1a194Pro + Va1199Asn + Pro204Asp + Tyr208G1u + Leu211G1y + Asn212G1n + G1y213Ser Pro195G1n + Va1197G1u + Va1199G1y + G1n200Ser + Thr207Ser + Ser210G1u + Leu211Asn Pro195Ser + Va1197Asp + Va1199Cys + Pro204G1y + Tyr208Ser + Ser210G1u + Asn212Ser A1a194G1n + Va1197Asp + Asn198Ser + Thr202Asn + Thr207Asn + Ser210G1u + Leu211His Va1197Asp + G1n200Asn + Tyr203Thr + Tyr208Va1 + Ser210Asp + Asn212Ser + Thr214Ser A1a194Pro + Va1197Asp + G1n200Ser + Thr202G1y + Leu211Cys + G1y213Asn + Thr214G1u Pro195Ser + Va1197G1u + Asn198G1n + Tyr208I1e + A1a209His + Leu211Va1 + Thr214G1u Pro195G1y + Va1197Asp + G1n200Ser + Thr202Pro + A1a209Thr + G1y213G1n + Thr214Asp Pro195Asn + G1y196Pro + Asn198Asp + Pro204Asn + G1y205Asn + A1a209Asn + G1y213Asp A1a194Pro + Asn198Asp + Va1199G1n + G1n200Ser + Thr207Pro + A1a209Asp + G1y213Asn Va1197Asp + Va1199His + G1n200G1u + Thr202Pro + Tyr203I1e + Thr207G1y + Thr214G1y Va1197G1u + G1n200Asp + Tyr203I1e + Pro204G1n + A1a209G1y + Leu211I1e + Thr214Pro Pro195Asn + G1y196Ser + Va1199Met + G1n200Asp + Tyr203A1a + Asn212Asp + G1y213Asn A1a194Thr + G1y196G1n + G1n200G1u + Tyr203Va1 + Tyr208A1a + Leu211A1a + Asn212G1u Va1197Asn + Asn198G1n + Pro204Asp + Tyr208Met + A1a209G1u + Asn212Ser + G1y213Asn Pro195Asn + Thr202G1y + Tyr203Asn + Pro204G1u + G1y205Asn + Thr207Ser + A1a209G1u A1a194Pro + G1y196Pro + Tyr203A1a + Thr207G1n + Leu211G1u + G1y213Pro + Thr219G1u Asn198G1n + Va1199Thr + Thr202Pro + Pro204G1n + A1a209Thr + Leu211G1u + Thr214Asp A1a194His + Va1199Cys + G1n200G1u + Tyr203I1e + G1y205Asn + Thr207G1u + Tyr208Met Va1199Pro + G1n200Asp + Pro204G1n + G1y205Asn + Thr207Asp + Tyr208Cys + Thr214Pro A1a199His + Pro195G1n + G1y196G1n + Va1199His + G1n200G1u + Thr207G1u + Asn212Ser A1a194G1y + Asn198G1n + G1n200Asn + Tyr203Leu + Ser210Asp + Leu211G1y + G1y213G1u ''"""' WO 95/30011 ~CTIUS95104760 Asn198G1n + G1n200Asn + Thr202Ser + Ser210Asp + Leu211Ser + G1y213G1u + Thr214Asn Pro195Asn + Va1197Met + Tyr203G1y + G1y205Pro + Tyr208His + Ser210Asp + G1y213Asp Pro195Ser + G1n200Asn + Thr207Pro + Ser210G1u + Leu211Met + G1y213G1u + 'rhr214G1n Va1197G1n + Va1199Ser + Thr202Asn + Tyr208G1y + A1a209G1y + Ser210G1u + G1y213Asp G1n200Asn + Pro204G1n + Thr207G1u + A1a209Ser + Ser210Asp l0 + Leu211Thr + G1y213Asn A1a194G1n + Thr202G1y + Tyr203Asn + Thr207Asp + Ser210Asp + Leu211Cys + G1y213Pro Pro195G1n + Va1199G1n + G1y205G1n + Thr207G1u + Tyr208I1e + Ser210Asp + Thr214G1y Pro195G1y + Va1197His + Va1199Met + Tyr203Thr + Thr207G1u + Ser210Asp + Asn212G1n A1a194G1n + Pro195G1n + Asn198Ser + Va1199G1n + Pro204G1n + Thr207G1u + Ser210G1u Va1197Pro + Asn198G1u + Va1199Thr + Pro204Asn + Leu211Cys + Asn212Ser + Thr219Asp A1a194Ser + Va1199Thr + G1n200G1u + Tyr203Cys + Tyr208G1y + Leu211G1y + G1y213G1u Va1199Pro + G1n200Asp + Tyr203Asn + Pro204G1y + Leu211Pro + G1y213G1u + Thr214Asn A1a194G1n + Asn198G1u + Thr207Ser + Tyr208G1u + Asn212G1n + G1y213G1n + Thr214G1n Pro195G1n + Asn198Asp + G1n200Ser + G1y205Ser + Tyr208Asp + Leu211A1a + Asn212Ser G1y196Pro + Va1197Asp + Asn198G1n + Va1199A1a + Pro204G1n + A1a209Asp + G1y213Asn Pro195Ser + Asn198G1n + Va1199G1y + Pro204G1u + A1a209G1y + Ser210Asp + Asn212G1n Pro195Ser + G1n200Ser + Pro204G1u + Thr207Pro + A1a209G1n + Ser210Asp + Asn212G1n A1a194His + G1y196Ser + Va1197G1y + Va1199Asn + G1n200G1u + Pro204G1u + A1a209Thr G1y196Pro + Asn198Ser + Pro204Asn + Tyr208G1y + A1a209Asp + Leu211Thr + G1y213Asp G1y196Pro + Va1199Thr + G1n200Asp + Thr202Asn + Tyr203Asn + Ser206G1u + A1a209Ser Pro195Ser + G1y196Pro + Va1199Pro + G1n200G1u + Pro204G1n + Ser206Asp + Leu211A1a Pro195Ser + G1n200Asp + Thr202G1n + Pro204G1n + Ser206G1u + Tyr208G1y + A1a209G1y G1y196Asn + Va1199Ser + G1n200G1u + Thr202Pro + A1a209Thr + Leu211Cys + Thr214Asp Va1197Asn + G1n200G1u + Tyr203G1n + G1y205Pro + A1a209Thr + Leu211Va1 + Thr214G1u Pro195G1y + Tyr203G1y + Ser206G1u + Tyr208G1y + A1a209G1n + Ser210G1u + Leu211I1e G1y196Asn + Va1197His + Va1199A1a + Pro209Asn + Ser206Asp + Ser210Asp + Leu211Thr A1a194G1n + Va1197Ser + Va1199Pro + Ser206Asp + Ser210G1u + Leu211Pro + Thr219G1v WO 95/30011 ~ ~ $ PC'TIUS95I04760 A1a194His + G1n200Ser + Thr202Asn + Ser206G1u + Tyr208Cys + Ser210Asp + Asn212Ser Va1197Thr + Thr202Ser + Pro209G1y + G1y205Asn + Ser206Asp + Ser210G1u + Leu211Pro Loop 6 - Octuple Mutation Variants Pro195Ser + Va1197Ser + Asn198Ser + Va1199G1y + Tyr203Thr + Thr207Asn + Tyr208Met + Asn212G1u Pro195G1n + G1y196G1n + Va1199G1n + Tyr203G1n + Tyr208Met + A1a209G1y + Leu211Va1 + G1y213Asp Pro195G1n + Asn198G1n + Va1199His + Pro209G1y + Ser206G1u + Thr207G1n + Tyr208Ser + Leu211G1n A1a194Thr + Va1197Met + Va1199His + Thr202Ser + Tyr203I1e + Thr207G1n + Tyr208Pro + Leu211G1y Pro195G1n + Va1197Asn + Va1199Cys + Tyr203Met + Pro204Asn + G1y205G1u + Thr207Pro + Tyr208Ser G1y196Ser + G1n200Asn + Pro204Asp + G1y205Ser + Thr207Pro + Tyr208Met + G1y213G1n + Thr214Ser Va1197G1n + G1n200Asn + Tyr203His + Pro204Asp + G1y205Ser + Tyr208Thr + A1a209G1n + Leu211Pro Pro195G1y + G1y196Ser + Va1197Pro + Va1199Ser + Thr202Asn + A1a209G1u + Leu211Ser + G1y213Pro A1a194His + G1y196Ser + Va1197Asn + Asn198Ser + Tyr203Leu + Pro204Asn + Thr207Ser + A1a209Thr A1a194Pro + G1y196Pro + Va1199Met + G1n200Asp + Thr202Asn + Thr207Asn + A1a209Pro + G1y213Pro Pro195Asn + G1y196Pro + Va1199Thr + Thr202G1n + Thr207G1y + Tyr208Ser + Ser210Asp + Asn212Ser A1a194Thr + Pro195Asn + G1y196G1n + Thr202Pro + Pro204Ser + G1y205Pro + Tyr208G1n + Leu211Asp A1a194G1y + Pro195Ser + Va1197A1a + Thr202G1y + Tyr203G1y + Pro204G1n + G1y205Ser + Leu211G1u Pro195Asn + Va1197G1u + Va1199Thr + G1n200Asn + Thr202Pro + Tyr203I1e + Pro209Ser + Leu211Cys A1a194His + Pro195G1n + Va1199Cys + G1n200Asp + Tyr203A1a + Thr207Ser + G1y213G1n + Thr219Pro A1a194G1n + G1y196G1n + Va1199His + Thr202Pro + Tyr203Thr + Pro204Asp + G1y205Ser + A1a209Asn Pro195Ser + G1y196Ser + G1n200Asn + Tyr203His + Thr207Pro + Leu211Met + G1y213G1u + Thr214G1n Va1197Cys + Asn198Ser + Va1199A1a + G1n200Asp + Tyr203His + Leu211Asn + Asn212G1n + G1y213Asn A1a194G1n + G1y196Pro + Asn198G1n + Va1199His + Tyr203Va1 + Thr207Ser + A1a209Ser + Leu211Asn Pro195G1y + G1y196Ser + Va1197Pro ~ Thr207G1y + Leu211Thr + Asn212Ser + G1y213Asn + Thr214G1u Va1197Asn + Thr202G1y + Tyr203A1a + G1y205G1n + Tyr208I1e + A1a209Thr + Ser210Asp + Leu211G1y A1a194Asn + Asn198G1n + Va1199Ser + Thr202G1y + Tyr203I1e + A1a209Thr + Ser210Asp + Thr214Ser Pro195Ser + G1y196Asn + Va1197Asn + Asn198Ser + Thr202Asn ..» w0 95/30011 ~~ 894z + Tyr203Cys + Tyr208Asn + Leu211Thr A1a194G1y + Pro195Ser + G1y196G1n + Va1199A1a + Thr202Pro + Thr207Ser + Asn212Ser + Thr214Asp Va1197Cys + Va1199Pro + Thr202Pro + Tyr203Ser + Ser206G1u + Thr207G1n + A1a209G1y + Thr214G1y A1a194G1y + Pro195G1n + G1y196G1n + Asn198Ser + Va1199Met + Thr202Pro + Thr207G1u + Thr214G1y A1a194His + Pro195G1n + Va1197G1n + Asn198G1n + Tyr203Ser + Ser206G1u + Tyr208A1a + Leu211A1a to A1a199G1y + G1y196Ser + Tyr203His + Pro204Asn + Ser206Asp + Tyr208I1e + Leu211G1n + Asn212Ser G1y196Pro + G1n200Ser + Tyr203G1n -~ Pro204Ser + G1y205Ser + Tyr208Cys + Leu211Met + G1y213G1n A1a194Asn + Tyr203Asn + G1y205G1u -+ Thr207G1n + A1a209Thr + Leu211His + G1y213Pro + Thr214G1n A1a194Ser + Pro195Asn + G1y196Asn -~ Va1199Ser + Pro204Ser + G1y205Asn + A1a209Asp + Thr214G1n Pro195Asn + G1y196G1n + Thr202Pro -~ G1y205Ser + Ser206G1u + Tyr208Ser + A1a209Pro + G1v213G1n A1a194Thr + Pro195G1y + Va1197Asp -F Asn198G1n + G1y205Ser + Tyr208Va1 + Asn212G1n + G1y213Ser Pro195G1y + Asn198Ser + Tyr203Thr -~ Pro204Asn + Tyr208Pro + A1a209Thr + Asn212Ser + G1y213Ser A1a199Thr + Pro195Ser + G1y196Asn -~ Va1197Met + G1n200Ser + Thr202Asn + Tyr203His + Tyr208G1n A1a194His + Asn198Ser + Tyr203G1n -~ Pro209G1n + A1a209Pro + Leu211Asp + Asn212G1u + G1y213.Asn A1a194Asn + Pro195Ser + Va1197G1y -~ Pro209G1y + G1y205Asn + A1a209Asp + Ser210Asp + Thr219G1n G1y196G1n + Va1197Pro + G1n200Asn - Thr202Asn + G1y205G1n + A1a209G1u + Ser210Asp + Thr219Pro Va1197Ser + Va1199A1a + Thr202Ser - Tyr203Pro + G1y205Asn + Tyr208I1e + A1a209Asp + Ser210~1u Asn198Ser + Va1199Met + G1n200Ser 1- Pro204Ser + Tyr208A1a + A1a209G1u + Ser210Asp + Leu211G1y A1a194G1y + Va1197Asn + Asn198Ser ~- G1n200Ser + Tyr203Thr + A1a209Asp + Ser210Asp + Thr214Pro A1a194His + Pro195Ser + Asn198Ser + Tyr203Cys + Thr207Ser + A1a209Asp + Ser210G1u + G1y213,Ser Pro195Ser + Va1199Asn + G1n200Ser ~- Pro204Ser + Thr207G1y + A1a209Asp + Ser210Asp + Asn212,5er A1a194G1n + G1y196Pro + Va1197Asp ~- Asn198G1u + Va1199Ser + G1y205G1n + A1a209G1y + Asn212;5er Pro195G1n + Va1197Asp + Asn198G1u ~- Va1199A1a + G1n200Ser + Thr202Ser + Thr207G1y + Tyr208A1a Pro195Ser + G1y196Pro + G1n200Asn ~- Thr202Asn + Tyr203Met + Thr207G1y + Asn212Asp + G1y213Asp G1y196Asn + Thr202Pro + Pro209G1y ~- Thr207Pro + Leu211Cys + Asn212G1u + G1y213G1u + Thr214:Pro G1y196Asn + Asn198G1n + G1n200Ser ~- Thr202Pro + Tyr203Cys + Pro204Asn + Asn212Asp + G1y213c.;1u Asn198Ser + Va1199G1y + Tyr203A1a ~~ Pro204Asp + G1y205G1u + Tyr208Met + A1a209Ser + Leu211~ro Va1197Cys + Va1199Asn + Thr2U2Gln + Pro204Asp + G1y205Asp + A1a209G1n + Leu211G1n'+: Asn212Ser Pro195Asn + Va1199Thr.+. Thr202G1y + A1a209His + Ser210G1u + Leu211Asp + Asn212Ser + G1y213Asn G1y196Asn + Va1199His + Tyr203A1a + G1y205Asp + Ser206Asp + Thr207G1n + Tyr208G1n + Leu211Asn A1a194G1y + G1y196G1n + Va1199G1n + G1n200Asn + Pro204G1y + G1y205Asp + Ser206Asp + Thr214Ser A1a194G1n + Pro195Asn + Va1197A1a + G1n200Asn + Tyr203Ser + G1y205Asp + Ser206Asp + Tyr208A1a A1a194G1n + Va1197G1n + Pro204Asn + G1y205Asp + Ser206G1u + Tyr208A1a + A1a209His + G1y213Asn Pro195Ser + G1y196Asn + Va1197Thr + G1n200Asn + Pro204Ser + G1y205G1u + Ser206G1u + Tyr208G1y Va1197G1y + Asn198G1n + Va1199Met + G1n200Asn + Tyr203His + Ser206Asp + Thr207G1u + Thr219Pro A1a194G1n + Pro195G1y + G1y196Pro + Thr202Pro + Ser206Asp + Thr207Asp + Tyr208Va1 + A1a209His A1a194Thr + Pro195Ser + Va1197G1n + Asn198G1n + Thr202Ser + Pro204Asn + Ser206G1u + Thr207Asp A1a194G1n + Va1199Met + Thr202Ser + Tyr203G1y + G1y205G1n + Ser206Asp + Thr207G1u + G1y213Ser A1a194G1y + Pro195Asn + G1y196G1n + Tyr203A1a + Ser206Asp + Thr207G1u + A1a209Thr + Asn212G1n G1y196Ser + G1n200Ser + Tyr203Leu + Pro204Ser + Ser206G1u 2,5+ Thr207G1u + A1a209Ser + G1y213Ser A1a194Ser + Pro195Asn + Thr202G1n + Ser206G1u + Thr207G1u + Tyr208G1y + Asn212Ser + Thr214G1y A1a199His + Pro195G1n + Va1197A1a + Thr202Asn + Tyr203Pro + G1y205Pro + Tyr208Asp + A1a209Asp A1a194G1n + Pro195G1n + Va1197Asn + G1n200Asp + A1a209G1y + Ser210G1u + Leu211A1a + Thr214Asn A1a194Pro + Va1199Met + G1n200G1u + Thr202G1y + Pro204G1n + G1y205Asn + Tyr208G1n + Ser210Asp G1y196Pro + Va1197Cys + Va1199Asn + G1n200G1u + G1y205Pro + Tyr208Va1 + Ser210G1u + Thr214Pro A1a194Thr + Pro195G1y + G1y196G1n + Va1197Asp + Va1199His + G1n200Ser + Thr202Ser + Asn212Asp Asn198Asp + Va1199Pro + G1y205Asn + Thr207G1y + Tyr208Pro + Ser210Asp + Leu211G1y + G1y213G1n A1a199G1n + G1y196Pro + Asn198G1u + Tyr203G1n + Thr207G1n + Tyr208Pro + A1a209Pro + Ser210G1u A1a194Pro + Asn198Asp + Va1199His + G1y205Asn + Ser210G1u + Leu211Asn + Asn212Ser + Thr214Pro Asn198G1u + Va1199Pro + Tyr203Asn + Thr207Pro + A1a209Asn + Ser210G1u + Asn212Ser + G1y213G1n A1a194G1y + Pro195Asn + Asn198Asp + Tyr203A1a + G1y205G1n + Thr207Ser + Ser210G1u + Leu211Va1 A1a194Thr + Asn198Asp + Thr202G1y + Pro204Ser + G1y205Ser + Thr207Asn + A1a209Pro + Ser210G1u SO A1a194G1n + Asn198Asp + Tyr203A1a + Thr207G1n + Tyr208Pro + Ser210G1u + Leu211Pro + Asn212Ser A1a194Thr + Pro195Ser + Asn198Asp + Tyr203Cys + Pro204Asn + Thr207G1n + Tyr208G1n + Ser210G1u Pro195Ser + Asn198Asp + Va1199Ser + G1n200Ser + Pro204Ser WO 95130011 . PCTIUS95I04760 + Ser210G1u + Leu211G1y + Asn212Ser A1a194G1n + Pro195Ser + G1y196Pro + G1n200G1u + Pro204Asn + G1y205Pro + A1a209G1u + Leu211Cys Va1199His + G1n200Ser + Pro204Asp ~~ G1y205Ser + Ser206Asp S + Tyr208G1y + G1y213G1n + Thr214G1y G1y196Asn + Asn198Ser + Thr202Ser ~~ Pro204Asp + G1y205Ser + Ser206Asp + Thr207Ser + Tyr208Va1 Va1197A1a + G1n200Asn + Pro204G1u -~ G1y205Ser + Ser206G1u + Tyr208Va1 + Leu211Ser + Asn212Ser l0 Pro195Asn + Va1197Thr + Thr202Ser -~ Tyr203I1e + Pro204Asp + Ser206Asp + Thr207G1n + A1a209G1n Pro195G1y + Va1197Asn + Va1199Ser -~ G1n200Asn + Tyr203Asn + Pro204G1u + Ser206G1u + Leu211Met G1y196Ser + Asn198Ser + Va1199Met -~ G1n200Asn + Pro204G1u 15 + Ser206Asp + Leu211Va1 + Thr214G1n A1a194G1n + G1y196Asn + Asn198Asp -~ G1n200Ser + Thr202Asn + Pro204Asn + Asn212G1u + Thr214.Asn Pro195G1y + Asn198G1u + Va1199Asn -r G1n200Ser + Thr202G1y + Thr207Asn + A1a209Thr + Asn212.Asp 20 Pro195Asn + G1y196Ser + Asn198G1u -- Thr207Ser + Tyr208Ser + A1a209Pro + Asn212Asp + Thr214.Asn A1a194Pro + Va1197Ser + Asn198G1u ~- G1n200Asn + Tyr203Cys + Thr207Pro + Tyr208Thr + Asn212-?asp Pro195G1y + G1y196Ser + Thr202Asn ~- Pro209Asn + G1y205G1u 25 + Thr207Asp + Tyr208Thr + Asn212Ser Va1197G1n + Va1199Asn + Thr202Pro ~- G1y205G1u + Thr207G1u + Tyr208A1a + Leu211Ser + G1y213:~sn A1a194Ser + Pro195Ser + Va1197G1y ~- Pro204Asp + G1y205Ser + Thr207G1u + Tyr208Va1 + Leu211G1y 30 A1a194G1n + Pro195G1y + G1y196Asn ~- Va1197Cys + Asn198G1n + Pro204G1u + Thr207Asp + G1y213G1n Pro195G1y + Asn198Asp + G1n200G1u + Tyr203Thr + G1y205Asn + Leu211Cys + Asn212Ser + G1y213G1n A1a194G1y + Pro195Asn + Asn198G1u -~- G1n200G1u+ Thr202G1n 35 + G1y205Ser + Thr207Pro + G1y213G1n Pro195G1y + Asn198G1u + Va1199G1n -+- G1n200Asp+ Thr202G1y + Pro209G1n + Thr207Pro + Leu211Cys Pro195Asn + G1y195G1n + Asn198G1u ~~ G1n200Asp + Thr202G1y + Tyr208G1n + Leu211Ser + Thr214Ser 40 Va1197Asn + Asn198Ser + G1n200G1u +~ Thr202Asn + Tyr203Cys + Pro204Ser + G1y205Pro + Tyr208c;1u A1a194His + Va1199His + Tyr203Asn + Tyr208His + A1a209G1u + Leu211Asp + Asn212G1n + G1y213c;1n G1y196G1n + Va1197Ser + G1n200G1u + Tyr203Va1 + Thr207Pro 45 + A1a209Thr + Leu211G1u + Asn212:3er Pro195G1n + Va1199G1y + G1n200Asp + Pro204Asn + G1y205Asn + Tyr208I1e + A1a209Pro + Leu211(~lu Pro195G1n + Asn198Ser + Va1199Asn + G1n200Asp + G1y205Pro + Tyr208Ser + Leu211G1u + Thr214Asn 50 A1a194His + Pro195Ser + Va1197G1u + Va1199Cys + Thr202Pro + Leu211G1u + Asn212G1n + Thr214Asn A1a194Ser + Pro195G1n + Asn198Ser + G1n200Asn + G1y205G1n + Leu211Asp + Asn212G1n + G1y213c;1u G1y196G1n + Va1199G1n + Tyr203G1y + G1y205Asn + A1a209His WO 95130011 218 9 .4. 2 ~ PCTIUS95I04760 7s + Leu211Asp + Asn212G1n + G1y213Asp Asn198G1n + G1n200Ser + Tyr203Leu + G1y205Asn + Thr207G1u + A1a209Asp + Leu211Ser + G1y213Asn A1a194Ser + Pro195Ser + Va1199G1n + G1n200Ser + G1y205G1n S + Ser210Asp + Asn212G1u + Thr214G1n Pro195Asn + Tyr203Cys + Pro209G1n + Tyr208Asn + A1a209His + Ser210Asp + Asn212Asp + Thr219G1y G1y196Asn + Va1199Ser + Thr202G1n + Pro204Ser + Thr207G1y + Ser210G1u + Leu211Thr + Asn212Asp G1y196G1n + Va1197Cys + Asn198Ser + Pro204G1n + Thr207Asn + A1a209G1n + Ser210Asp + Asn212Asp Pro195G1y + G1y196Pro + Va1197Cys + Asn198Ser + G1n200Asn + Tyr208His + Ser210Asp + Asn212G1u Pro195G1n + Asn198Ser + Va1199G1y + Thr202Pro + Thr207Ser + Tyr208I1e + Ser210Asp + Asn212Asp A1a194Asn + Va1197Met + Va1199Thr + A1a209Asn + Ser210Asp + Leu211Va1 + Asn212G1u + G1y213G1n A1a194G1n + Pro195Asn + Va1199Asn + Tyr203Ser + Thr207Asn + Ser210Asp + Leu211G1y + Asn212Asp Va1199Cys + G1n200Ser + Thr202Asn + G1y205Asn + Thr207G1y + Tyr208Asn + Ser210G1u + Asn212Asp A1a194Thr + Va1199Cys + G1n200Ser + G1y205Ser + Ser206G1u + Tyr208Asp + Leu211A1a + G1y213Asn A1a194Pro + Va1197Pro + G1n200Ser + Ser206Asp + Thr207Pro + Tyr208Asp + A1a209Pro + Thr214G1y G1y196G1n + Asn198Ser + Va1199Met + Thr202Ser + Pro204Asn + Ser206G1u + Tyr208G1u + Asn212G1n Pro195Asn + G1y196Asn + Va1199A1a + G1n200Asn + Ser206Asp + Thr207Asn + Tyr208Asp + A1a209Ser G1y196Asn + Tyr203A1a + Pro204Ser + Ser206Asp + Tyr208Asp + A1a209Ser + Asn212Ser + Thr214G1n Va1197G1y + Asn198Ser + Tyr203Asn + Pro204G1y + G1y205Ser + Thr207Pro + Asn212Asp + Thr214Asp A1a194G1n + Va1197Asp + Asn198G1n + Tyr203His + Ser210G1u + Leu211A1a + G1y213Ser + Thr219Asn A1a199Pro + Va1197G1u + Va1199His + G1n200Ser + Tyr203His + Pro204Asn + Tyr208Thr + Thr214G1u G1y196Asn + Asn198Asp + Va1199G1n + G1n200Ser + Thr202Ser + Pro204Ser + Leu211Cys + G1y213G1u Asn198Asp + Tyr203Met + Pro209G1y + Thr207Asn + Tyr208G1n + A1a209G1u + Asn212Ser + Thr214Ser Asn198G1u + Va1199G1y + Thr202G1y + Thr207G1n + Tyr208Cys + A1a209Asp + Leu211Thr + Asn212G1n A1a194His + Va1197Asp + Asn198G1n + G1n200G1u + Thr202Asn + Tyr208Pro + Leu211Ser + Asn212Ser Pro195G1y + G1n200Asp + Tyr203G1y + G1y205Asn + Leu211Ser + Asn212G1u + G1y213G1n + Thr214G1n A1a194Asn + G1y196Ser + Va1199Pro + G1n200G1u + Thr207G1y + Tyr208Asn + A1a209Thr + Asn212G1u A1a194Thr + Va1199Asn + G1n200G1u + Tyr203G1n + Pro204G1y + A1a209His + Asn212G1u + Thr214Ser A1a194Asn + G1y196Pro + Va1197Ser + Asn198G1n + Tyr203Pro + Pro204Asp + Tyr208Asn + A1a209Asp Pro195Ser + Va1199Cys + G1n200Ser + Pro204G1u + G1y205Ser "' WO 95/30011 PCTIUS95104760 + Tyr208A1a + A1a209G1u + Asn212G1n Asn198G1n + Va1199His + Thr202G1y -~ Pro209G1u + G1y205Pro + Tyr208A1a + A1a209G1u + Asn212G1n G1y196Ser + Asn198Ser + Va1199G1y -~ G1n200Ser + G1y205Ser S + Tyr208Asp + Leu211Asp + Thr214Ser Pro195G1n + G1y196Asn + Va1197G1y -~ G1n200Asn + Pro204Asn + Thr207G1y + A1a209Asp + Asn212G1u A1a194Ser + G1n200Ser + G1y205Ser -~ A1a209Asp + Leu211G1y + Asn212Asp + G1y213G1n + Thr214.Asn A1a194G1n + Pro195G1n + G1y196Pro -r Tyr203Leu + Pro204Ser + Ser206Asp + A1a209G1u + Leu211Pro Thr202Asn + Tyr203A1a + Pro209Ser -- Ser206Asp + Tyr208Cys + A1a209G1u + Leu211Cys + Asn212Ser G1y196Ser + Va1199His + G1n200Asn ~- G1y205Asn + Ser206G1u + A1a209Asp + Leu211His + Asn212Ser A1a194Asn + Va1197A1a + G1n200Ser ~- Ser206G1u + Thr207Pro + A1a209G1u + Asn212G1n + G1y213.~sn Pro195G1n + Va1197Met + Va1199Thr + G1n200Ser + Tyr203A1a + A1a209Ser + Ser210G1u + G1y213G1u Asn198Ser + Va1199Asn + A1a209Pro ~- Ser210G1u + Leu211G1y + Asn212Ser + G1y213Asp + Thr214G1y A1a194G1y + Asn198G1n + Tyr203A1a ~- G1y205Asp + Tyr208G1n + A1a209Asp + Leu211A1a + Asn212G1n A1a194G1y + G1y196G1n + Thr202G1n ~- Tyr203Thr + Thr207Asp + Tyr208Thr + Ser210G1u + Asn212Ser A1a194Pro + Va1197Thr + Tyr203Pro 1- Pro209Ser + Thr207Asp + Ser210Asp + Leu211Pro + Thr214Ser Pro195Asn + Va1199A1a + Thr202Ser ~- G1y205G1n + Thr207Asp + A1a209His + Ser210G1u + G1y213:~1sn Pro195G1y + G1y196Ser + Va1197Pro ~- Pro209G1y + Thr207G1u + Tyr208G1n + A1a209His + Ser210:asp A1a194Asn + Pro195Asn + Asn198Ser ~- Va1199His + Thr207Asp + Tyr208Va1 + A1a209Pro + Ser210G1u A1a194G1y + G1y196Ser + Asn198Asp ~- Thr207Asn + A1a209G1n + Leu211Pro + G1y213G1n + Thr214:~sp A1a194G1y + Pro195Ser + Va1197His ~- Asn198Asp + G1n200Asn + Pro204Ser + Asn212Ser + Thr214:~sp Va1199Asn + G1n200G1u + Pro209G1y a- G1y205Asn + Tyr208Ser + Leu211A1a + Asn212Ser + G1y213G1u A1a194G1n + Pro195Ser + G1y196Asn -+- Asn198G1n+ G1n200G1u + Tyr203Cys + Leu211Ser + G1y213G1u A1a194Ser + G1y196Asn + Va1197Thr 1- Asn198G1n + G1n200G1u + Thr207Asn + Tyr208His + G1y213Asp A1a194G1n + Va1197G1n + Asn198Asp -~- G1n200Asn+ G1y205G1n + Tyr208Asp + Leu211G1y + Thr214Ser A1a199Ser + Asn198Ser + Thr202Pro -+- Pro204Asp+ G1y205G1n + Thr207Asn + Ser210Asp + Asn212:~er Pro195G1n + G1y196Asn + Pro204G1u ~~ G1y205Asn + Thr207Asn + Tyr208Ser + A1a209Pro + Ser210:~sp 5o A1a194Thr + G1y196Pro + Va1199Pro + G1n200Asn + Pro204G1u + A1a209Thr + Ser210Asp + Leu211:~1a A1a194Ser + G1y196G1n + Va1199Thr + Pro204G1u + Tyr208I1e + A1a209G1n + Ser210Asp + Thr214G1y A1a194His + Va1197Ser + Va1199Ser ~ G1n200Ser + Pro204G1u ~I89~2'~

+ Thr207G1y + A1a209Pro + Ser210Asp A1a194G1n + Va1197A1a + Asn198Ser + Pro204Asp + Tyr208Cys + A1a209G1y + Ser210Asp + Asn212Ser G1y196Ser + Va1197Thr + Asn198Ser + G1n200G1u + Thr202Pro 5 + Pro204G1u + A1a209Thr + G1y213Ser Pro195Asn + Va1197Pro + Va1199G1y + G1n200Asp + Pro204G1u + Thr207G1y + Leu211Thr + G1y213Ser A1a194G1n + Va1197Asn + G1n200Ser + Thr202G1n + Thr207G1u + A1a209G1y + Leu211G1u + G1y213Asn 10A1a194Ser + G1y196Ser + Va1197Ser + Asn198G1n + G1y205G1n + Ser210G1u + Asn212Ser + Thr219Asp G1y196Ser + Va1199Cys + G1n200Ser + Thr207Pro + Tyr208Pro + A1a209G1y + Ser210Asp + Thr219Asp Va1197Cys + Va1199G1y + Tyr203G1y + Tyr208Met + A1a209Ser 15+ Ser210G1u + Leu211A1a + Thr214G1u A1a194Pro + Pro195Ser + G1y196Ser + Va1199Asn + Thr202G1y + Ser210Asp + Asn212G1n + Thr219G1u A1a194Asn + G1y196Asn + G1n200Ser + Pro204Asn + G1y205Ser + A1a209Ser + Ser210Asp + Thr214Asp 20A1a194His + Va1197Cys + Tyr208I1e + Ser210G1u + Leu211G1y + Asn212Ser + G1y213Asn + Thr219Asp G1y196G1n + G1n200Asn + Thr202Pro + Tyr203Cys + G1y205G1n + Tyr208Ser + Ser210G1u + Thr219Asp Asn198G1n + Va1199Pro + G1n200G1u + Thr202Pro + Pro204Ser 25+ Ser206G1u + Tyr208G1n + Thr214Asn Pro195G1y + G1n200Asp + Tyr203Va1 + Ser206Asp + Tyr208Thr + Leu211G1y + Asn212G1n + G1y213Asn G1y196G1n + G1n200G1u + Thr202G1n + Tyr203Va1 + Ser206G1u + Tyr208Va1 + A1a209Asn + Leu211G1y 30Pro195G1y + G1n200G1u + Thr202Pro + G1y205Ser + Ser206G1u + A1a209G1n + Leu211His + Asn212G1n Asn198G1n + G1n200G1u + Pro204G1y + G1y205G1n + Ser206Asp + Tyr208Met + Asn212Ser + Thr214Asn A1a194Pro + Pro195G1n + G1y196Ser + Va1197His + G1n200G1u 35+ G1y205Ser + Ser206G1u + Leu211Ser A1a194G1n + G1n200Asp + Tyr203G1n + Pro204Asn + Ser206G1u + Tyr208I1e + A1a209G1y + G1y213Asn A1a194G1n + Pro195G1n + Va1197His + G1n200G1u + Pro204Ser + Ser206Asp + Tyr208G1n + A1a209Asn 40Pro195G1n + Va1197His + Va1199Asn + G1n200Asp + Thr202Pro + Ser206G1u + Leu211Thr + Thr214Pro Pro195G1y + Asn198G1n + Va1199Cys + G1n200Asp + Thr202G1y + Ser206G1u + Tyr208Cys + G1y213Ser A1a194G1y + Pro195Asn + G1y196Asn + Pro204Asp + Thr207Ser 45+ Leu211G1u + Asn212G1n + Thr214Asn Loop 6 - Nonuple Mutation Variants A1a194G1y + Pro195Ser + Va1197Cys + Va1199Asn + G1n200Asp 50 + Tyr203Pro + Pro204Ser + G1y205Ser + Leu211G1n A1a194Pro + G1n200Asn + Thr202Asn + Tyr203Asn + Pro204Ser + Tyr208Asn + A1a209Asn + Ser210Asp + Thr214G1n ~~ss~z~
~" WO 95130011 PCT/US95/04760 s~
Pro195G1y + Va1199A1a + G1n200Ser + Thr202G1y + G1y205Pro + Ser206G1u + A1a209His + Leu211A1a + G1y213Asn Pro195G1n + G1y196G1n + Va1197Thr ~- Va1199His + Thr202G1n + Tyr203G1y + Pro204G1y + G1y205G1u + G1y213G1n S Pro195G1y + Va1197G1y + Va1199Asn ~- Pro204Asn + Ser206Asp + Thr207Ser + Tyr208G1n + A1a209G1y + Thr214Asn A1a194Thr + Va1197Asn + Va1199Pro ~- G1n200Asp + Thr202Ser + Tyr203Asn + Thr207Asn + Tyr208Leu + A1a209G1y Pro195Asn + G1y196Asn + Asn198G1n ~- Tyr203G1n + Thr207Ser + A1a209Thr + Leu211A1a + Asn212Asp + Thr214Asn A1a194G1n + Pro195G1y + G1y196Pro + Va1197G1y + G1n200Asp + Thr202Ser + Pro204G1y + Tyr208Thr + Thr214G1n G1y196Asn + Va1199G1y + Tyr203Ser 1- Pro209Ser + Tyr208G1n + A1a209His + Asn212Ser + G1y213.Asp + Thr214G1n IS Va1197His + G1n200Ser + Thr202Asn -+- Pro204Ser + Thr207Pro + Tyr208G1y + A1a209His + Ser210G1u + Leu211A1a Pro195Ser + Va1197His + G1n200G1u +~ Thr202G1n + Pro204Ser + Tyr208Va1 + A1a209His + Leu211Thr + Thr214Pro Pro195G1y + G1y196Pro + Va1199Thr + Thr202Pro + Tyr203I1e + Thr207G1y + A1a209Pro + Ser210.Asp + Asn212G1n A1a199G1y + Pro195G1n + Va1197Asp +~ Tyr203Cys + Tyr208Va1 + A1a209G1n + Leu211G1y + Asn212G1n + G1y213G1n G1y196Asn + Asn198G1n + Thr202G1y +~ Thr207Asp + Tyr208G1y + A1a209Ser + Leu211Thr + Asn212Ser + Thr214Pro A1a194Pro + G1y196Asn + Va1197Met + G1n200G1u + Tyr203G1n + G1y205Asn + Tyr208Asn + A1a209'Thr + Thr214G1n A1a194Pro + G1y196G1n + Va1197Asn + G1y205Ser + Ser206Asp + Thr207Asn + A1a209Ser + Leu211Cys + Thr214Pro G1y196G1n + Va1197Thr + G1n200Ser + Tyr203Asn + Pro204Asn + G1y205Pro + Tyr208Ser + Ser210G1u + Asn212Ser A1a194Thr + Pro195Ser + G1y196Pro + Va1197Cys + G1n200G1u + Pro204Ser + G1y205Asn + A1a209:His + Leu211Pro Pro195G1n + G1y196Asn + Asn198G1n + Va1199G1n + G1n200Asp + Pro204Ser + G1y205Asn + Thr207;Ser + Tyr208Ser A1a194His + Va1197Asn + Tyr203G1y + Pro204G1n + Thr207G1y + A1a209G1n + Leu211His + Asn212G1n + G1y213G1n A1a194Ser + Pro195G1n + Va1197Met + Va1199His + Thr207Pro + Tyr208A1a + Asn212G1n + G1y213G1u + Thr214Asn G1y196Pro + Va1197His + Asn198G1n + G1n200G1u + Tyr203Ser + G1y205Pro + Thr207Ser + Leu211'Thr + G1y213Asn G1y196Ser + Va1197Asn + Va1199Thr + Thr202G1y + Tyr203Asn + Pro204Asn + A1a209G1n + Leu211:Pro + Thr214Asp A1a194Thr + Pro195Ser + Va1197Asn + Tyr203Met + Tyr208A1a + A1a209Ser + Asn212G1n + G1y213;~sp + Thr214Asp A1a199Asn + G1y196G1n + Va1197Thr + Va1199A1a + G1y205Asn + A1a209Pro + Leu211Asn + G1y213G1u + Thr214Asp Pro195Asn + Va1199Met + G1n200Asn + Thr202Asn + Tyr203Cys + A1a209Ser + Leu211G1n + G1y213Asp + Thr214G1u Pro195Asn + Va1197Met + Asn198Ser + Va1199Asn + G1n200Asn SO + Tyr208G1n + Leu211His + G1y213G1u + Thr214Asp Va1197His + Va1199A1a + G1n200Ser + Pro209Ser + A1a209G1u + Ser210G1u + Asn212G1n + G1y213:Pro + Thr219Ser G1y196G1n + Va1197Asn + Asn198G1n + Thr202G1y + G1y205G1n + A1a209Asp + Ser210G1u + Leu211Met + Thr219G1y WO 95130011 ~ ~ PCT/US95104760 sz Va1197Cys + Asn198Ser + Va1199G1n + Tyr203G1y + Pro209G1n + A1a209G1u + Ser210G1u + G1y213Pro + Thr214G1y Pro195Asn + Va1199His + G1n200Ser + Pro204G1n + G1y205G1n + A1a209G1u + Ser210Asp + Leu211G1n + Asn212G1n S Va1197Asp + Asn198G1u + Va1199Met + Thr202G1n + Pro204Ser + G1y205G1n + Thr207G1n + Tyr208His + Asn212Ser A1a194Thr + Va1197G1u + Asn198Asp + Va1199His + Tyr203Asn + Tyr208His + A1a209Ser + G1y213Ser + Thr214Asn Va1197Cys + Asn198Ser + Va1199Pro + G1n200Ser + Tyr203A1a + Tyr208Asn + Asn212Asp + G1y213G1u + Thr214Ser G1y196Asn + Va1197Met + Asn198Ser + Thr202G1y + Pro204G1n + A1a209Ser + Leu211His + Asn212Asp + G1y213Asp A1a194Thr + Pro195Ser + G1n200Asn + Thr202Asn + Tyr203G1n + Tyr208Ser + Leu211A1a + Asn212Asp + G1y213Asp Pro195G1y + Asn198Ser + Va1199Pro + G1y205Asp + Ser206Asp + Thr207Pro + Tyr208A1a + Asn212Ser + G1y213Ser Pro195G1n + Asn198Ser + G1n200Asn + Thr202Ser + Tyr203Met + G1y205Asp + Ser206G1u + Thr207Pro + Thr219Ser G1y196Ser + Va1199Thr + G1n200Asn + Tyr203A1a + G1y205Asp + Ser206Asp + Tyr208Leu + A1a209Thr + G1y213Pro A1a194Pro + G1y196Ser + Va1197Asn + Va1199His + G1n200Asn + G1y205G1u + Ser206G1u + Asn212G1n + Thr214G1n A1a194Thr + Va1199Ser + Thr202Asn + G1y205G1u + Ser206Asp + A1a209Pro + Leu211Pro + Asn212G1n + G1y213G1n Asn198Ser + Thr202Ser + Tyr203Ser + Pro209G1y + G1y205Asn + Ser206Asp + Thr207G1u + A1a209Pro + Thr214Ser Pro195Asn + G1y196Asn + Va1197Pro + Va1199Ser + Thr202Ser + Ser206G1u + Thr207G1u + Leu211I1e + G1y213Ser Thr202Ser + Tyr203Thr + Thr207Asp + Tyr208G1u + A1a209Pro + Leu211Va1 + Asn212Ser + G1y213Ser + Thr214Asn Va1199Pro + G1n200Ser + Thr202Ser + Thr207Asp + Tyr208Asp + A1a209G1y + Leu211Met + Asn212Ser + Thr214Pro A1a194G1n + Pro195G1y + Va1197A1a + G1n200G1u + Tyr203Thr + G1y205Ser + A1a209Pro + Ser210Asp + Thr214Asn A1a194Pro + Pro195Ser + Va1199Met + G1n200G1u + Thr202G1y + Pro204G1n + G1y205Asn + Tyr208G1n + Ser210Asp A1a194G1n + Va1199Pro + G1n200Asp + Pro204G1y + G1y205G1n + Thr207Pro + Tyr208Asn + A1a209G1n + Ser210Asp A1a194Pro + G1y196Pro + Asn198G1n + G1n200Asp + Pro209G1y + G1y205Asn + Tyr208I1e + Ser210Asp + Asn212Ser Prol955er + Asn198Ser + Va1199A1a + G1n200G1u + Tyr203Thr + G1y205G1n + Tyr208Met + Ser210G1u + G1y213Pro Pro195Asn + Asn198Ser + G1n200Asp + Thr202G1n + Tyr203A1a + G1y205Pro + Ser210Asp + Leu211Ser + G1y213Ser Pro195Asn + G1y196Asn + Va1197A1a + Asn198Ser + G1n200Asp + Tyr203G1n + Pro204Asn + A1a209Ser + Ser210Asp Pro195Ser + Va1197Pro + Asn198G1n + Va1199Pro + G1n200G1u + Thr202G1n + Thr207Asn + A1a209His + Ser210G1u Pro195Ser + G1y196Asn + Asn198G1n + G1n200G1u + Thr202Ser + Pro204G1y + Thr207Asn + Ser210G1u + Thr219Asn A1a194Ser + Pro195Ser + Va1197His + Va1199Thr + G1n200G1u + G1y205G1n + Thr207G1y + Ser210G1u + G1y213Pro Pro195Asn + Va1197Asp + Asn198Ser + Va1199Asn + Thr207Pro + Tyr208A1a + Leu211Va1 + Asn212G1u + G1y213Ser Pro195Asn + G1y196Asn + Va1197G1n + Asn198G1u + Va1199Thr + Tyr203Asn + Thr207Pro + Ser210G1u + G1y213G1n Va1197Cys + Asn198Asp + Va1199Ser + Tyr203G1n + G1y205Ser + Thr207Ser + Tyr208Leu + Ser210Asp + Leu211G1y A1a194Pro + G1y196G1n + Va1197G1y + Asn198G1u + G1n200Ser + Tyr203Thr + Pro204Ser + A1a209His + Ser210G1u G1y196Ser + Va1197His + Asn198G1u + Thr202Pro + Tyr203I1e + Thr207Asn + Tyr208Va1 + Ser210G1u + Thr219Ser G1y196Asn + Va1197Thr + Asn198Asp + Va1199A1a + Pro209Asn + Ser210Asp + Leu211Va1 + G1y21:3G1n + Thr219Pro G1y196Ser + Asn198G1u + Thr202Asn + G1y205Ser + Thr207G1n + Tyr208I1e + Ser210G1u + Leu2l:LSer + Asn212Ser A1a199Asn + G1y196Pro + Asn198Asp + Thr202G1y + Tyr208Ser + A1a209Pro + Ser210G1u + Leu2l:LAla + Thr214Asn A1a194His + Asn198G1u + Va1199G1y + G1n200Asn + G1y205Asn + Ser210G1u + Leu211His + Asn2l:?Gln + Thr214Ser A1a194G1y + Asn198G1u + G1n200Asn + Thr202Pro + Tyr203Cys + Thr207G1n + Tyr208Met + Ser210Asp + Asn212G1n Pro195G1y + G1y196G1n + Asn198G1u + G1n200Ser + Pro204G1n + Tyr208Leu + A1a209Thr + Ser210G1u + Leu211I1e A1a194Pro + Pro195G1n + Va1197His + Asn198G1u + Thr202G1n + G1y205Pro + Thr207Asn + Tyr20E3Va1 + Leu211Asp Pro195G1n + Asn198G1u + Thr202Ser + Tyr203G1y + Pro204Ser + Thr207G1n + A1a209Pro + Leu211Asp + G1y213Asn Va1199Asn + G1n200Asp + Thr202G1n + Tyr203G1y + Pro209Asn + Thr207G1n + Tyr208A1a + A1a209G1u + Asn212Ser G1y196Pro + Va1197Thr + Pro204Asp + Ser206G1u + A1a209G1n + Leu211G1n + Asn212Ser + G1y213Ser + Thr214Pro G1y196Pro + Asn198Ser + Va1199Thr + Pro204G1u + Ser206Asp + Thr207G1n + A1a209G1y + Asn2la?Ser + Thr214G1y A1a199Asn + Va1197G1y + G1n200Ser + Thr202Ser + Pro204Asp + Ser206Asp + Tyr208G1n + A1a209G1n + Leu211G1y A1a194Pro + Thr202G1n + Tyr203Asn + Pro204Asp + Ser206G1u + Tyr208G1y + A1a209Ser + G1y213Pro + Thr214Pro A1a194Asn + Pro195G1n + Va1197Thr + Asn198Ser + Pro209Asp + G1y205Ser + Ser206Asp + Asn212Ser + Thr214Asn Pro195G1y + G1n200Ser + Thr202G1n + Tyr203Pro + Pro204Asp + G1y205Asn + Ser206G1u + Thr20~~Asn + Leu211His A1a194His + G1y196Asn + Va1197Met + Asn198Ser + Pro204Asp + Ser206Asp + Leu211Asn + Asn212Ser + Thr219G1n Asn198G1n + Va1199Cys + G1n200Ser + Thr202G1n + Pro204Asp + G1y205G1n + Ser206Asp + Tyr208Leu + Thr214Pro A1a194His + Pro195Ser + G1y196Pro + Asn198G1n + Pro204G1u + Ser206Asp + Thr207Pro + Tyr208His + A1a209Asn A1a194Ser + Asn198Asp + G1n200Ser + Thr202G1y + Thr207Pro + Tyr208Asn + A1a209G1n + Leu217.Ser + Asn212G1u G1y196Asn + Asn198G1n + G1n200Asn + Tyr203Thr + Pro204Ser + G1y205Asp + Thr207G1u + A1a20~~Ser + Thr214Asn Va1197His + Asn198G1n + Va1199Met + G1n200Ser + Pro204G1u + G1y205Ser + Thr207G1u + Tyr20~3Va1 + Thr214G1y Va1197Asn + Asn198Asp + Va1199A1a + G1n200G1u + Tyr203Cys + Pro209G1y + G1y205Asn + Thr207G1n + G1y213Asn Pro195Asn + G1y196G1n + Asn198Asp + Va1199Pro + G1n200G1u + Tyr203Va1 + G1y205Asn + Tyr20~~Cys + A1a209Pro Pro195G1y + G1y196Pro + Va1197Asp + Thr202Ser + Pro204G1n + Thr207G1y + Tyr208I1e + Asn211'Gln + G1y213G1u A1a194Pro + Va1197G1u + Tyr203His + G1y205Pro + Thr207G1y + A1a209Asn + Leu211His + G1y213Asp + Thr214G1y Pro195G1n + Va1197G1u + Asn198G1n + Va1199A1a + Pro2045er + G1y205G1n + A1a209Ser + G1y213G1u + Thr214Ser Pro195Asn + Va1197Asp + Asn198G1n + Va11995er + G1n200Ser + Thr207Ser + Tyr208I1e + Asn212Ser + G1y213Asp G1y196Pro + Va1197G1u + Asn198G1n + Va1199Asn + Pro204G1y + Thr207Pro + Leu211His + G1y213Asp + Thr214Asn A1a194Ser + Asn198G1n + Thr202Pro + Tyr203Met + Pro2045er + Thr207G1n + A1a209Asp + Leu211G1u + Thr214G1n G1y196Pro + Va1197Pro + Thr202Asn + Tyr203Cys + Tyr208G1n + Ser210Asp + Asn212Asp + G1y213Pro + Thr214Ser Pro195G1n + Tyr203Thr + G1y205G1n + A1a209G1n + Ser210Asp + Leu211Asn + Asn212G1u + G1y213Asn + Thr214G1n A1a194G1y + Pro195Ser + Va1197His + Thr202G1n + Pro204G1y + Ser210Asp + Leu211His + Asn212G1u + Thr214G1y Pro195Asn + G1y196Asn + Va1197Asn + Thr202Ser + Tyr203Va1 + G1y205Ser + Ser210Asp + Asn212G1u + G1y213Pro G1n200Ser + Thr202G1y + Tyr203Leu + Thr207Pro + Tyr208Pro + Ser210Asp + Leu211Pro + Asn212G1u + Thr219G1y A1a194G1n + G1y196Ser + Va1197Cys + Va1199Met + Tyr203Leu + A1a209Thr + Ser210Asp + Asn212G1u + Thr219Ser Va1197Met + Va1199A1a + G1n200Asn + Tyr203Thr + Thr207G1n + Tyr208G1u + Ser210G1u + Asn212Ser + Thr214G1y G1y196Ser + Va1197A1a + Va1199Ser + Thr202G1y + Tyr203Cys + G1y205Asn + Ser206G1u + Tyr208Asp + Leu211I1e A1a194G1y + Pro195Ser + Va1199His + Thr202Asn + G1y205G1n + Ser206Asp + Tyr208G1u + Asn212Ser + Thr214Pro G1y196Pro + Va1199G1n + Thr202Ser + G1y205Pro + Ser206Asp + Thr207G1n + Tyr208G1u + Leu211His + Asn212Ser Va1197His + Asn198Ser + G1y205Ser + Thr207Pro + Tyr208Va1 + Leu211Va1 + Asn212G1u + G1y213Ser + Thr214G1u Pro195Ser + G1y196Pro + Va1199Thr + Tyr203Ser + Tyr208G1y + A1a209Asn + Asn212G1u + G1y213Ser + Thr219G1u Pro195Asn + Asn198G1n + G1n200Asn + Thr202Asn + Thr207Asn + Tyr208G1n + Asn212G1u + G1y213Ser + Thr214G1u A1a194Asn + Va1199Ser + Thr202G1y + Pro204Asn + G1y205Ser + A1a209Asn + Leu211Asn + Asn212Asp + Thr214G1u A1a194Ser + G1y196G1n + Pro204Asp + G1y205Pro + Thr207Pro + Tyr208G1u + Leu211I1e + G1y213Asn + Thr214Ser Pro195Ser + Va1197Asp + Asn198G1n + G1n200Asn + Tyr203Pro + G1y205Asn + Ser210Asp + Asn212Ser + Thr214Asn A1a194His + G1y196Ser + Va1197Asp + Asn198Ser + G1n200Asn + Thr2025er + G1y205G1n + Ser210G1u + Asn2125er A1a194Thr + Va1197G1u + Thr202G1n + Pro204G1n + Tyr208G1y + A1a209Thr + Ser210G1u + Asn212Ser + Thr214G1n G1y196Ser + Va1197Asp + Va1199Pro + Tyr203Cys + Pro204Ser + Thr207Asn + Tyr208Asn + Ser210Asp + Asn212Ser A1a194Pro + Prol955er + G1y196G1n + Va1197Asp + Va1199Met + G1n200Asn + Leu211Met + G1y213Pro + Thr214Asp Pro195Asn + G1y196G1n + Asn198G1n + Va1199A1a + G1n200Ser + Tyr203Pro + G1y205G1u + Thr207Asn + Tyr208Asp ° r'"~ WO 95130011 PCT/US95104760 8~
G1y196Pro + Asn198Ser + Va1199Asn + Thr202G1y + Pro204G1n + G1y205Asp + Thr207G1n + Tyr208Asp + Thr214Asn Va1197Ser + Asn198G1u + G1n200Asn + Tyr203G1n + Pro204Asn + G1y205Pro + Thr207G1y + A1a20~3Thr + G1y213G1u A1a194Thr + G1y196G1n + Va1197His + Asn198Asp + Va1199Ser + Tyr203Pro + Tyr208Cys + G1y21_iAsp + Thr214Pro Asn198Asp + G1n200Ser + Tyr203G1n + Pro204Ser + G1y205Asn + Thr207Asn + A1a209G1u + Leu217.G1n + Asn212Ser Pro195G1y + G1y196Asn + Asn198G1u + Thr202G1n + Tyr203A1a + Pro204Ser + Thr207Pro + A1a209G1u + G1y213G1n A1a194Pro + Asn198G1u + Va1199Thr + Pro209G1y + Thr207G1y + Tyr208I1e + A1a209Asp + Leu211.Met + Asn212Ser A1a194Ser + Va1197G1u + Va1199Thr + G1n200Asp + Tyr203G1y + G1y205Ser + Tyr208His + A1a209Thr + Asn212Ser G1y196G1n + Va1197G1u + Va1199Pro + G1n200Asp + G1y205G1n + Thr207Ser + A1a209Thr + Leu211Pro + Thr219G1y Va1197G1y + G1n200G1u + Thr202G1y + Tyr203Leu + G1y205G1n + Thr207G1y + Tyr208Leu + A1a209Asn + Asn212Asp Pro195Ser + G1y196Asn + Asn198Ser + Va1199His + G1n200G1u + Tyr203Pro + A1a209Ser + Asn21%Asp + Thr214Asn Asn198Ser + Va1199Met + G1n200G1u + Thr202Ser + Tyr203I1e + G1y205Pro + A1a209Pro + Asn212G1u + G1y213G1n G1y196Pro + Va1197Ser + Va1199Pro -+- G1n200G1u + A1a209G1y + Leu211G1n + Asn212Asp + G1y213Pro + Thr219Asn A1a194G1n + G1y196Ser + Va1197Pro -+- Thr202Pro + Pro204Asp + Tyr208G1y + A1a209G1u + G1y213Asn + Thr214Pro A1a194Pro + Va1197Ser + Va1199His -~ G1n200Asn + Pro209G1y + Ser206G1u + Thr207Asn + Tyr208Leu + A1a209G1u G1y196Asn + Thr202Asn + Pro209Asn -~ G1y205Pro + Ser206G1u + Tyr208Asn + A1a209Asp + Leu211Met + Thr219G1n Pro195G1y + Asn198G1n + Va1199Ser -~ G1n200Ser + Tyr203Thr + G1y205Pro + Ser206G1u + A1a209G1u + Thr219Asn A1a194Thr + Va1197A1a + Thr202Ser -F Tyr203Pro + G1y205Asn + Ser206G1u + A1a209G1u + Leu211G1n + Thr214Asn Pro195Ser + Va1197A1a + Thr202Asn -~ Pro204G1y + Ser206Asp + Thr207Asn + A1a209G1u + Asn212Ser + Thr214Ser A1a194Thr + Va1199Asn + Thr202Pro -F Tyr203I1e + Pro204Asn + Ser206Asp + Thr207Asn + A1a209G1u + Thr214Asn A1a194Ser + Pro195Asn + Va1197G1y -~ Va1199G1y + G1n200G1u + Thr202G1n + Pro209Asn + Thr207Asp + Leu211G1n A1a194G1y + Va1197His + G1n200Ser -. Pro209Asn + Thr207Ser + Ser210G1u + Leu211G1y + G1y213G1u + Thr219Asn A1a194G1n + G1y196G1n + Va1197Met -- Va1199His + Thr202Pro + G1y205Asn + A1a209Ser + Ser210G1u + G1y213G1u Pro195Asn + Va1197Thr + Va1199A1a ~- G1n200Asn + Tyr203Asn + Thr207G1y + Ser210Asp + Asn212G1n + G1y213G1u A1a194His + Va1199Pro + G1n200Asn ~- Thr202Pro + Tyr203Asn + G1y205G1u + A1a209G1u + Asn212G1n + Thr214Asn G1y196Pro + Asn198G1n + G1n200Asn ~- Tyr203G1n + Pro204G1n + G1y205Asp + A1a209G1u + Asn212G1n + Thr214G1n Pro195G1n + Asn198G1n + Va1199A1a -~- G1n200Asn + Thr207G1u + Tyr208Leu + A1a209Ser + Ser210Asp + G1y213Pro Pro195G1y + G1y196G1n + Va1199Ser ~- G1n200Asn + Pro204Ser + G1y205Ser + Thr207G1u + Ser210G1u + Thr214Pro s~
G1y196G1n + G1n200Ser + Thr202G1y + Tyr203l1e + Thr207Asp + Tyr208I1e + Ser210Asp + Leu211Asn + Asn212Ser A1a194His + G1y196G1n + Va1197G1y + Thr207G1u + Tyr208Va1 + A1a209G1n + Ser210Asp + Leu211His + G1y213Ser A1a194G1n + Asn198Asp + Va1199Ser + G1n200Ser + Tyr203I1e + G1y205Asn + Thr207Asn + Asn212G1n + Thr214Asp Va1197Pro + Asn198G1u + Va1199Thr + G1n200Ser + Pro204Asn + G1y205Pro + Leu211Cys + Asn212Ser + Thr214Asp A1a194Ser + Va1199Thr + G1n200G1u + Tyr203Cys + Tyr208G1y + A1a209His + Leu211G1y + Asn212Ser + G1y213G1u G1y196G1n + Asn198G1n + Va1199Met + G1n200Asp + Pro204G1n + Tyr208Pro + Asn212G1n + G1y213Asp + Thr214G1n Va1197Asp + Va1199G1n + G1n200Ser + Thr202Pro + Thr207G1n + A1a209G1u + Leu211G1y + Asn212Ser + G1y213G1n A1a194Ser + G1y196Pro + Va1197G1u + Asn198Ser + Va1199Met + Pro204G1y + Thr207G1n + A1a209G1u + G1y213G1n Va1197Asp + Asn198Ser + Pro204G1n + G1y205G1n + Thr207G1y + Tyr208Met + A1a209G1u + Asn212G1n + Thr214G1n A1a194Ser + Va1197G1u + Asn198G1n + Tyr203Asn + G1y205Asn + A1a209G1u + Asn212G1n + G1y213Ser + Thr214Pro Pro195G1y + Va1197Ser + Asn198Ser + Tyr203Leu + Pro204G1u + Ser210Asp + Leu211Ser + Asn212Ser + Thr214G1n A1a194Asn + Pro195G1n + Asn198G1n + Va1199Asn + G1n200Asn + Pro204Asp + Thr207Pro + Ser210G1u + G1y213G1n A1a194Pro + Va1197Ser + Asn198Ser + Thr202Pro + Tyr203Va1 + Pro204G1u + Ser210Asp + Asn212Ser + Thr214Asn A1a194G1n + Pro195Ser + G1y196Pro + Va1199Ser + Pro204Asp + Tyr208Cys + A1a209Thr + Ser210G1u + G1y213Asn Pro195Ser + Tyr203Pro + Pro209Asp + G1y205Pro + Thr207G1n + A1a209G1y + Ser210Asp + Leu211Asn + Thr214Asn G1y196Ser + Asn198Ser + Thr202Pro + Pro204Asp + Tyr208Va1 + A1a209G1n + Ser210Asp + Leu211Asn + Asn212G1n G1y196Ser + Va1199Met + Tyr203A1a + Pro204G1u + G1y205Ser + Thr207Ser + Tvr208Leu + A1a209G1n + Ser210Asp Pro195G1y + Va1197Pro + Thr202Asn + Pro204G1u + G1y205Ser + Thr207Asn + Tyr208Pro + Ser210G1u + Asn212Ser A1a194Asn + G1y196Ser + Asn198Ser + Va1199Met + G1n200Asn + Thr202G1y + Thr207G1n + A1a209Asp + G1y213Asp A1a194G1n + Pro195G1n + Thr202Ser + Pro204Asn + A1a209Thr + Ser210G1u + Leu211Asn + G1y213G1n + Thr219G1u G1y196G1n + Va1197Pro + Asn198G1n + Va1199G1y + A1a209Pro + Ser210Asp + Leu211Cys + Asn212Ser + Thr214G1u A1a194Asn + Pro195G1y + Asn198G1n + Va1199His + Tyr203Pro + Thr207G1y + Tyr208Met + Ser210Asp + Thr214Asp A1a194Pro + Pro195Ser + G1y196Asn + Thr202G1y + Pro204Ser + A1a209His + Ser210G1u + Leu211Ser + Thr214Asp A1a194Thr + G1y196Pro + Asn198Ser + Thr207Asn + Tyr208I1e + Ser210Asp + Leu211Va1 + G1y213G1n + Thr214G1u A1a194G1y + Pro195Ser + Asn198Ser + Thr202Ser + G1y205Ser + A1a209G1n + Ser210Asp + Asn212Ser + Thr214Asp A1a194Ser + Pro195G1y + G1y196Ser + Va1199A1a + Tyr203G1n + Pro204G1n + Thr207Ser + Ser210G1u + Thr214G1u G1y196Ser + G1n200Asn + Thr202Pro + Tyr203Leu + Thr207G1n + Ser210Asp + Leu211A1a + G1y213Pro + Thr214Asp s7 A1a194G1n + Asn198Ser + Va1199Thr ~- G1n200Ser + Tyr203Thr + A1a209Pro + Ser210Asp + Leu211Asn + Thr214G1u Pro195G1n + G1y196G1n + Tyr203Cys ~- Pro204Ser + G1y205Pro + Tyr208His + A1a209Thr + Ser210Asp + Thr214G1u G1y196G1n + Asn198Ser + Va1199Thr ~- G1n200Ser + Thr202Ser + Thr207Ser + A1a209His + Ser210G1u + Thr214G1u Pro195Asn + G1y196Pro + Asn198G1n ~- Tyr203A1a + Thr207Ser + A1a209Pro + Ser210Asp + Leu211Thr + Thr214Asp A1a194Ser + Pro195Asn + Va1199Pro ~- Pro204Asn + G1y205G1n + Thr207G1n + Ser210G1u + G1y213Ser + Thr214Asp G1y196Ser + Va1197Asn + Thr202G1n ~- Tyr203Ser + Pro204Ser + G1y205Ser + Ser210G1u + Leu211Cys + Thr214Asp Va1197Asn + G1n200Asp + Tyr203Cys ~- Pro204Asn + G1y205Asn + Ser206Asp + Thr207Ser + Tyr208Leu + Asn212G1n Pro195Asn + G1n200Asp + Thr202G1n - Tyr203G1n + Pro204Ser + Ser206G1u + Thr207Pro + Asn212Ser + G1y213G1n A1a194G1n + Pro195G1n + G1y196Asn - Va1199Ser + G1n200Asp + Thr202Ser + G1y205Ser + Ser206G1u + A1a209Ser Pro195Asn + G1y196Pro + Va1197Cys -~ Va1199Met + G1n200Asp + Ser206G1u + Thr207Ser + A1a209G1n + Leu211Va1 Pro195G1y + Va1199Pro + G1n200Asp -~ Thr202Ser + Tyr203G1y + Pro204G1n + Ser206Asp + Leu211Met + Asn212Ser G1y196Pro + Va1197Cys + Asn198G1n -~ G1n200G1u + Thr202Asn + Ser206Asp + A1a209Asn + Leu211Cys + G1y213Ser Pro195G1n + Asn198Ser + G1n200G1u -~ Thr202Asn + Pro204G1y + Ser206Asp + Thr207Ser + Tyr208Ser + Asn212G1n G1y196Asn + Asn198Ser + Va1199Met -~ G1n200Asp + Thr202Ser + Tyr203His + Pro209Asn + G1y205G1n + Ser206G1u A1a194Thr + Asn198G1n + Va1199A1a -~ G1n200Asp + Thr202Asn + Ser206G1u + Thr207Ser + Tyr208His + G1y213G1n A1a194G1n + G1y196G1n + Asn198G1n -~ G1n200Asp + Thr202Asn + Ser206G1u + Thr207Ser + Tyr208Thr + G1y213G1n Loop 6 - Decuple Mutation Variants A1a194Ser + G1y196G1n + Va1197A1a -~ Asn198G1n + Thr202Pro + Pro204Ser + G1y205Ser + Thr207Pro + Leu211Va1 +
Asn212Asp Pro195Ser + Va1197G1n + Va1199His +- Thr202Asn + Tyr203Pro + G1y205G1n + A1a209Thr + Ser210G1u + Asn212Ser +
Thr214G1n A1a199G1y + Asn198Ser + Va1199A1a ~~ Thr202G1n + Tyr203Leu + Pro209G1y + G1y205Pro + A1a209His + G1y213Ser +
Thr214Asp A1a194His + G1y196G1n + Thr202G1y ~~ Tyr203Va1 + G1y205Asn + Thr207G1n + Tyr208Cys + A1a209Ser + G1y213G1n +
Thr214Asn A1a194Thr + Pro195Ser + G1y196Asn -+ Asn198G1u + G1n200Asn + Thr202G1y + Pro209Ser + Asn212G1n + G1y213Pro +
Thr214G1y A1a194Ser + Pro195Asn + G1y196Asn + Va1199Thr + Thr202G1n + Pro204Asp + Thr207Asn + Tyr208Ser + Leu211Asn +

ss Asn212G1n Pro195Asn + G1y196Ser + Va1197G1y + Asn198Ser + Thr202G1y + G1y205Pro + Thr207Pro + Tyr208Asp + A1a209Ser +
Thr214Asn A1a194His + Pro195G1n + G1y196Asn + Asn198G1n + Va1199His + Pro204Ser + G1y205Asn + Thr207Ser + Tyr208Ser +
Thr214Ser A1a194Pro + Pro195Asn + Va1197Asn + Va1199Cys + G1n200Ser + Thr202Pro + Pro204Ser + Ser206G1u + Tyr208His +
Leu211I1e A1a194Thr + Va1197Met + Asn198Asp + Va1199G1y + G1n200Ser + Tyr203G1n + Pro204Asn + Thr207G1n + A1a209Pro +
Leu211A1a A1a199Pro + Pro195G1y + Va1197Asn + Asn198Ser + Va1199His + Tyr208His + A1a209Asp + Leu211Thr + Asn212G1n +
Thr214G1n Pro195Ser + Va1199Met + Thr202Ser + Tyr203I1e + G1y205G1n + Ser206Asp + Tyr208His + Leu211Pro + Asn212G1n +
G1y213Pro A1a194Thr + Va1197Ser + Va1199Asn + Thr202G1n + Tyr203Met + G1y205Pro + Ser206G1u + Tyr208I1e + A1a209Asn +
G1y213Ser A1a194Thr + Va1197G1n + Asn198G1n + Va1199G1y + Thr202Ser + Pro204Ser + G1y205Pro + Thr207Asn + A1a209Pro +
Thr214Asn A1a194His + Pro195Asn + G1y196Asn + Va1199Thr + G1n200Asn + Thr202Asn + Tyr203A1a + G1y205Asp + Thr207G1y +
Thr214Pro A1a194Ser + Pro195G1y + G1y196Ser + Va1197Pro + Asn198G1n + Va1199Thr + Thr202Pro + Tyr203Met + Pro209G1n +
G1y205G1n A1a194Thr + Pro195G1n + Va1197Asp + G1n200Ser + Thr202Pro + Pro209Ser + G1y205G1n + Thr207G1y + Tyr208His +
A1a209G1y A1a194Pro + Pro195Ser + Asn198Ser + Va1199G1n + G1n200Asp + Thr202G1n + Tyr203Asn + Pro204G1y + Leu211Va1 +
Thr214Pro Pro195Ser + G1y196G1n + Va1199Pro + G1n200Asn + Tyr203G1n + Thr207Asp + A1a209His + Leu211His + Asn212G1n +
Thr214G1y G1y196Pro + Asn198Ser + Va1199G1y + G1n200Ser + Thr202Asn + Tyr203Ser + Tyr208Leu + A1a209His + Leu211Asp +
Asn212Asp G1y196Asn + Va1199A1a + G1n200Ser + Thr202G1n + Tyr203His + Pro204Asn + G1y205Pro + Asn212G1n + G1y213G1u +
Thr214G1u G1y196Pro + Va1199Ser + G1n200Ser + Thr202Pro + Pro204G1n + A1a209G1u + Ser210G1u + Asn212Ser + G1y213Ser +
Thr214Ser Va1197Thr + Asn198G1n + Va1199G1n + Pro204G1y + Thr207Pro + A1a209Asp + Ser210G1u + Asn212Ser + G1y213Ser +
Thr214Ser A1a194Pro + Pro195G1n + Va1197A1a + Va1199G1n + G1n200Ser + Pro204G1n + G1y205Pro + Tyr208Met + A1a209Asp +

s9 Ser210G1u Pro195G1y + Va1197G1y + Asn198G1n + Thr202G1y + Tyr203A1a + G1y205Pro + A1a209G1u + Ser210G1u + Leu211Ser +
G1y213G1n S Pro195Ser + G1y196Asn + Asn198G1n + G1n200Ser + Thr202G1y + Pro209G1n + Tyr208Thr + Leu211Cys-+ Asn212G1u +
G1y213G1u G1y196G1n + Va1197Ser + G1n200Ser + Thr202Pro + Pro204Asp + G1y205Asp + Tyr208G1n + A1a209Pro + Asn212Ser +
Thr219G1n A1a194Pro + Pro195G1n + G1y196G1n + Pro209Asp + G1y205G1u + Tyr208A1a + A1a209Ser + Leu211Asn + G1y213Asn +
Thr214Ser A1a194Thr + Va1197Cys + Va1199Met ~+ G1n200Ser + G1y205Pro + Tyr208Thr + Ser210G1u + Leu211Asp + Asn212Ser +
G1y213Pro A1a199G1y + Va1197His + Thr202Ser -~ Tyr203Asn + Thr207Pro + Tyr208Cys + Ser210G1u + Leu211G1u + Asn212G1n +
G1y213Pro Va1197His + Asn198Ser + G1n200Ser -~ Thr202Asn + G1y205Ser + Tyr208G1n + Ser210G1u + Leu211G1u + Asn212Ser +
Thr214Pro A1a194Thr + G1y196Asn + Asn198G1n -~ G1n200Asn + Thr202Pro + Tyr203Thr + G1y205Asn + Ser210G1u + Leu211G1u +
Asn212Ser A1a194G1y + G1y196G1n + Asn198Ser -~ Va1199Cys + Pra204Asn + G1y205Asp + Ser206Asp + Thr207G1n + A1a209G1y +
G1y213Asn Pro195G1n + Va1197Met + Va1199A1a -~ G1n200Ser + G1y205Asp + Ser206Asp + Thr207Asn + Tyr208G1n + Leu211Pro +
Asn212Ser A1a194His + Pro195G1n + Asn198G1n + Thr202Pro + G1y205G1u + Ser206Asp + Thr207Pro + Leu211Thr + G1y213Ser +
Thr214G1n G1y196Pro + Asn198G1n + Va1199G1y -~ G1n200Ser + Thr202Asn + Pro204Asn + G1y205G1u + Ser206Asp + Thr207G1y +
Thr214G1n Pro195G1n + Va1197His + Asn198G1n - Thr202G1y + G1y205Asp + Ser206G1u + Thr207Ser + Tyr208A1a + Asn212G1n +
G1y213Asn A1a194Asn + G1y196Asn + Va1197Ser + Asn198Ser + Va1199Met + Thr202Asn + Tyr203A1a + G1y205Asp + Ser206Asp +
Thr219Asn A1a194Thr + Pro195Asn + G1y196G1n ~- Va1199Thr + G1n200Asn + Thr202Ser + Pro204Asn + G1y205Asp + Ser206G1u +
Thr207Asn Pro195Ser + G1y196Ser + Va1197Pro ~- Asn198G1n + G1n200Asn + Tyr203Leu + G1y205Asp + Ser206G1u + Asn212Ser +
Thr214Pro Pro195Ser + G1y196Pro + Va1197Ser ~- G1n200Ser + Thr202Pro + Tyr203Va1 + Pro204G1y + G1y205G1u + Ser206G1u +
Tyr208Met A1a194G1y + G1y196G1n + G1n20USer ~- Thr202Ser + Pro204Asn + Ser206Asp + Thr207Asp + A1a209G1n + Leu211Met +

PCTlUS95104'760 Thr214Pro G1y196Asn + Va1197Ser + Va1199A1a + Tyr203I1e + Ser206Asp + Thr207G1u + A1a209Ser + Leu211A1a + Asn212Ser +
Thr214Asn 5 A1a194Ser + Pro204Asn + G1y205Ser + Ser206Asp + Thr207G1u + Tyr208Met + A1a209Asn + Leu211I1e + Asn212G1n +
G1y213Asn A1a199G1n + Asn198G1n + Va1199Asn + Thr202G1n + Tyr203I1e + G1y205G1n + Ser206Asp + Thr207G1u + Tyr208Ser +
10 G1y213Ser G1y196G1n + Asn198G1n + Thr202G1n + Pro204Ser + G1y205Ser + Ser206G1u + Thr207Asp + Leu211I1e + G1y213Pro +
Thr214Pro Pro195Asn + G1y196G1n + Va1197G1n + Thr202Asn + G1y205Ser 15 + Ser206G1u + Thr207G1u + Tyr208I1e + G1y213G1n +
Thr214Asn Asn198Ser + Thr202Ser + Tyr203I1e + G1y205Asn + Thr207Asn + Tyr208Asp + A1a209G1u + Leu211I1e + G1y213Ser +
Thr214Ser 20 Va1199His + G1n200G1u + Tyr203Met + Pro209G1y + Thr207Pro + A1a209Thr + Ser210G1u + Leu211Asn + Asn212Ser +
G1y213Pro A1a194His + G1y196Ser + Asn198Ser + G1n200G1u + Thr202G1n + Tyr203G1n + G1y205Pro + Thr207G1y + Ser210G1u +
25 G1y213Asn G1y196Pro + Va1199Asn + G1n200G1u + Pro204G1y + Tyr208His + Ser210Asp + Leu211G1n + Asn212Ser + G1y213G1n +
Thr214Asn Pro195G1y + G1y196Pro + G1n200Asp + Tyr203Asn + Pro209Asn 30 + G1y205Asn + Thr207Asn + Tyr208I1e + Ser210Asp +
G1y213G1n A1a194G1n + Pro195G1n + G1y196G1n + Va1197G1n + G1n200Asp + Thr207G1y + A1a209Asn + Ser210Asp + Leu211G1y +
G1y213Asn 35 Va1197Thr + Va1199Ser + G1n200G1u + Tyr203Asn + Pro204G1y + Thr207Asn + Tyr208G1y + Ser210G1u + Asn212Ser Thr214Asn G1y196G1n + Va1197G1n + Va1199Asn + G1n200G1u + Tyr203Asn + Thr207G1n + A1a209G1y + Ser210G1u + Asn212G1n +
40 G1y213G1n Pro195Asn + Va1197Met + Va1199Cys + G1n200Asp + Thr202Asn + Thr207Ser + Tyr208G1y + A1a209G1n + Ser210G1u +
Leu211Asn A1a194Asn + Va1199G1n + G1n200Asp + Tyr203G1y + Pro204Ser 45 + G1y205Pro + Tyr208I1e + Ser210G1u + Leu211Rsn +
G1y213Ser A1a194His + Pro195G1n + Va1197G1u + Asn198Ser + Va1199Ser + Tyr203His + Thr207Pro + Leu211G1n + Asn212G1u +
G1y213Ser 5o G1y196Ser + Va1197G1u + Asn198G1n + Va1199Cys + Tyr203Pro + Pro204G1y + A1a209G1n + Asn212G1u + G1y213G1n +
Thr214Asn G1y196Ser + Va1197G1y + Asn198Asp + G1n200Ser + Tyr203Pro + Tyr208A1a + A1a209G1y + Ser210G1u + Leu211I1e +

Thr214Asn G1y196Pro + Asn198Asp + Va1199G1n + G1n200Asn + Pro204G1n + G1y205Pro + Thr207G1y + Ser2lc)Asp + Asn212G1n +
G1y213Ser S Pro195Asn + G1y196Ser + Va1197G1n + Asn198G1u + G1n200Ser + Pro204G1y + Tyr208G1n + A1a20~aAsn-+ Ser210G1u +
G1y213Pro Asn198Asp + Va1199Met + G1n200Asn + Thr202G1n + Pro204Asn + G1y205G1n + Tyr208A1a + A1a20~aHis + Ser210G1u +
l0 Thr214Pro A1a194G1n + Va1197A1a + Asn198G1u + G1n200Asn + Thr202G1y + Pro204G1n + G1y205G1n + Tyr20~3Thr + Ser210Asp +
Leu211Va1 Va1197Ser + Asn198Asp + Va1199Asn + G1n200Ser + Pro204Ser 15 + Thr207Pro + Tyr208Leu + Ser210G1u + Asn212G1n +
Thr214Ser A1a194Thr + G1y196Asn + Va1197Ser + Asn198G1u + Va1199Asn + G1n200Ser + Thr202Ser + Thr20~~Ser + Ser210G1u +
G1y213Ser 20 G1y196Pro + Va1197Met + Asn198Asp + Va1199G1y + G1n200Asn + Tyr203Pro + G1y205Asn + Tyr208Va1 + Ser210Asp +
Leu211His G1y196G1n + Asn198Asp + G1n200Asn + Tyr203Asn + G1y205Pro + Thr207Ser + Tyr208G1n + A1a209Pro + Leu211G1u +
25 Asn212G1n Pro195Asn + Va1197Pro + Asn198G1u + Va1199Cys + Tyr203Asn + Thr207G1y + Tyr208G1n + A1a20~~Ser + Leu211G1u +
G1y213Asn A1a194G1n + G1y196G1n + Va1199Met + G1n200Asp + Tyr203Cys 30 + Thr207Ser + A1a209Asp + Asn212Ser + G1y213G1n +
Thr214G1n Pro195Asn + G1y196Pro + Asn198Ser + G1n200G1u + Thr202Ser + Thr207G1n + Tyr208A1a + A1a209G1u + Leu211A1a +
Asn212G1n 35 Pro195G1y + G1y196Asn + Va1199G1n + Thr202G1y + Pro204G1u + G1y205G1n + Ser206G1u + Thr207Ser + Leu211G1n +
Asn212G1n Pro195G1y + G1n200Asn + Tyr203Leu + Pro204G1u + Ser206Asp + Tyr208G1n + A1a209G1n + Asn212G1n + G1y213Pro +
40 Thr214G1n A1a194His + Pro195Ser + Va1199Thr + Thr202G1y + Tyr203Cys + Pro204G1u + Ser206G1u + Leu211.His + Asn212Ser +
G1y213Pro Pro195G1n + Va1197G1n + Asn198G1n + G1n200Ser + Pro204G1u 45 + G1y205Ser + Ser206Asp + A1a20~3G1y + Asn212Ser +
Thr214G1n A1a194Thr + Asn198Asp + Va1199A1a + G1n200Asn + G1y205Pro + Tyr208Ser + A1a209G1y + Leu211.Va1 + Asn212G1u +
G1y213G1n 50 A1a194G1y + Pro195G1n + Va1199Cys + Tyr203Leu + pro204Ser + G1y205G1u + Thr207Asp + A1a20~3G1n + Asn212G1n +
Thr214G1y Pro195G1n + Va1197G1n + G1n200Ser + Thr202G1n + Pro204G1u + G1y205Ser + Thr207Asp + Leu211His + Asn212Ser +

WO 95/30011 ~~

Thr214Ser A1a194Asn + Pro195Ser + Va1197Ser + Asn198G1n + Thr202Asn + Pro204Asp + Thr207G1u + Leu211His + Asn212Ser +
G1y213Ser A1a194Thr + Pro195Ser + Va1197G1n + Asn198G1u + Va1199Met + G1n200Asp + Pro204G1y + G1y205Ser + A1a209Asn +
Thr214Asn Pro195G1n + G1y196Pro + Va1197Met + Asn198Asp + Va1199A1a + G1n200G1u + Thr202Pro + Tyr203I1e + Tyr208Cys +
Leu211Thr A1a194G1n + G1y196Asn + Asn198G1u + Va1199A1a + G1n200Asp + Thr202Ser + Tyr203Cys + Thr207Asn + Tyr208Met +
G1y213Asn A1a194Thr + Va1197Pro + Va1199His + G1n200Asp + Thr202G1n + Tyr203Met + G1y205Ser + Thr207Asn + Tyr208Asp +
Asn212Ser Pro195Asn + Va1197Asp + Asn198G1n + Va1199G1y + G1n200Ser + Thr207Ser + Tyr208I1e + A1a209Asn + Asn212Ser +
G1y213Asp Pro195Asn + Va1197Pro + Asn198G1n + G1n200G1u + G1y205G1n + Tyr208G1y + A1a209G1y + Leu211G1u + Asn212Ser +
Thr214Ser A1a194Thr + G1y196Ser + Asn198Ser + G1n200Asp + Thr202G1y + Tyr203Met + G1y205G1n + Tyr208A1a + Leu211Asp +
Thr214Asn Pro195Ser + Asn198Ser + Va1199Thr + Thr202Asn + G1y205Asn + Thr207Ser + A1a209G1y + Leu211G1u + Asn212G1n +
G1y213Asp Pro195G1n + G1y196G1n + Asn198Ser + Thr202Pro + Pro204Asn + Thr207Asp + Tyr208G1y + A1a209Asp + Leu211I1e +
Asn212Ser A1a194G1y + Pro195G1y + Asn198Ser + G1n200Asn + Thr202Asn + Tyr203A1a + Tyr208Va1 + Ser210G1u + Asn212Asp +
Thr214G1y A1a194G1n + Pro195G1n + Asn198G1n + G1n200Asn + Thr202G1y + Tyr203Leu + G1y205G1n + A1a209G1n + Ser210G1u +
Asn212G1u Pro195G1n + Va1197G1y + Va1199G1y + Thr202Asn + G1y205Pro + Thr207Asn + A1a209Asn + Ser210G1u + Leu211Met +
Asn212G1u A1a194Thr + Pro195G1y + G1y196Pro + Asn198Ser + Thr207Pro + Tyr208Cys + A1a209Ser + Ser210G1u + Leu211Ser +
Asn212Asp A1a194His + Pro195Ser + G1y196G1n + Va1197A1a + Va1199Asn + Tyr203G1n + Thr207Asn + Tyr208His + Ser210Asp +
Asn212G1u A1a194G1n + Pro195Asn + G1y196Ser + Va1197Met + Pro204Ser + G1y205Pro + Thr207Ser + Tyr208A1a + Ser210G1u +
Asn212G1u A1a194Ser + Va1199Thr + Thr202Ser + Tyr203A1a + Pro204Asn + G1y205Pro + Ser210Asp + Leu211A1a + Asn212Asp +
Thr214Pro Pro195Asn + Thr202G1n + Pro209Ser + Thr207G1y + Tyr208G1u + A1a209Asn + Ser210G1u + Leu211Asn + Asn212G1n +

°~"'"~ WO 95/30011 TIUS95104760 PC

G1y213Asn G1y196Asn + Va1197Thr + Va1199Thr + Pro204Ser + Thr207G1n + Tyr208Asp + Ser210Asp + Leu2l:LThr + Asn212G1n +
Thr214Asn A1a194Asn + Pro195Asn + Va1199Cys + G1n200Asn + Pro204G1n + Thr207Ser + Tyr208G1u + Ser210Asp~+ Leu211Asn +
Asn212G1n A1a194Thr + G1y196Pro + Va1199His + G1n200Ser + G1y205Asn + Ser206G1u + Tyr208G1u + A1a20~)Thr + Leu211Met +
l0 Thr214Ser A1a194ser + Pro195G1n + Asn198G1n + Va1199His + Pro204Asn + G1y205G1n + Ser206G1u + Tyr208Asp + A1a209Thr +
G1y213Asn G1y196G1n + Va1197Met + Va1199Ser + G1n200Ser + Thr202Asn + Pro204Asn + Tyr208Thr + A1a20~aGln + Asn212G1u +
Thr219Asp Va1197A1a + Asn198Ser + G1n200Ser + Thr202G1y + Pro204G1n + Thr207G1n + Tyr208G1n + A1a20~3His + Asn212G1u +
Thr214Asp A1a194Pro + G1y196Ser + Va1197Asp + Va1199Asn + Thr202Asn + Tyr203Va1 + Thr207G1n + A1a20~~Asn + Ser210G1u +
Asn212G1n Pro195ser + Va1197G1u + Asn198G1n + Va1199G1n + Tyr203Leu + Tyr208Va1 + A1a209Asn + Asn212G1n + G1y213Pro +
Thr219G1u Pro195G1n + G1y196Pro + Va1197Asn + Asn198G1u + G1n200Ser + Pro204Asn + G1y205Asn + Leu217.Cys + Asn212G1n +
G1y213Asp Pro195G1n + G1y196Pro + Va1197Thr + Asn198Asp + Thr202Pro + Tyr203G1n + Thr207G1y + A1a20~~G1u + G1y213Ser +
Thr214Ser A1a194ser + Pro195G1n + Va1197Pro + Asn198Asp + Va1199A1a + Tyr203Thr + Pro204G1n + A1a209Asp + Leu211Va1 +
Asn212Ser Pro195G1y + Va1197G1u + Asn198G1n + G1n200Asp + Tyr203His + Pro204Ser + Thr207G1y + Tyr208His + G1y213Pro +
Thr214G1y Pro195G1y + G1y196Pro + Va1199Thr + G1n200G1u + Tyr203Met + Pro204G1n + G1y205Pro + Tyr208Ser + Asn212Asp +
G1y213Pro G1y196Pro + Va1197Ser + Va1199Pro + G1n200G1u + Thr207G1y + A1a209G1y + Leu211G1n + Asn2l~:Asp + G1y213Pro +
Thr214Asn A1a194His + Pro195G1n + G1y196Asn + Va1197Ser + G1n200G1u + Tyr203A1a + Pro204Asn + G1y205Ser + Thr207Pro +
Asn212G1u A1a194Ser + Pro195Asn + G1y196Pro + Asn198Ser + Va1199Thr + Tyr208I1e + Leu211G1u + Asn212G1n + G1y213Ser +
Thr214G1u A1a199Pro + Pro195Asn + Asn198Ser + Va1199Met + Tyr203Met + G1y205Pro + Thr207Pro + Tyr20ESer + Leu211G1u +
Thr214G1u A1a194His + Pro195G1n + G1y196G1n + Va1199Pro + Tyr203His + Thr207Asn + A1a209Asp + Leu211A1a + Asn212G1u +

9.~
Thr214Asn A1a194His + G1y196G1n + Thr202Ser + Tyr203G1y + Pro204G1y + G1y205Ser + Ser206Asp + Thr207G1n + A1a209Asp +
Thr214Pro S Pro195G1n + Thr202Ser + Pro204G1n + G1y205Pro + Ser206Asp + Tyr208Met + A1a209G1u + Asn212G1n + G1y213Pro +
Thr214Ser G1y196G1n + Va1197Pro + Thr202Asn + Tyr203G1y + G1y205Pro + Ser206G1u + Thr207Ser + A1a209G1u + Leu211G1n +
Asn212Ser A1a194Asn + Pro195Ser + G1y196Pro + Va1199A1a + Thr202G1n + Pro204G1n + Ser206Asp + A1a209G1u + Leu211His +
Asn212Ser G1y196Ser + Asn198Ser + Va1199Ser + G1n200Asn + Tyr203Asn + G1y205G1n + Ser206G1u + A1a209G1u + Leu211Ser +
G1y213G1n A1a194Thr + Va1197Cys + Va1199Asn + Pro209G1y + Ser206Asp + Thr207G1y + Tyr208G1y + A1a209G1u + Leu211I1e +
Thr214G1n A1a199G1y + Pro195Ser + G1n200Ser + Thr202G1y + Tyr203Leu + Pro204G1y + G1y205Pro + Ser206Asp + A1a209Asp +
Thr214G1y Pro195Ser + G1y196Pro + Va1199His + G1n200Asp + Thr202Pro + G1y205Pro + Thr207Asp + A1a209Thr + Asn212G1n +
G1y213G1n A1a194G1n + G1y196Asn + Asn198G1n + Va1199Pro + G1n200Asp + Thr202G1y + Tyr203Thr + G1y205Pro + Thr207Asp +
A1a209G1n Pro195Asn + Va1197Thr + Va1199A1a + G1n200Asn + Tyr203Asn + G1y205Pro + Thr207G1y + Ser210Asp + Asn212G1n +
G1y213G1u A1a194Asn + Pro195G1n + Va1197Asn + Va1199Asn + Pro204G1y + Thr207Pro + Ser210Asp + Asn212Ser + G1y213G1u +
Thr214Pro A1a194G1n + Pro195Asn + Va1197G1y + Tyr203Met + Pro204Ser + Tyr208G1n + A1a209Asn + Ser210G1u + Asn212Ser +
G1y213G1u A1a194His + G1y196Ser + Tyr203Asn + Pro209G1y + G1y205Pro + A1a209Ser + Ser210G1u + Leu211Pro + Asn212Ser +
G1y213Asp G1y196Pro + G1n200Asn + Thr202Asn + Tyr203His + G1y205Asp + Tyr208I1e + A1a209G1u + Leu211Met + G1y213Pro +
Thr214Pro Va1197His + Va1199Asn + G1n200Asn + Thr202Ser + Tyr203Ser + G1y205G1u + Tyr208Ser + A1a209Asp + G1y213Pro +
Thr214Pro A1a194Asn + Va1197Pro + G1n200Ser + Thr202G1n + Tyr203Met + G1y205G1n + Thr207Asp + Ser210Asp + Leu211Ser +
Asn212Ser A1a194Thr + Asn198Ser + Tyr203Asn + G1y205Pro + Thr207Asp + A1a209G1n + Ser210G1u + Leu211Va1 + Asn212Ser +
Thr214Ser Asn198G1n + Va1199Pro + G1n200Asn + Thr202G1n + Tyr203Asn + Thr207Asp + A1a209Pro + Ser210G1u + Asn212Ser +

~"" WO 95/30011 ~ PCTlUS95/04760 9~
G1y213Asn A1a194Ser + Pro195Asn + Asn198G1n -~ Va1199A1a + G1n200Asp + Tyr203His + A1a209Thr + Leu211Ser + G1y213Asp +
Thr214Ser A1a194Pro + Pro195Asn + G1y196Pro -- Va1197Ser + G1n200Asp + Tyr208Cys + Leu211G1n + Asn212Ser ~+ G1y213Asp +
Thr214G1y A1a194G1n + Pro195Ser + G1y196G1n ,- Asn198Asp + G1n200Ser + Thr202Ser + Tyr208Asp + A1a209Thr + Leu211His +
l0 Thr214Asn A1a194Asn + Pro195G1n + Va1197Thr ~- Asn198Asp + Pro209Asn + Thr207Ser + Tyr208Asp + Leu211I1e + G1y213G1n +
Thr214G1n A1a194G1n + Pro195Asn + Va1199Asn ~- Tyr203Ser + Pro204Asp IS + Thr207Asn + Ser210Asp + Leu211G1y + Asn212Ser +
G1y213Ser A1a194Asn + Pro195Asn + Thr202G1y + Pro204Asp + Tyr208I1e + A1a209G1y + Ser210Asp + Leu211Va1 + Asn212G1n +
Thr214Ser 20 A1a194Asn + G1y196Ser + Asn198Ser ~- Va1199Pro + Tyr203His + Pro204G1u + G1y205G1n + Tyr208Met + A1a209Thr +
Ser210G1u A1a194Thr + Asn198Ser + Va1199Thr ~- Thr202Asn + Pro204G1u + G1y205Asn + Thr207G1n + A1a209His + Ser210G1u +
25 Thr214G1y A1a194His + Pro195G1n + Asn198Ser +- Va1199G1y + Tyr203G1n + Pro204Asp + Thr207G1y + Ser210G1u + Asn212G1n +
G1y213Ser Pro195Asn + Tyr203Ser + Pro204G1u +- G1y205Pro + Tyr208His 30 + A1a209G1y + Ser210G1u + Leu211Pro + G1y213Pro +
Thr219Asn G1y196Asn + Asn198Ser + G1n200Ser +- Thr202G1y + Pro204Asp + G1y205Asn + A1a209Thr + Ser210G1u + G1y213Pro +
Thr219Ser 35 G1n200Ser + Thr202G1n + Tyr203Met +~ Pro204Asp + G1y205Pro + Tyr208Cys + A1a209G1y + Ser210.Asp + Leu211Thr +
G1y213G1n A1a194Pro + G1y196Asn + G1n200Ser + Tyr203Ser + Pro204G1u + Thr207G1y + A1a209Asn + Ser210.Asp + Leu211Va1 +
40 Thr214Ser Va1199His + G1n200Ser + Thr202Ser + Pro204Asp + G1y205Asn + Tyr208Pro + Ser210Asp + Leu211Pro + G1y213Pro +
Thr214Pro A1a194Ser + Pro195Asn + G1y196Ser + Va1197Ser + Asn198G1n 45 + Va1199Ser + Pro204G1u + Tyr208Leu + Ser210Asp +
Leu211Ser A1a194His + G1y196Pro + Va1199G1y + G1n200Asp + Pro204G1u + Tyr208G1n + A1a209G1y + Leu211,Ser + Asn212Ser +
Thr214Asn 50 A1a194G1n + Va1197Pro + Va1199Asn + G1n200Asp + Thr202G1n + Pro204Asp + G1y205Asn + Tyr208:His + A1a209Asn +
Leu211I1e Pro195Ser + Va1197Met + Asn198Ser + G1n200Asp + Thr202G1y + Tyr203G1n + Pro209G1u + Thr207Pro + Asn212Ser 1-WO 95130011 PCTIiJS95104760 Thr214G1y Pro195Ser + Va1199Met + G1n200Ser + Pro209G1n + Thr207Ser + A1a209G1u + Leu211A1a + Asn212G1n + G1y213G1u +
Thr214G1y Pro195G1n + G1y196Pro + Asn198Ser + Va1199G1y + Tyr203Asn + G1y205Asn + Thr207Pro + A1a209G1u + Leu211Va1 +
G1y213Asp A1a194Asn + Pro195G1n + Asn198Ser + Va1199Ser + G1y205Asn + Thr207G1u + A1a209Ser + Leu211G1u + Asn212G1n +
Thr214Ser A1a194Ser + Asn198Ser + G1n200Ser + Thr202G1y + Tyr203Leu + Thr207G1y + A1a209His + Ser210G1u + Asn212Ser +
Thr214Asp G1y196Ser + G1n200Asn + Thr202Pro + Tyr203Leu + Pro209Asn + Thr207G1n + Ser210Asp + Leu211A1a + G1y213Pro +
Thr214Asp G1y196Ser + Asn198G1n + Tyr203G1y + Pro204G1n + G1y205Pro + Thr207Pro + Tyr208His + Ser210G1u + G1y213G1n +
Thr214Asp Va1197Thr + Va1199Thr + G1n200Ser + Thr202Ser + G1y205Asn + Thr207Pro + Tyr208Cys + Ser210G1u + G1y213Ser +
Thr214Asp G1y196G1n + Va1197Thr + G1n200Asp + Thr202Asn + Tyr203His + Ser206G1u + Thr207Ser + Tyr208Pro + Leu211Pro +
Asn212G1n Va1197Met + Va1199G1n + G1n200G1u + Thr202G1n + Pro204G1n + G1y205Asn + Ser206Asp + Thr207G1n + Leu211Va1 +
Thr214Ser A1a194Thr + Pro195G1y + G1y196Asn + Va1197Ser + G1n200G1u + Thr202Pro + G1y205Pro + Ser206G1u + Leu211Met +
Asn212Ser Pro195Ser + G1y196G1n + Va1199Cys + G1n200G1u + Ser206G1u + Tyr208Thr + Leu211G1n + Asn212G1n + G1y213Pro +
Thr214Asn G1y196G1n + Va1197G1y + Asn198G1n + G1n200G1u + G1y205Asn + Ser206G1u + Thr207Pro + A1a209Asn + Asn212G1n +
G1y213Ser Pro195G1n + Va1197G1y + G1n200G1u + Thr202G1n + Tyr203Va1 + G1y205G1n + Ser206Asp + Tyr208Met + A1a209Pro +
Thr214G1y A1a194Ser + Pro195Asn + Va1197Thr + Va1199Thr + G1n200G1u + Thr202Pro + Tyr203Ser + G1y205Pro + Ser206G1u +
Thr214G1y Pro195Ser + G1n200G1u + Thr202G1n + Tyr203G1y + G1y205Pro + Ser206Asp + Thr207G1y + A1a209His + Leu211His +
Thr214G1y A1a194Asn + Pro195G1y + G1y196Asn + Va1197Cys + Va1199Asn + G1n200G1u + Tyr203Pro + Pro204G1n + Ser206Asp +
Leu211A1a A1a194Thr + Pro195Asn + G1y196G1n + Va1197Asn + G1n200G1u + Thr202Ser + Ser206G1u + A1a209His + Leu211A1a +
Thr214Ser Va1199G1y + G1n200G1u + Thr202G1n + Pro204Asn + G1y205Pro + Ser206Asp + Thr207Ser + A1a209G1y + Leu211Cys +

Asn212Ser A1a194Thr + Va1197Ser + Va1199G1n + G1n200Asp + Thr202G1n + Pro204G1n + Ser206G1u + A1a209G1y + Leu211His +
G1y213G1n A1a194G1y + G1y196Asn + Asn198G1n + G1n200G1u + G1y205Pro + Ser206Asp + A1a209His + Leu211Pro + G1y213G1n +
Thr214G1y G1y196Ser + Va1199Ser + Thr202Ser + Pro209Asp + Thr207Asn + Tyr208Leu + Leu211G1u + Asn212G1n + G1y213Ser +
Thr219Pro A1a194Pro + Pro195Asn + Va1197Pro + Thr202Pro + Tyr203Thr + Thr207G1n + Tyr208G1u + Asn212G1u + G1y213Ser +
Thr219Ser G1y196Asn + G1n200Ser + G1y205Pro + Thr207G1n + Tyr208Asp + A1a209Ser + Leu211Ser + Asn212G1u + G1y213Pro +
Thr214Pro A1a194G1y + Va1199Pro + G1n200Asp + Thr202Pro + Tyr203Cys + Pro204G1y + A1a209G1n + Leu2l:LCys + Asn212G1n +
Thr214Asp G1y196Ser + Va1197G1y + G1n200Asp + Tyr203Cys + Pro204Ser + Thr207G1n + Leu211A1a + Asn2l:?Gln + G1y213Ser +
Thr214Asp A1a194Pro + Va1197Thr + Thr202G1n + Tyr203Met + Pro204Asn + Ser206Asp + Tyr208His + A1a209His + Ser210Asp +
Thr214Asn G1y196Asn + Va1197Cys + Thr202Asn + Tyr203Met + G1y205Pro + Ser206Asp + Thr207G1y + A1a209Pro + Ser210Asp +
G1y213G1n A1a194Thr + Va1197Ser + Va1199His + Thr202G1n + Tyr203Ser + Ser206Asp + Thr207Ser + Ser210Asp + G1y213Pro +
Thr214Pro A1a194Thr + Pro195Ser + G1y196Asn + Va1197Pro + Asn198G1n + Thr202G1n + Ser206G1u + Ser210G1u + Asn212Ser +
Thr214G1y Pro195Asn + G1y196Ser + Va1197Ser + Asn198G1n + Thr202Asn + Tyr203Met + Ser206G1u + Ser210Asp + Asn212G1n +
Thr214G1n 4o Multi-loop Double Mutation Variants Leu 99G1y + G1n200G1u Gln 57Ser + Asn 60Ser Val 93G1n + G1y213Asp Tyr102Cys + Thr207G1y Ser159G1u + Asn:198G1n Leu124I1e + Asn:198G1n A1a209G1y + Ser:?lOGlu Gln 57Asp + Leu 94G1y Leu 94A1a + Ser:L54Glu SerlOlAsp + Leu:?llThr A1a131G1u + Gly:L96Gln Ser128G1u + Pro:?09Asn ~s Ser103Asp + Pro129Asn G1y157Asn + Thr207Ser Gly 98G1u + G1y155G1n G1n200Asp + A1a209Pro Asn 60G1u + Asn198G1n Leu 94I1e + Ser 99Asp Leu124I1e + Ser210Asp Ser126G1u + Thr202Ser Val 93A1a + Pro127Asp G1y157Pro + Ser210Asp Va1197G1y + Thr207Asp G1n185G1u + A1a199Thr Ser 97G1u + Tyr203Asn Glyl00Pro + Thr207Asp Ser210Asp + Leu211Met Tyr161Asn + Thr207Asp Leu124Ser + Pro127Asn Gly 6lSer + Thr207Pro Gly 98G1u + G1n200Ser G1n200Asn + Asn212G1u Asp 58G1u + I1e105Cys Gly 6lPro + A1a209Thr Leu124Asp + Pro204Asn Gln 57G1u + Asn198Ser G1n185Ser + Asn212Asp Phe183His + Va1197G1u Asn 60G1u + G1n200Ser Gly 59Asn + G1y 6lGlu G1y155Asp + I1e159Cys Gly 63G1n + Leu211G1y Thr 64Pro + Tyr161Asp Leu 94G1n + Pro127G1y Thr207G1y + G1y213G1u G1n185Asp + Tyr203Ser Gly 63Asn + A1a209Asn Tyr208Met + G1y213Asn Ser210G1u + Leu211Asn Tyr102A1a + Pro129Asn G1y125G1u + Leu211G1y Ser103Asp + Thr207Ser Ser128G1u + Leu211His G1y155G1n + Leu211His Gln 57Asn + G1n200Asp Ser103Asp + G1y157G1n G1y125Ser + Thr207G1y I1e105Pro + Va1197G1u Pro204Asp + A1a209G1n Tyr161Asp + Thr207Asn Asn198Ser + Ser210Asp SO Gly 95Ser + G1n200Asp Gly 59G1n + A1a156G1u Gly 98G1n + Tyr102Cys A1a181His + Thr207G1u Ser154G1u + Pro204Ser ~~~
"'"' WO 95130011 PCT/US95104760 G1y205Asn +Thr207G1n Ser210G1u +Leu211Pro Leu 94G1n +Ser210G1u A1a209Asp +Leu211Pro Asn 60G1u +Leu211His Ala 96Asn +Thr214G1y Asn 60G1u +A1~i194G1y Ser103Asp +A1~i181His G1y125G1u +A1a209Asn l0 Ser158Asp +Asn198Ser G1y155Ser +Asn198Ser Ser109Asp +G1y152Asn Ser182Asp +Leu211Cys Val 93Ser +G1y125G1u Asn198G1u +Leu211Ser G1yl00Ser +Ser103Asp A1a181G1n +G1y213G1n Thr 64G1y +Ser126Asp I1e159Cys +G1n200Asp Thr207G1u +Leu.211Asn Ser104G1u +Pro209G1y Ser130Asp +Pro209Asn Pro129G1n +Glr..185Asn Asn 60G1u +G1y157Ser G1y125Pro +Va1197Met Asn 60G1u +I1e105Cys Pro127G1n +Va1197Thr Ser101Asp +Leu.211A1a Gln 57Ser +Ser 99G1u Phe183Pro +Asn198G1u Asn198G1u +Thr202Pro Va1197G1n +Leu211His Gly 6lSer +G1n200Ser Gly 6lAsp +Asn198G1n Gly 6lGlu +Phe183Ser Thr207Asn +Asn212G1u Tyr203Thr +Tyr208G1y Asn198Ser +Leu211Cys Asn153Asp +Leu211Ser Gln 57Asn +G1y213Pro Thr 64Ser +Ser210Asp Pro204Asp +Leu211I1e Gln 57Asp +Gly 6lPro G1y155G1n +Ser210Asp A1a156G1y +Thr207Asn Gly 6lGln +A1a181Thr I1e159Cys +Asn198G1n Pro129G1y +Leu211G1u Asn198G1u +Thr207Ser Gly 98Ser +Ser128Asp A1a131Asn +Va1193Ser G1n185Ser +G1n200Asn Ser210Asp +Leu211His G1y125G1u +A1a156G1n PCTlUS95104760 W095/30011 zl~~~z~

Ser 97Asp + G1y125G1n Asn 60G1n + G1v155Pro Ser126G1u + Thr'?C7Ser Phe183Met + Thr207Pro Ser154G1u + Phe183I1e Val 93G1n + Ser210Asp Val 93G1n + Tyr208His Asn 60Ser + Asn198G1u G1y152G1n + G1n200Asp Pro127Asn + G1y152Asn G1y155G1n + Thr214Asn Ala 96Ser + Tyr208His Tyr161Thr + Ser210Asp Glyl00Pro + Pro204G1n Gln 57Asp + A1a194Asn Asn 60G1n + Thr202Asn G1y157Pro + G1y213Asn Val 93Thr + Leu211A1a Ser103G1u + Leu211Thr Ser101Asp + Leu124I1e Leu 94G1u + Pro204G1n G1n200G1u + A1a209His SerlOlGlu + G1y152G1n Asn198Asp + G1y205Pro Gly 6lSer + G1n200Asn Ser158Asp + Va1197Asn Gly 6lGlu + Phe183l1e Asp 58G1u + Tyr203Ser G1y213Ser + Thr219G1y Gln 57Asp + Asn198G1n Tyr102Leu + Ser158G1u Ala 96G1n + Leu211Asn Val 93Asp + Thr202Asn Leu 94Ser + Asn198Asp G1y196Ser + Asn212Asp G1y155Asn + G1y157Asp Val 93Asn + Ser130Asp Leu 94I1e + Ser210Asp Va1197A1a + Ser210Asp Ser104Asp + G1y205Pro Asn153Asp + A1a181G1y G1n200Asn + Leu211G1u Leu 94Asn + Ser210G1u G1n185Asp + Va1197G1n Tyr102Ser + Thr207Asp Gly 6lGlu + Gly 95Asn Gly 6lAsp + Pro129G1n Ser 99Asp + Thr207Ser Ser126G1u + G1y152G1n SO Va1197His + Ser206G1u Gly 6lAsn + G1y155G1u G1y155Asn + Asn198G1u A1a209Asn + Ser210G1u Ser128Asp + Thr207G1y '~""° W~ 95/30011 k PCT/US95/04760 A1a209Thr +Ser210Asp G1n185Asn +G1;y213Pro Ser126G1u +Leu211G1y Ser 97Asp +G1y205Ser Leu 94Thr +Leu129Thr Asn 60G1n +Leu211G1y Pro129Asp +G1:,~155Pro Asp 58G1u +G1y157Ser Thr202Pro +Thr207G1u A1a156Ser +Tyr208Asp Gly 98G1u +Tyr161G1n I1e105Cys +G1n200Asn G1n200G1u +Pro209G1y Gly 98Pro +A1a131Asn Gly 59Asp +Thr207G1n Tyr102G1n +A1a209G1n Gly 98G1u +I1e105His SerlOlAsp +Pro129G1n Pro127Asp +G1y213Asn Ser104G1u +Asn198G1n Gln 57Ser +G1n200Asn Va1197Cys +G1n200Ser Val 93A1a +A1~~156G1u G1n200Asn +G1y205G1u Leu 99A1a +Thr207Asn A1a131Thr +G1n200Ser Leu 94G1u +Tyr161Thr G1n200Asn +Ser210Asp Gln 57Asp +Thr207Asn Ser 99G1u +A1a156Pro Leu124I1e +Asn198Ser Tyr161G1y +Thr207G1n I1e105Cys +Thr214Asp I1e159His +Ser206Asp Gly 63G1n +G1y125Asn Asn198Asp +G1y205Ser Ser104G1u +Leu.124G1n A1a209Pro +Ser210G1u Tyr161Va1 +Asn.198Asp Pro129Ser +G1n.200Asp Ser 97Asp +Tyr203Ser Ser103Asp +Phe183Thr Tyr161G1y +Va1197A1a Asn198G1n +Pro204G1u Leu211G1y +Asn212G1u Ser206G1u +Leu211Thr G1y152Pro +Va1197Ser I1e105A1a +A1a156G1n Gly 63Asn +Tyr102Pro Ser182G1u +Va1197Asn Thr 64G1u +Ala 96G1n Asn 60G1n +Ser206G1u Asn 60G1n +Ser128Asp SerlOlAsp +I1e159Pro WO 95!30011 G1yl00Ser + Thr207Ser Gly 63Asn + G1y205Ser A1a181G1u + Thr207Asn G1y125Ser + Tyr161Ser G1n200G1u + Leu211A1a SerlOlAsp + Tyr102L'eu Ser126G1u + Thr207Pro Gly 59Asp + Tyr102Thr G1yl00Gln + Tyr102Met Gly 59Ser + Asn198Asp Asn 60Ser + Leu 99Cys Pro127G1u + G1y205Pro Gln 57G1u + A1a156Asn A1a131Asn + Thr207Asp A1a131Asp + Thr202Ser Leu129His + A1a131Asn G1y152G1u + Leu211His SerlOlGlu + A1a181Thr Asp 58G1u + G1y205Ser Gly 6lAsn + G1n200Asp Pro129G1y + Va1197Asp Thr 64G1n + Asn198Ser Val 93A1a + Ser154G1u Ser130G1u + Tyr161Va1 ~5 Phe183Asp + Leu211Thr Ser206G1u + Asn212G1n Gly 63Ser + A1a131G1u Pro127Asn + G1n200Asp Ser130Asp + Phe183Thr A1a181Thr + Pro195Ser Gly 95Ser + A1a156G1u Tyr161G1n + Tyr208I1e Gln 57Asp + Leu211Cys Asn153G1n + G1y213Asn Val 93Thr + Leu124Ser Ser101G1u + Asn212G1n G1y157Ser + Asn198G1u G1y152Ser + Ser182G1u Tyr161G1n + G1y213Ser Pro195G1y + G1n200Asn Asn 60Asp + Leu211Pro Pro195Ser + Tyr208G1u G1n200Asp + Leu211Asn Leu124Va1 + Thr207Pro Thr 64Pro + Ser126G1u Thr 64Ser + Tyr203Va1 Ser 97G1u + Asn198Ser G1y152G1n + Leu211Pro Asn198Asp + Leu211Asn Val 93G1u + G1y125G1n Gly 98Asn + G1n200Asp G1n200Asn + Thr207Asp Asn198Ser + G1n200G1u Tyr102Leu + Asn198G1n Leu124Cys + Asn153Ser Asn198Asp + Leu211Pro Ser126G1u + Tyr~08Met Ala 96Asn + G1y100Ser Ser 99G1u + Leu~llCys Ser 97Asp + Leu~llAla Ser182G1u + Leu211His I1e159Cys + Leu211G1u G1y152Asn + Ser210Asp to Va1197A1a + Gln:?OOGlu Asn 60Ser + Ser:158G1u Ser104G1u + Tyr208Asn Pro204Asp + Leu:?llGly Asp 58G1u + Va1:L97Gly 15 Ser182G1u + Tyr:?08Ser Ser103Asp + Ala:l94Ser A1a156G1n + Thra?07Asp Asn198Ser + Thr=?07Ser Gln 57Asp + Thr_?07Ser 20 G1y152G1u + Thr~?07G1y A1a131Ser + Ile.59Asn Pro127Asn + Gly:.52G1u G1n200Asn + Gly?l3Glu Leu124Asp + Leu~'_llAla Gly 98G1u + Glyl_25Asn I1e159G1y + Thr207Asn Pro127G1n + Asn1.53Asp Ser103G1u + Phe7.83Tyr Ser160G1u + Pro_'04Ser 30 G1y157Ser + Tyr1.61His A1a156Pro + Ser1.58G1u G1yl00Glu + Pro1.27Asn Gln 57Asn + Asn 60G1u G1y125Ser + Ser210G1u 35 G1y125Pro + Ser1.28Asp G1y125G1u + Va11.97G1y Va1193Met + Asn1.98Ser Ser160G1u + Gly~:l3Asn Ser206G1u + Ala~:09His 40 Ser 97G1u + Gly~:l3Ser G1yl00Asp + Leu~:llGly Pro129G1n + Thr~:07G1y Gly 95G1n + Ser210Asp G1n185Ser + Pro2:04G1u 45 Pro129G1u + A1a156Ser Ser126Asp + Asn212Ser Gly 59Ser + I1e159A1a Ala 96Asn + Ser 99G1u Ser104Asp + I1e159Va1 50 Gly 63Asn + Ser210G1u Phe183Pro + Ser210G1u Ala 96Asp + Tyr208I1e Gly 98Asp + Thr207G1n Val 93G1n + G1y125G1u 2189~2'~
.o I1e159G1u + Thr207G1n Leu 94I1e + Leu211Va1 G1y157Asp + Leu211His Gly 6lAsp + Leu211Cys Gln 57Asn + G1y157Asn Glyl00Glu + Va1197Ser G1n200G1u + Pro204Asn A1a131G1y + Leu211His Ser206Asp + Leu211Asn Asn153Asp + I1e159Leu G1y152Ser + Asn198G1u Leu 94Met + Ser130G1u Gln 57G1u + Asn198G1n Ser182G1u + Leu211Thr Va1199A1a + Ser210G1u Thr207G1y + Ser210G1u Thr 64G1y + Gly 98Asp Gly 6lGln + Ser 99Asp G1y155G1u + Leu211Ser Leu124Ser + Thr207Asp Val 93Ser + Asn198G1n Ser 99Asp + G1y125Asn Gln 57Asn + Asn198G1u Ser 99G1u + I1e159Met Multi-loop Triple Mutation Variants Gln 57Ser + Leu 94G1y + G1n200G1u Asn 60Ser + Val 93G1n + G1y213Asp Tyr102Cys + Asn198G1n + Thr207G1y Leu124I1e + Ser154G1u + Asn198G1n Leu 94G1y + A1a209G1y + Ser210G1u G1y155Asp + A1a209G1n + Asn212G1n Pro129Asn + G1y157Asn + Thr207Ser Asn198G1n + G1n200Asp + A1a209Pro Val 93A1a + Pro127Asp + Thr202Ser Tyr161Asn + Thr207Asp + Leu211Met Gly 6lSer + Pro127Asn + Thr207Pro Gly 6lPro + Leu124Asp + Pro204Asn Gln 57G1u + G1n185Ser + Asn198Ser Gly 59Asn + Asn 60G1u + G1n200Ser Gly 63G1n + G1n200Asp + Leu211G1y Tyr203Ser + Thr207G1y + G1y213G1u Gly 63Asn + G1n185Asp + A1a209Asn Pro129Asn + Ser210G1u + Leu211Asn Tyr102A1a + G1y125G1u + Leu211G1y Gln 57Asn + G1y155G1n + Leu211His I1e105Pro + G1y125Ser + Thr207G1y Asn198Ser + Thr207Asn + Ser210Asp Gly 59G1n + Gly 95Ser + A1a156G1u Tyr102Cys + Ser210G1u + Leu211Asn G1y205Asn + Thr207G1n + Ser210G1u Leu 94G1n + A1a209Asp + Leu211Pro Asn 60G1u + Leu211His + Thr214G1y Asn 60G1u + A1a181His + A1a194G1y Ser104Asp + Glyl555er + Asn198Ser G1y152Asn + Ser182Asp + Leu211Cys G1yl00Ser + Ser103Asp + Leu211Ser Thr 64G1y + Ser126Asp + I1e159Cys Ser130Asp + A1a181Asn + Pro204G1y Pro129G1n + G1y157Ser + G1n185Asn Asn 60G1u + G1y125Pro + Va1197Met I1e105Cys + Pro127G1n + Va1197Thr Gln 57Ser + Ser 99G1u + Phe183Pro Gly 6lSer + Va1197G1n + Leu211His Gly 6lAsp + Asn198G1n + G1n2005er Tyr203Thr + Thr207Asn + Tyr208G1y Asn153Asp + Asn198Ser + Leu211Ser Gln 57Asn + Ser210Asp + G1y213Pro Thr 69Ser + Pro204Asp + Leu211I1e SerlOlAsp + A1a156G1y + Thr207Asn I1e159Cys + A1a181Thr + Asn198G1n Gly 98Ser + Asn198G1u + Thr2075er Ser128Asp + A1a131Asn + Va11935er G1n185Ser + G1n200Asn + Leu211His Ser126G1u + Phe183Met + Thr2075er Ser154G1u + Phe183I1e + Thr207Pro Val. 93G1n + Tyr208His + Ser210Asp Val. 93His + Pro127Asn + G1y152G1n G1y155G1n + G1y213G1u + Thr219Asn G1yl00Pro + Tyr161Thr + Ser210Asp Gln 57Asp + A1a199Asn + Pro209G1n G1y157Pro + Thr202Asn + G1y213Asn Thr 64G1n + Val 93Thr + Leu211A1a Gly 6lSer + G1n200Asn + G1y205Pro Ser158Asp + Phe183I1e + Va1197Asn Asn198Asp + G1y2135er + Thr219G1y G1y152Ser + G1y155Ser + Ser206Asp G1y152G1n + G1n1855er + Va1197G1n Leu 94I1e + Va1197A1a + Ser210Asp A1a181G1y + G1n200Asn + Leu211G1u Leu 99Asn + Va1197G1n + Ser210G1u Gly 6lAsp + Pro129G1n + Thr2075er Gln 57Asn + Gly 6lRsn + G1y155G1u Gly 6lPro + Ser128Asp + A1a209Thr Thr207G1y + A1a209Thr + Ser210Asp Ser128Asp + G1n185Asn + G1y213Pro Leu 94Thr + Leu124Thr + G1y205Ser Asn 60G1n + G1y155Pro + Leu211G1y Asp 58G1u + G1y157Ser + Thr202Pro Gly 98G1u + I1e105Cys + G1n200Asn Gly 98Pro + G1n200G1u + Pro204G1y Gly 59Asp + A1a131Asn + Thr207G1n Gly 98G1u + Tyr102G1n + A1a209G1n SerlOlAsp + I1e105His + Pro129G1n Pro127Asp + Asn198G1n + G1y213Asn WO 95130011 21 g g 4 2 ~ PCTJUS95/04760 IOG
I1e159G1y + Va1197G1y + Asn198G1n Gln 57Ser + G1y155G1u + G1n200Asn Va1197Cys + Asn198G1u + G1n2005er Leu 94A1a + G1n200Asn + G1y205G1u A1a131Thr + G1n200Ser + Thr207Asn Leu124I1e + A1a156Pro + Asn198Ser Val 93G1u + Tyr161G1y + Thr207G1n Gly 63G1n + G1y125Asn + A1a209G1u Tyr161G1y + Va1197A1a + Asn198G1n Ser158Asp + Asn198Ser + Leu211G1y I1e105A1a + G1y152Pro + Va1197Ser Gly 63Asn + Tyr102Pro + A1a156G1n Ala 96G1n + Ser182G1u + Va1197Asn Asn 60G1n + SerlOlAsp + I1e159Pro G1yl00Ser + G1y205Sez + Thr207Ser Tyr161Ser + A1a181G1u + Thr207Asn G1y125Ser + G1n200G1u + Leu211A1a Gly 59Asp + Tyr102Thr + Thr207Pro Gly 59Ser + G1yl00Gln + Tyr102Met Asn 60Ser + Leu 94Cys + Asn198Asp Pro127G1u + A1a156Asn + G1y205Pro Asp 58G1u + A1a181Thr + G1y205Ser Thr 64G1n + Val 93A1a + Asn198Ser Phe183Asp + Leu211Thr + Asn212G1n Pro127Asn + Ser130Asp + Phe183Thr Tyr161G1n + Tyr208I1e + Leu211Cys Leu124Ser + Asn153G1n + G1y213Asn Val 93Thr + Ser101G1u + Asn212G1n Tyr161G1n + G1n200Asn + G1y213Ser G1n200Asp + Thr207Pro + Leu211Asn Thr 69Pro + Leu124Va1 + Ser126G1u Thr 64Ser + Ser 97G1u + Asn198Ser G1y152G1n + Ser210Asp + Leu211Pro Val 93Pro + Asn198Ser + G1n200G1u Thr 64G1n + Tyr102Leu + Asn153Ser Ala 96Asn + Glyl00Ser + Tyr208Met I1e159Cys + Ser182G1u + Leu211His Asn 60Ser + Ser158G1u + Tyr208Asn Ser103Asp + A1a194Ser + Tyr208Ser A1a156G1n + Asn198Ser + Thr207Asp A1a131Ser + G1y152G1u + I1e159Asn Pro127Asn + G1n200Asn + G1y213G1u Gly 98G1u + G1y125Asn + Leu211A1a I1e159G1y + Thr207Asn + Ser210G1u Ser103G1u + Phe183Tyr + Pro204Ser G1y157Ser + Ser160G1u + Tyr161His Gln 57Asn + Glyl00Glu + Pro127Asn G1y125Pro + Va1197G1y + Ser210G1u Va1193Met + Asn198Ser + G1y213Asn Gly 95G1n + Pro129G1n + Thr207G1y Gly 59Ser + Ser126Asp + Asn212Ser Gly 63Asn + I1e159Va1 + Ser210G1u Phe183Pro + Tyr208I1e + Ser210G1u Leu 94I1e + I1e159G1u + Thr207G1n t07 Gln 57Asn + G1y157As:n +Leu211Cys A1a131G1y + Pro204As:n +Leu211His I1e159Leu + Ser206As +Leu211Asn Leu 99Met + G1y152Ser +Asn198G1u Ser182G1u + Va1199A1~~ +Leu211Thr Leu124Ser + Thr207Asp +Leu211Ser Val 93Ser + G1y125Asn +Asn198G1n Tyr102Pro + A1a156Se:r +Phe183His Gly 98G1n + Ser128G1u +I1e159Cys Ser104Asp + I1e105Pro +Tyr161Thr Tyr102Pro + I1e105Th:r +A1a181G1u Thr 64Ser + I1e105G1u +G1n200Asn Ser109Asp + G1n200Se:r +Thr214Pro Thr 64Pro + A1a156G1u +Thr202G1y Gly 63G1n + G1yl00As~~ +Leu211His Asn 60Ser + Gly 98Asp +Leu211Thr Tyr161G1y + Va1197Cy;~ +Ser206G1u Thr207G1n + Tyr208G1n +Ser210G1u Gly 98Ser + I1e105he1i +Ser210G1u Thr 64G1y + Pro195Se~_-+Thr214Ser Val 93Met + Ser159As~~ +Leu211Met Leu124Ser + Asn198Ser +Leu211Asp Thr 64G1y + A1a156Ser +G1y157Pro Gly 59Ser + G1n200Asn +G1y213G1u Gly 6lAsn + Leu124I1e +A1a181Pro Ala 96His + G1n185Asp +Leu211A1a Ser101Asp + Asn198Ser +Thr214Ser Leu 99Met + Asn153Asp +Pro195G1n Pro127G1n + G1y152Asp +Va1193His Ser 97Asp + I1e105Thr +Thr207G1n Gly 6lPro + Ser160G1u +Thr207G1n Leu129Va1 + Pro204G1v +Leu211I1e Ser 99Asp + Asn198G1n +G1n200Ser Ser101G1u + A1a181Asn +A1a209Pro Gly 6lAsn + Ala 96Thr +Gly 98Ser Val 93Pro + Asn198Asp +Thr207G1n Thr 64Pro + Ala 96Pro +G1y155G1u Thr 69G1y + A1a181Thr +Thr214Pro Thr 64Pro + Leu 99Cys +Tyr208Met Gln 57Asn + Leu 99G1n +Thr214Asn Leu 94G1n + Tyr161Pro +Thr207G1y Val 93His + G1n185Asn +Thr207Pro G1y125Pro + Asn198G1u +G1n200Ser Asn153Ser + I1e159Cy:; +Ser160Asp Thr 64G1y + G1n200G1u +Leu211Cys Leu 94Va1 + Pro195G1n +Leu211Asp Gly 59Pro + G1yl00Ser +Thr207Pro Tyr102Cys + G1y157G1n +Thr207Pro Ser 99Asp + Leu124Ser +Thr219Asn Gly 59G1n + G1n200Asp +Thr207Ser Ala 96His + A1a181Asp +Leu211Cys Val 93Met + A1a181Hi~> +Thr207Ser Pro129G1n + Asn198Ser +Thr207Ser Asp 58G1u + Gly 95Ser +A1a194G1y W095/30011 ~~$94~v' ios Ser160G1u + Thr207G1n + Thr214G1y Thr 64Asn + Phe183Asp + Thr207Asn Ser158Asp + Phe183I1e + Va1197His Gly 95Pro + Asn153Ser + G1n200G1u G1y157Asp + Pro204G1y + Thr207Ser Va1197Pro + Asn198G1n + 'I'hr207G1n Gly 6lPro + Leu 99G1u + Tyr161I1e Pro129G1n + G1y155Pro + Ser210G1u Val 93Met + Ser182Asp + G1n200Ser Pro127Ser + Leu211Met + G1y213Ser Ala 96His + Ser160Asp + Tyr208G1y Gly 98Pro + Pro129Ser + Pro204Asp Gly 59Ser + Gly 63Pro + Pro129Asp G1yl00Pro + Leu124Pro + Thr207Asn Ser103Asp + I1e159A1a + Thr202G1n Gly 59Asn + Ser130G1u + Leu211Thr Ala 96G1y + A1a131Asn + G1y155Pro Asn153Ser + G1n200Asn + Ser210G1u Asn153G1u + Thr207Pro + Leu211A1a Pro127Asn + Va1197His + Thr207Asp G1yl00Asp + A1a131G1n + Tyr208Thr G1y125Pro + Phe183Pro + Asn198G1u Asn 60Ser + I1e105Thr + Thr207G1u Thr 64Ser + Pro127Asp + A1a131Thr Ser 99Asp + Leu124Asn + Phe183Pro Thr 64G1n + SerlOlAsp + Leu211Ser Pro195G1n + Va1197G1y + Leu211Ser G1yl00Asn + Tyr102G1y + Thr207Pro Asn198Ser + G1n200Asp + Leu211Pro Tyr102Met + Ser182G1u + Leu211A1a Gly 6lAsn + G1n200Asn + G1y213Asn Ser 99Asp + G1y152Pro + G1y157Ser Leu124Ser + Tyr161Met + G1n200G1u Tyr102Met + Leu211G1u + Thr214Ser Asp 58G1u + Gly 98Asn + Tyr161Asn I1e159Ser + G1n200Asn + Tyr208Asn Thr 64Asn + A1a156G1y + Leu211Asp G1n200Asn + Ser210G1u + Leu211Thr Asn 60Asp + G1y155Pro + Va1197Asn A1a156G1n + Asn198G1u + G1y205G1n Gln 57Asp + G1n200Ser + Tyr208Thr Leu124I1e + A1a209G1u + Ser210Asp Gly 59G1u + Asn 60Asp + Leu211A1a A1a131G1y + G1y152Asp + Asn153G1u Tyr203Va1 + Ser210G1u + Leu211G1u G1y152Asn + Ser210Asp + Leu211G1u Phe183Va1 + Ser210Asp + Leu211Asp Tyr102Leu + Ser210Asp + Leu211G1u G1n200Asn + Thr207G1u + Tyr208G1u SO A1a156Ser + Thr207G1u + Tyr208Asp Pro129Asp + Ser130Asp + Asn198Ser Asn198Ser + G1n200G1u + Ser210Asp Leu129Cys + G1n200G1u + Ser210G1u Thr 64G1y + G1n200Asp + Ser210Asp ..
W0 95/30011 ~~~~ ~ ' PCTIUS95104760 Gly 6lAsn +G1n200Asp + Ser210G1u Phe183His +Va1197G1u + Asn212Asp Ala 96G1y +SerlOlGlu + Ser103G1u Ser126G1u +Ser128G1u + Asn198Ser Gly 6lPro +Ser154Asp + A1a156G1u Val 93Asp +Ser104Asp + Leu211A1a Ala 96Ser +G1y125Asp + Tyr161Asp Ser 97Asp +Ser 99G1u + Leu211Cys Ser 97Asp +Ser 99Gltz+ Asn153G1n Ala 96Pro +G1y152Asp + Ser159Asp Gly 98Asn +Asn198Asp + G1n200Asp Asn 60Ser +Asn198G1u + G1n200Asp Pro129Ser +Asn198Asp + G1n200Asp Gln 57G1u +Gly 59G1u + Leu211G1n Ser128Asp +Ser130G1u + A1a209His Ser158Asp +Ser160G1u + G1n185Asp Pro127G1n +Ser210G1u + Asn212Asp Ser210G1u +Leu211Asn + Asn212Asp Va1197Asp +G1n200G1u + Ser210Asp Phe183G1u +Tyr208Va)_+ G1y213Asp Pro127G1u +G1y152G1u + Tyr161Asp Leu 94G1u +SerlOlAsp + Leu129I1e Leu 94G1u +SerlOlAsp + G1n200Asn Pro127G1n +Asn153G1u + A1a156G1u Asp 58G1u +Gly 6lGln + Gly 95Asp Pro129G1y +Va1197Asp + 5er210G1u Asp 58G1u +Gly 6lGlu + Gly 95G1u Va1197G1u +Ser210Asp + G1y213Asp Ser154G1u +Ser182G1u + Pro204Ser SerlOlGlu +G1y125G1u + Ser126G1u Ser 99Asp +Ser104Asp + I1e105Leu Ser128G1u +A1a131G1u + G1y196G1n Gly 6lGlu +Ser 97Asp + A1a131His Ser 97Asp +G1yl00Asp + Ser104G1u Asp 58G1u +Pro204Asn + G1y205Asp G1n185G1u +Leu211Cy~>+ G1y213Asp G1n200Asp +Thr207G1u + Leu211Asn G1y125Ser +G1n200Asp + Thr207G1u G1n200G1u +Thr207Asp + Leu211Cys Asp 58G1u +Val 93G1~~+ Ser 97G1u Asp 58G1u +Ser 97G1u + Tyr203Asn Gly 6lGlu +Gly 95Asn + Thr207Asp Ala 96Pro +Thr207Asp + Ser210Asp Pro204Asn +Thr207G1u + Ser210Asp G1yl00Pro +Thr207Asp + Ser210Asp Gly 6lAsp +Thr207Asp + Ser210Asp Asp 58G1u +Thr 69Asp + Ser 97G1u Asn153Asp +G1y157G1u + A1a181G1u Ser130Asp +Ser160As~>+ G1y205G1n Tyr102Asp +Ser128As~>+ Ser160Asp Gln 57Asp +Ser206Asp + Thr207G1u Ser 99Asp +Ser126G1u + G1y152G1n A1a181His +Asn198G1u + Thr214G1u Gln 57Asn +Thr 64G1u + G1n200G1u PCTlUS95/04760 W095130011 2t89 ~.2~

Thr 64Asp + Leu124Thr + G1n200Asp Gly 98Asp + G1y125G1u + Thr207G1n Asn198G1u + G1n200G1u + Pro209G1u Asp 58G1u + Gly 59Asp + G1n200Asp Leu124Asp + Va1197Asp + Asn198Asp Pro129G1u + Asn212Asp + G1y213G1u Ser158Asp + Ser210Asp + Leu211G1u Ser101G1u + Tyr102G1u + G1n200Asp Ser103Asp + Ser109Asp + Asn198Asp Asn 60G1u + Ser103G1u + Ser104G1u Ser182Asp + G1n200Asp + Ser210Asp G1y157G1u + G1n200G1u + Ser210Asp Gln 57Asp + G1n200Asp + Ser210G1u Ser 99G1u + G1n200Asp + Ser210Asp Ser182G1u + Asn198Asp + G1n200G1u Gln 57Ser + G1n200Asp + G1y213G1u Ser 99G1u + Ser103Asp + I1e159Met Ser 99Asp + Ser103Asp + G1y213G1n G1y155G1u + G1n185G1u + Asn198G1u G1y155Asp + G1n185G1u + Ser210Asp Gln 57Asp + Thr 69G1u + Ser210G1u Thr207G1u + Leu211G1u + G1y213Asp Ser126Asp + Ser160Asp + Ser210Asp Ser130G1u + Asn198Asp + Ser210Asp 2,5 Ser126Asp + Asn198Asp + Ser210G1u SerlOlAsp + Asn198G1u + Leu211G1u G1y155G1u + Asn198Asp + Leu211G1u Asp 58G1u + Asn 60G1u + Ser103Asp Ser 99G1u + Ser109G1u + Ser126G1u Gly 59Asp + Gly 6lGln + Ser 99Asp Asn 60G1u + Ser158G1u + G1n185G1u Glyl00Glu + Ser104Asp + Ser210G1u G1yl00Asp + Ser104Asp + Ser182G1u Ser128G1u + Ser130Asp + Asn198G1u Ser159G1u + G1n200Asp + Leu211G1u Ser103Asp + G1n200G1u + Leu211Asp Ser103G1u + G1n200G1u + Leu211Asp Ser126Asp + A1a156G1u + Ser182Asp Ser158Asp + Ser160G1u + Va1197G1u Ser158Asp + Ser160Asp + Va1197G1u Val 93G1n + G1y125G1u + Ser158G1u Gln 57Asp + Leu124Asp + Ser126Asp Asn 60Asp + Ser210G1u + Asn212G1u G1y157Asp + Ser210G1u + Asn212Asp Ser 99G1u + Ser101G1u + Ser210G1u Asp 58G1u + Asn198G1u + Pro209G1u Tyr102Asp + Ser130G1u + Ser182Asp Asn 60Asp + Gly 95G1u + Ser128Asp Leu 99G1u + Ser 9~Asp + Ser206G1u Gln 57Asp + Ser126G1u + G1y152Asp Leu 94G1u + SerlOlGlu + Leu211Asp Gly 59G1u + Ser 97G1u + Ser182G1u G1yl00Asp + Ser103G1u + Ser206Asp Asn 60G1u + G1n185G1u + Thr214G1u WO 95130011 "~" PCTIUS95104760 Asn 60G1u + Ser130G1u + Pro204Asp Asn 60Asp + Asn198Asp + Pro204Asp Ser 99Asp + Ser182G1u + G1n185Asp Pro204Asp + Ser210G1u + Leu211G1y Tyr161Va1 + Pro204G1u + Ser210G1u Ser101G1u + Asn198Asp + G1y213G1u Leu129Asp + Ser154G1u + Va1193Ser Ser 99G1u + A1a181G1u + Asn212Asp Ala 96Asp + Glyl00Asp + Ser154G1u Gly 6lAsp + Ser 99Asp + Ser210G1u Asp 58G1u + Thr207G1u + Ser210Asp Multi-loop Quadruple Mutation Variants Gln 57Ser + Asn 60Ser + Leu 94G1y + G1n200G1u Val 93G1n + Tyr102Cys + Thr207G1y + G1y213Asp I1e105Va1 + Leu124I1e + Ser154G1u + Asn198G1n Leu 94G1y + Ser103G1u + G1n200Asn + A1a209G1y A1a156His + G1n200Ser + Thr207Pro + Leu211Thr Pro129Asn + G1y155Asp + A1a209G1n + Asn212G1n Leu124Pro + A1a194Asn + Asr~l9eGlu+ G1n200Asn Gly 6lSer + Asn198G1n + G1n200Ser + Leu211His Tyr203Thr + Thr207Asn + Tyr208G1y + Asn212G1u Gln 57Asn + Asn153Asp + Leu211Ser + G1y213Pro A1a131Asn + G1n185Ser + Va1.193Ser+ G1n200Asn A1a181G1y + A1a194Pro + Asn198Ser + Asn212Asp Ser101Asp + Leu129Va1 + Pro195G1n + Thr207Pro Gly 95Asp + Asn198G1n + G1n200Ser + Thr207Ser Ala 96G1n + Tyr102Pro + Ser182G1u + Va1197Asn Asn 60G1n + G1yl00Ser + SerlOlAsp + I1e159Pro Gly 63Asn + Asn153Asp + G1y205Ser + Thr207Ser Ser126G1u + A1a131Pro + Asr.153Ser+ Phe183Thr Asn153G1n + G1y205Pro + Ser210G1u + G1y213Pro I1e159G1u + A1a194Ser + Pro204G1y + Leu211I1e Asp 58G1u + Val 93Ser + A1a194Pro + G1n200Ser Gly 95Asp + I1e105G1n + Phe183Cys + Asn198G1n Gly 59Pro + Gly 98Asn + Leu.211Va1+ Thr214Asp Gly 6lPro + Val 93Pro + Prc129Asn + G1y213Pro Gln 57Asn + A1a156Pro + A1a181Pro + Phe183G1y Gly 95G1n + G1y152Asp + Phe183Met + Asn198G1n Gly 59Ser + Gly 95Asn + Pro127G1n + Ser210G1u Va1197Cys + Asn198G1n + Thr207G1u + Leu211Cys Pro129Asn + Ser159Asp + Phe183Pro + Leu211Cys Gly 59G1n + G1yl00Glu + A1a131G1y + A1a181G1n Gly 6lGlu + Tyr161Leu + Pro204Ser + G1y213Pro Asp 58G1u + I1e159Met + Tyr161G1n + G1y213Asn I1e105Pro + Leu124Asn + I1e159Ser + Asn198Asp Tyr102Pro + A1a156Ser + Phe183His + Pro204Asp Tyr102Pro + I1e105Pro + G1n200Asn + Pro204G1u Thr 64Ser + I1e105G1u + G1n200Ser + Thr214Pro Gly 63G1n + G1yl00Asp + Asn198Ser + Leu211His Asn 60Asp + I1e105Leu + G1y152G1n + A1a209G1n W O 95130011 ~ 1 g g ~ ~'~~.

G1y152G1n + G1n185Asn + G1y205Ser + Leu211A1a Gln 57Ser + G1yl00Pro + Pro127G1y + Asn198G1u Leu 94Cys + Pro127G1y + Va1197G1y + Asn198Ser Leu 99Thr + Tyr102Cys + Leu124Cys + Tyr208Ser Ala 96Ser + A1a181Asn + Pro204Ser + Tyr208I1e Leu124Va1 + Asn198G1n + Pro204G1y + Leu211I1e Gly 6lAsn + Ala 96Thr + Gly 98Ser + Ser210Asp Thr 64Pro + Ala 96Pro + G1y155G1u + Leu211Cys G1y157Asp + Va1197Pro + Asn198G1n + Pro204G1y Pro129G1n + G1y155Pro + Va1197His + Ser210G1u Val 93Met + Ser182Asp + G1n200Ser + G1y213Ser Leu 94G1u + Pro127Ser + G1n200Asn + Leu211Met Ala 96His + Gly 98Pro + Pro129Ser + Pro204Asp Ser 97Asp + Glyl00Pro + Leu129Pro + Thr207Asn Gly 59Asn + Thr207Pro + Ser210G1u + Leu211Thr Ala 96G1y + A1a131Asn + G1y155Pro + Ser210G1u Asn153Ser + Va1197Thr + G1n200Asn + Ser210G1u Asn 60Ser + I1e105Thr + Ser182G1u + Thr207Ser Gly 98Asn + Tyr161Asn + G1n200Asn + Tyr208Asn Leu 94G1y + Ser 99G1u + Leu124Ser + A1a181Pro Ser104G1u + A1a131Pro + Leu211Ser + G1y213Ser Tyr102Asp + Asn198Ser + Thr207G1n + Leu211Asn Gly 98Pro + Ser206G1u + Leu211G1y + G1y213Pro A1a131Asn + A1a181Thr + Tyr208G1n + Ser210Asp G1y155G1n + Tyr161Cys + G1n185Ser + A1a209G1n Thr 64Asn + Val 93Asn + Ser126G1u + G1n200Asn A1a131Pro + Phe183A1a + Thr207Asp + Thr219G1n Ser 97G1u + Leu124Pro + A1a131Ser + Pro204G1y Gly 59Asp + Tyr161His + Thr207G1y + Leu211Met Gly 95Pro + Gly 98Pro + SerlOlGlu + Leu211Cys Gly 59Ser + Thr 64G1y + Leu124I1e + G1y125G1u Thr 64Pro + SerlOlGlu + Tyr161G1y + G1n200Asn G1yl00Glu + Pro195Ser + Va1197Ser + Thr207Asn Ser160G1u + Tyr161G1n + Pro209Ser + Leu211G1n Leu124Met + Tyr161Leu + Ser182G1u + Leu211His I1e105Thr + Pro127Asn + G1y157Asn + Tyr161Pro G1n185Asn + Thr207G1n + Asn212Ser + Thr219G1n Val 93G1n + Leu 94Ser + Gly 98G1u + Thr219G1y Asn153G1n + Ser159G1u + A1a181Asn + Leu211Va1 G1y125Asn + Ser154Asp + Va1197Ser + Leu211Va1 I1e159Asn + G1n185G1u + Va1197Met + Asn212G1n Ala 96Pro + G1y196G1n + Va1197A1a + Asn198G1n A1a131G1u + G1y152G1n + A1a194Ser + Leu211I1e Phe183Ser + Asn198Asp + G1n200Ser + Thr207Pro Gly 95G1n + G1yl00Ser + Ser130G1u + Thr207G1n Leu129Cys + G1y152Ser + Tyr161Met + Thr207G1n Leu 94Met + Leu129Met + Leu211A1a + Asn212Ser Gly 6lAsn + Ala 96Ser + G1y125Pro + G1n200Asp I1e159G1n + Va1197G1u + G1n200Asn + Tyr208His Tyr161Asn + Ser182G1u + A1a209Ser + Asn212Ser Gln 57Asn + G1y152Ser + Phe183G1u + G1n185Asn Asn198G1n + Thr207G1u + A1a209G1y + G1y213Pro Va1197G1u + Tyr208G1y + A1a209Pro + Leu211Pro A1a131His + G1y205G1n + Tyr208Asp + Leu211G1n w'WO 95130011 ~ PCT/US95104'760 Gly 6lAsn +Val 93Cys + Gly 98G1n + Leu211G1u Leu124A1a +Va1197G1n + Ser210G1u + Asn212Ser Pro127G1n +I1e159Leu + Ser160G1u + A1a209Ser Asp 58G1u +Gly 59G1n + G1y205G1n + Leu211Cys Asp 58G1u +Phe183Tyr + Asn198Ser + Thr207G1n Pro204G1y +Ser206Asp + Thr207G1n + Leu211His Gln 57Asp +Gly 98Ser + I1e105G1n + Tyr208Met Ser103G1u +G1y155Ser + A1a209His + Leu211Thr Ser104G1u +A1a181Asn + Asn198G1n + Leu211Thr G1n185Asn +Va1197Cys + G1y205Asp + Leu211Cys Gly 59G1n +Tyr102G1u + Pro127G1y + A1a209Thr A1a131Thr +Asn153Ser + Va1.193G1n+ Va1197Asp A1a131Ser +Asn153Ser + Phe183Tyr + A1a194Ser Pro129G1n +A1a181Pro + A1a.209His+ Leu211A1a Pro127G1y +G1y196Asn + Ser210G1u + Leu211Met Leu 94I1e +G1yl00Asp + Asn.198Ser+ Tyr208Leu Asn153Ser +G1y155Pro + G1y205Asp + Leu211Ser Gly 63Asn +G1y152Pro + Asn.198G1n+ Thr207G1u G1y152Ser +Ser160G1u + Tyr161His + Leu211Met Pro129Asn +Asn153G1n + G1y155G1n + Ser210Asp Gln 57Ser +Thr 64G1n + Va1197Asn + G1n200Asp Ser126Asp +Asn153G1n + Prc209G1n + Leu211G1n Gly 6lGln +Ala 96G1n + Phe183Leu + Leu211Thr Gly 63Asn +A1a156Asn + Asn198Ser + Ser210G1u Gly 59Asp +G1y205Pro + Thr207G1n + Leu211Asn Gly 98Ser +Leu124Pro + I1e159Thr + Va1197G1u G1y205Pro +A1a209G1n + Ser210G1u + Leu211Pro Pro129Ser +Thr207G1y + Ser210G1u + Leu211Asn Gln 57Asp +A1a131G1n + Leu211Met + Asn212G1n Thr 64Asn +Tyr161I1e + Phe183Asn + Ser210Asp G1y155G1n +Pro204Ser + A1a209His + Leu211Asp G1yl00Gln +G1y157Pro + I1e159Leu + Ser160Asp Gly 98Pro +Ser103Asp + A1a131G1y + Leu211Asn Gln 57Ser +Gly 6lPro + I1e159G1n + Leu211Asn G1y155Ser +G1n185Asp + Va1199Met + Thr207G1y Asp 58G1u +Ala 96G1y + Phe183Thr + Thr207G1y Val 93A1a +Ser126G1u + A1a131Thr + Thr207Pro Thr 64G1n +Gly 98Pro + Va1197Thr + G1n200G1u G1y152G1u +Asn153G1n + G1y155Pro + G1y213Pro Ser159G1u +Asn198Ser + G1n200Ser + Leu211Ser I1e105Thr +I1e159Thr + Phe183Va1 + Leu211Asp Leu124Pro +Ser128G1u + I1e159Met + Leu211Pro Gln 57Asp +Asp 58G1u + Leu211Thr + Thr219Ser Gly 59Asp +Asn 60Asp + Va1197Pro + Va1199A:La Ala 96Thr +Leu129Cys + Va1197Asp + Asn198G1u Thr 64Asn +A1a156G1y + Ser210Asp + Leu211Asp Leu124Ser +G1n200Asn + G1y205Asp + Ser206G:1u Gly 63Pro +A1a156Asp + G1y157Asp + I1e159G1n Thr 64G1y +G1yl00Glu + Ser101G1u + Leu211Asn Gly 6lAsn +Asn153G1n + G1n200Asp + Ser210Asp Leu124Ser +G1n200G1u + Ser210G1u + Leu211G1n Va1197G1n +G1n200G1u + Thr207Ser + Ser210G1u A1a209G1u +Ser210G1u + Leu211G1u + G1y213Ser Thr207Pro +Ser210G1u + Leu211Asp + Asn212G1u WO 95!30011 lid Ser101Asp + Ser104Asp + Asn198G1n + Leu211Ser Thr 64Asn + Ala 96Asp + Gly 98Asp + G1y213Asn Asn198G1u + G1n200Asn + Ser210Asp + Leu211Thr Tyr161Va1 + Asn198G1u + Ser210G1u + G1y213Asn Val 93Pro + Asn198G1u + G1n200Ser + Ser210G1u Va1193Pro + Asn198Asp + Thr207S~er+ Ser210Asp G1n185Ser + Asn198Asp + G1n200Asp + Ser210G1u Gly 95Ser + Gly 98G1n + Asn198Asp + Leu211Asp Val 93His + Ala 96His + G1n200G1u + A1a209Asp Leu 94Cys + I1e159Leu + G1n200Asp + A1a209G1u Gln 57Asn + Gly 6lAsp + Thr 69Asp + Leu 94Va1 A1a156G1n + Asn198G1u + G1y205G1n + Asn212Asp Tyr102Asp + Ser104Asp + Phe183G1n + Va1197His Ala 96Ser + Asn153Ser + G1y155Asp + Ser182Asp Ala 96Asn + Asn153G1u + G1n185G1u + Thr214G1y Asp 58G1u + Gly 6lGlu + Asn153Ser + Leu211Cys Pro129Ser + Asn198Asp + G1n200Asp + Leu211Pro G1y125Ser + Asn198Asp + G1n200G1u + Leu211A1a Asn198Asp + G1n200Asp + Thr207Pro + Leu211Pro Gly 98Pro + Asn198Asp + G1n200G1u + Tyr203Ser Asn198G1u + G1n200G1u + Tyr203Leu + Thr214Asn G1yl00Asp + Tyr102Asp + Asn153Ser + Tyr208Leu Thr 64G1n + Thr207Asn + Ser210G1u + Asn212G1u Pro127G1n + I1e159Cys + Ser210G1u + Asn212Asp Val 93A1a + Ser 99Asp + SerlOlGlu + I1e105Asn G1yl00Gln + G1y155G1n + Asn212Asp + Thr214Asp Gly 95Ser + Tyr102Leu + Pro129G1u + Ser160Asp Gly 59Ser + Pro129Asp + Ser160Asp + Leu211Pro Thr 69Pro + Ala 96Thr + Asn153G1u + Ser182Asp Asp 58G1u + Gly 6lGln + Gly 95Asp + A1a181Thr Gly 63Pro + Ser 99Asp + SerlOlGlu + Ser103G1u Asn153G1n + Va1197Asp + Leu211G1u + Thr219G1u G1y152Ser + A1a181Pro + Asn198Asp + G1y213Asp Ser159G1u + G1y155Pro + Ser160Asp + Asn198G1n G1y152Asn + Ser159G1u + Ser158G1u + Leu211Asn Asn 60G1u + Leu 94Asp + Tyr208A1a + G1y213Pro Thr 64Asp + G1n200Asp + Ser210Asp + G1y213Pro Ser103Asp + Ser130G1u + I1e159A1a + Thr202G1n Ser103G1u + Ser130G1u + Asn198G1n + Pro204Asn Thr207Pro + Ser210Asp + Leu211G1u + Thr214G1u Ser210G1u + Leu211Met + G1y213G1u + Thr214G1u G1y125Ser + G1n200Asp + Ser206G1u + Thr207G1u Ala 96Pro + G1y125Asp + Ser154Asp + A1a156Asp Ser 99Asp + Ser104Asp + I1e105Met + G1n200Ser Gly 59G1n + Ser128Asp + Ser154G1u + Ser160Asp Asp 58G1u + Gly 98Asp + Leu124Ser + Tyr161Met G1n185G1u + Asn198Ser + Leu211Cys + G1y213Asp Gly 6lGln + Gly 98Pro + Glnl'OOAsp+ Thr207G1u G1yl00Gln + G1n200G1u + Tyr203Pro + Thr207G1u SO Va1197Pro + G1n200Asp + Thr207Asp + A1a209G1n Asp 58G1u + Val 93G1y + Ser 97G1u + Tyr161I1e Asp 58G1u + Ser 97Asp + G1n200Ser + Tyr208Thr G1y152Asp + G1y157G1u + Va1199Ser + Thr207Ser Va1197GTn + Pro204Ser + Thr207Asp + Ser210Asp WO 95130011 . PCT/US95104760 ~ 1 G1y125Asn + Pro129Asn + Thr207G1u + Ser210G1u Ser101Asp + Ser128Asp + Prc>129G1u+ G1y152Ser Leu 94A1a + Asn198G1u + G1n200Asp + Thr214G1u Ala 96Ser + I1e105G1u + Ser126G1u + Ser160Asp Ser130Asp + Ser158G1u + Ser160Asp + Asn198G1n Leu124Asp + Phe183G1u + Tyr208Va1 + G1y213Asp Ser101Asp + Ser126G1u + A1a156G1y + Thr207Asn Asp 58G1u + Asn 60Asp + Glyl00Asp + A1a181G1n Asp 58G1u + G1y205Ser + Thr207Asp + Tyr208G1u Thr 64G1n + Leu 94G1u + Ser 97Asp + G1y125Asp Ser158G1u + I1e159Asp + Leu211Va1 + G1y213G1u Gln 57Asp + Thr 64Asp + Leu124Thr + G1n200Asp Gly 6lGlu + G1n200Ser + G1y205G1u + Ser210G1u Asp 58G1u + I1e105G1y + LeL.211Asp+ Asn212G1u Gln 57Asp + Asp 58G1u + G1r200G1u + Pro209Ser Gln 57Asp + Asp 58G1u + Ala 96His + Ser160G1u G1y157Asp + G1n200Ser + A1a209G1u + Ser210G1u Gly 59G1u + Asn 60Asp + Gly 95Asn + G1n185G1u Ser101Asp + Tyr102His + Va1197G1u + Asn198G1u G1y152Asp + Asn153G1u + Ser210Asp + Leu211Met Gly 59Asp + Tyr102I1e + Ser210Asp + Leu211Asp Ser101G1u + Tyr102Asp + G1n.200G1u+ Leu211Asn Gly 59Asn + Ser130G1u + G1y205Asp + Ser206Asp Ser128Asp + Tyr161G1y + Ser206Asp + Thr207G1u Asn 60G1n + Ser 97G1u + Ser182Asp + Phe183Asp Gly 98G1u + Ser 99Asp + Ser160G1u + Thr207G1n Gly 98Asp + Thr202G1n + Thr207G1u + Tyr208Asp Ser154G1u + Tyr161Leu + Ser210G1u + Asn212G1u Asn 60Asp + I1e105Ser + G1n200Asp + Ser210G1u A1a131Asp + G1n200G1u + Ser210Asp + Leu211Pro Gln 57Asp + G1n200G1u + Ser210Asp + G1y213Pro Pro129G1n + Ser160G1u + G1n200Asp + Ser210G1u Pro129Asp + Asn198G1n + G1n.200Asp+ Ser210G1u Gly 59G1u + Thr 64G1y + G1n200G1u + Ser210G1u Asn 60G1u + Ala 96G1u + Asn198G1n + Ser210G1u Asn 60Asp + Ala 96Asp + Ser130G1u + A1a156Pro Gln 57Ser + I1e159Ser + G1n200Asp + G1y213G1u G1y155Asp + G1n185G1u + ASn198G1u + Tyr203His Ser109G1u + Leu124Asp + G1n185Ser + Va1197G1y Gly 59G1n + Ser101G1u + Ser103Asp + Asn198Asp Asn 60G1n + Ser126Asp + Ser128Asp + Ser210G1u Ser126G1u + Ser128Asp + G1y155Asn + Asn212G1u Ser103Asp + Asn198G1u + Ser210G1u + Thr219G1n Ser160G1u + Asn198G1u + Ser210Asp + Leu211Ser Asp 58G1u + Asn198Asp + Thr207Pro + Ser210Asp Gly 98Asn + Ser103G1u + Asn198G1u + Ser210G1u Ser128Asp + G1y155Pro + Asn198Asp + Ser210Asp G1y152G1u + Asn198Asp + Thr207Asn + Ser210Asp Ser101Asp + Pro129G1y + Asn198G1u + Leu211G1u Asn198G1u + Tyr208Asp + G1y213Ser + Thr214Ser Ser 97G1u + G1y152G1u + Ser154Asp + G1n185Ser Thr 64Asp + Ser210Asp + Leu211Va1 + G1y213G1n Thr 64G1u + G1y205Ser + Ser210Asp + Leu211Asn Leu 94Thr + Ser126G1u + Pro204G1u + Thr207Asp G1yl00Gln +Tyr102Asp + Pro209Asp +Thr207Asp SerlOlGlu +Asn198G1u + G1n200G1u +Leu211A1a Thr 64Asp +Ser 99G1u + Pro209G1u +Thr207Asn G1y155Pro +Ser182Asp + G1n200Asp +Leu211G1u S G1yl00Asn +Ser160G1u + G1n200G1u +Leu211G1u Asp 58G1u +Val 93Asn + Ser206G1u +Leu211Met A1a131Pro +Asn153G1u + Ser182Asp +Asn198G1u Asp 58G1u +Ala 96G1u + Pro127Ser +G1y157G1u Leu 94Asp +Tyr102Leu + Thr207G1u +A1a209G1u Ala 96G1n +Ser158Asp + Ser160G1u +Va1197G1u G1yl00Asp +Ser126G1u + Ser154Asp +I1e159G1y Tyr102Asn +G1n200G1u + Thr207G1u +G1y213Asp Val 93G1u +Pro127Ser + Ser210Asp +Asn212Asp I1e105Asp +A1a131G1u + A1a156G1n +Thr202Pro Gln 57Ser +Ser126G1u + G1y152Asp +Ser210G1u Ser126G1u +G1y152G1u + Asn198G1u +Thr207Asn Leu 94G1u +Ser101Asp + Tyr102Leu +Thr207G1u Leu 94Asp +SerlOlAsp + Ser154Asp +G1y213Asn Asn 60Asp +Thr 64G1u + Ser103G1u +Thr214Asn Asp 58G1u +Va1197Asp + Ser210G1u +Leu211Pro Ser160Asp +Phe183Tyr + Va1197Asp +Ser210Asp Pro129G1u +Va1197G1u + Ser210G1u +Leu211His Ser126Asp +Ser130G1u + Ser182G1u +G1n200Ser Ser126Asp +Ser130G1u + Asn198Asp +Leu211His Thr 69Ser +Pro209Asp + Ser210Asp +Leu211I1e Ser 97Asp +G1yl00Glu + G1y152Pro +Asn198G1u Ser 97Asp +G1yl00Glu + G1n200G1u +Tyr208G1y Asp 58G1u +Gly 59Pro + Ser159Asp +G1y157Asp G1y155G1n +A1a181G1u + G1n185Asp +Ser210Asp G1y125G1u +Asn153Asp + G1n200Asp +Tyr208Pro Gln 57Ser +Ser101Asp + Ser154Asp +Ser160Asp Ser104Asp +Asn198G1u + A1a209Asp +Leu211Met Leu124I1e +G1y125Pro + Pro129G1u +Ser158G1u Ser154G1u +Ser158Asp + G1y205Pro +Thr207G1u G1y152G1u +Ser158Asp + G1n200Asp +Thr207Pro A1a131Asp +G1y152Asp + Ser158Asp +Va1197Thr Ser103G1u +G1n200G1u + Thr207Pro +Asn212Asp Ser130G1u +G1n200G1u + Thr202Ser +Asn212G1u Asn 60G1u +Leu 94G1u + I1e105Leu +Ser130G1u Ser 99G1u +Pro127Asp + Pro129Ser +Ser154G1u Asn153G1u +I1e159G1y + Leu211G1u +G1y213Asn A1a156Asp +Ser160G1u + G1y205G1n +Leu211Asp Ser109Asp +Ser130Asp + G1y155Pro +G1y157Asn Gly 98Asp +SerlOlAsp + Ser128Asp +A1a181G1n Asn 60Asp +Tyr102G1y + G1n200G1u +Thr207Asp Gly 59Pro +A1a131Asp + Ser154Asp +Thr219Asp Ser103G1u +Ser130G1u + Asn198G1u +Asn212G1n Asn 60Asp +Gly 98Asn + Ser103G1u +Ser130Asp Ser160Asp +Phe183Ser + Ser210G1u +Thr219Asp Asp 58G1u +Ser 99Asp + Va1197Pro +Pro204Asn Asp 58G1u +Gly 98G1u + G1y155G1n +Leu211Asp Thr 64G1u +Leu 99Asp + G1n185Ser +Ser210Asp Asn 60Asp +Ser 99G1u + A1a181G1u +Va1197Asn Ser128G1u +Ser206Asp + A1a209G1u +Asn212G1n "~- WO 95/30011 PCT/US95104760 G1yl00Asp + G1n185G1u + Leu211Ser + G1y213G1u Ser126G1u + Ser154G1u + Asn198G1n + G1y205G1u G1y125G1u + Va1197Thr + G1n200G1u + Thr207Asp Asn 60G1u + I1e105Pro + I1e159Va1 + Leu211G1u Pro129Asn + G1n200G1u + Pro204Asp + Thr207G1n Asn198Ser + G1n200Asp + Tyr203h1e + Pro204G1u Gly 98Ser + Ser103G1u + A1a131G1u + Ser210Asp Ser103G1u + A1a131Asp + Tyr161His + Ser210G1u Gly 6lGlu + G1y125Pro + Ser126G1u + Thr207G1u G1y152Asp + G1y157Asp + Pro209Asp + A1a209Thr Pro127Asp + Asn153G1u + Pro204Ser + Ser206Asp Gln 57Asp + G1n200Ser + Thr207G1u + Ser210G1u Ser154Asp + Thr207G1u + Ser210G1u + Thr214G1n Ser130G1u + Pro204Asn + Thr207G1u + Ser210Asp Asp 58G1u + Val 93A1a + Ser101G1u + Ser130Asp G1y125G1u + Ser130Asp + G1n200Asp + Leu211Thr Leu 94G1u + Ser182Asp + Thr207G1n + Asn212G:Lu Ser 97G1u + I1e159Asp + Phe183I1e + Thr214Asp Gly 95G1u + Ser103Asp + Pro129Asn + A1a131Pro Asn 60G1n + G1y 6lGlu + Ser130Asp + Ser210Asp SerlOlGlu + Ser126Asp + A1a209G1u + Asn212Ser Ser101G1u + Ser126Asp + Tyr161Leu + G1y205G1u Asn 60Ser + Gly 98Asp + Ser126G1u + Leu211Thr Val 93G1u + Ser 97G1u + Pro129Ser + G1n200G1u Thr 69Asn + G1n200Asn + Thr207Asp + Leu211Asp Gly 98G1u + Ser109Asp + Ser126Asp + Phe183G1y G1y125Asp + G1y157Asp + Thr207G1n + G1y213G1u Leu 94Asp + Ser128Asp + G1y152Asp + Leu211Met Ser158G1u + Ser182G1u + Thr207G1y + Tyr208Cys G1n200Asn + Pro204G1n + Ser210G1u + Thr214G1u Thr 64G1u + A1a131Asp + Ser210Asp + Leu211A1a Asp 58G1u + Gly 59Pro + G1yl00Pro + Thr207Asp Asn198G1n + G1n200G1u + Ser206G1u + Tyr208His Gly 95Pra + Ser126Asp + A1a131G1u + G1n200G1u Ser 97G1u + Ser101G1u + G1n200Asp + Pro204Asn Ser 97Asp + Ser101Asp + G1y125G1n + Ser210Asp A1a131G1y + Ser154Asp + Tyr161G1u + Ser210Asp Ser126G1u + Pro204Asp + Thr207G1y + Ser210Asp Gly 98G1u + Ser104Asp + Va1197G1u + Asn198Ser Ser104Asp + Ser130G1u + A1a156Thr + Thr207Asp Ser104Asp + Ser130Asp + A1a131Thr + Thr207Asp Pro127Ser + Ser158Asp + G1n200Ser + Thr219G1u Asn 60Asp + Tyr102Ser + Va1197Asn + Thr207Asp Ser101Asp + Pro127Ser + A1a131G1u + A1a156G1u Asp 58G1u + Ser 99Asp + G1n200G1u + Tyr208Ser Asp 58G1u + Ser 99Asp + Leu124His + Asn198G1u Gly 6lGlu + Thr 64Ser + Val 93Asp + Tyr208G1y Asn 60G1u + G1n185Ser + Asn198Ser + Ser206Asp Gly 6lPro + Ser158Asp + A1a181G1u + Leu211Asp Tyr161G1u + G1n200G1u + Tyr203His + Pro204G1u Gly 6lGlu + G1y157Asp + Leu211Met + Asn212Asp Gly 59Ser + Gly 98G1u + Tyr102G1u + Ser158Asp us Multi-loopQuintuple Mutation Variants Val 93G1n + Tyr102Cys + Ser154G1u + Asn198G1n + Thr207G1y Leu 94G1y + Leu124I1e + Asn198G1n + A1a209G1y + Ser210G1u Leu124Asp + A1a156His + G1n200Ser + Thr207Pro + Leu211Thr Pro129Asn + G1y155Asp + G1y157Asn + Thr207Ser + Asn212G1n Gly 6lSer + Gly 98G1u + Pro127Asn + G1n200Ser + Thr207Pro A1a156G1n + G1n185Ser + G1n200Asn + Ser210Asp + Leu211His Gly 63Asn + Tyr102Pro + A1a156G1n + Ser182G1u + Va1197Asn Ala 96Pro + I1e105Pro + Thr207Ser + Leu211I1e + Thr214Pro Val 93Asn + Va1197G1y + Asn198G1n + Ser210Asp + Leu211Ser Val 93Pro + Leu 94I1e + I1e159Asn + G1n200Ser + Thr207Ser Gly 6lPro + Gly 95Pro + Ser154G1u + A1a181His + G1y213Pro G1y157Ser + Ser182G1u + Pro195G1n + Thr207Pro + Leu211Ser Ser 97G1u + Tyr102Met + Asn198Ser + A1a209G1n + Leu211A1a Leu124His + A1a131His + G1y157Pro + Asn198Asp + Thr202Ser Gly 95Asp + A1a181Pro + G1n200Asn + Leu211A1a + G1y213G1n Leu 94I1e + Gly 95Asn + Ser158G1u + Leu211Met + G1y213Ser Gly 98Asn + G1y125Pro + Pro127G1y + Tyr161I1e + Tyr208Met Ala 96Ser + Ser154G1u + Tyr161G1y + G1y196G1n + Va1197G1n Leu 945er + Gly 95Asn + Pro127Asn + Va1197Ser + Thr214Asn Asp 58G1u + Val 93G1n + Leu 99Met + G1n200Asn + Thr207G1n Gly 95Asp + G1y155Ser + Va1197Thr + Thr207G1y + Leu211Pro Gly 98Ser + A1a131G1u + Va1197G1n + G1n200Ser + Pro204G1y Gln 57Asn + Val 93Thr + Tyr102Ser + Leu124Pro + Ser126Asp Ser101Asp + Tyr102Cys + Leu124Va1 + Phe183G1y + Va1199Asn Asp 58G1u + Thr 69G1n + I1e105Pro + G1y125G1n + Asn198Ser G1y155G1u + Tyr161Va1 + Pro209G1y + Thr207Pro + Leu211I1e G1yl00Gln + Ser182Asp + Phe183Asn + Asn198Ser + Thr207G1n Gly 98G1u + Pro129G1y + G1y155Pro + Thr207G1y + Thr214G1y Leu 94Met + G1yl00Ser + G1y196G1n + G1n200Asn + Asn212Asp Ser104G1u + G1y152G1n + Asn153G1n + A1a194G1n + Asn212Ser Pro129G1n + Va1197Thr + Tyr208I1e + Ser210G1u + Thr214Ser Val 93G1y + Ala 96Asn + Pro129G1n + Ser160Asp + Pro204G1y Pro127G1n + A1a131Asn + G1y196Ser + Va1197Thr + Ser206G1u Pro127Ser + Ser128Asp + A1a156His + A1a199G1n + Leu211A1a Gly 95G1n + Pro127G1y + Va1197G1y + G1y205Asn + Ser210G1u A1a131His + Pro204G1y + Thr207Asn + Leu211Thr + G1y213Asn Leu 94G1y + Tyr102G1n + Phe183Va1 + Thr207Ser + Leu211Asp Gly 6lAsn + I1e105Met + G1y125Ser + Va1193G1y + Asn198G1u Gly 59Pro + Ala 96G1y + SerlOlAsp + Tyr208His + Leu211His Tyr102Va1 + G1n200Asp + Pro204Ser + Leu211I1e + Thr214Pro Val 93Thr + Ser104G1u + Pro195Ser + Va1199Pro + Leu211Va1 I1e159G1n + Tyr161A1a + Phe183Leu + Ser210G1u + Thr214G1y Gly 59Asn + Pro127Ser + I1e159A1a + G1n200Asn + Leu211Met Gln 57G1u + Val 93His + G1y125G1n + A1a131Thr + A1a156Asn Ser126Asp + G1y157Ser + Asn198G1n + G1n200Ser + Thr207Ser G1y125Asn + Pro129Ser + Phe183Asp + Thr207G1y + Leu211G1y Asn 60Asp + Leu 99Met + Tyr102Pro + G1y152Asn + Thr207G1n I1e105Leu + G1y152Pro + Ser158G1u + Asn198Ser + Thr214G1y Ala 96Asn + A1a131Thr + G1y157Asn + Thr207G1y + Ser210G1u Val 93G1n + Leu124Met + A1a181G1n + Asn198G1u + G1n200Ser Pro127Ser + I1e159Thr + Pro195G1y + Asn198Ser + G1n200G1u ~''' WO 95130011 ~~'~'' PCTIUS95104760 Ala 96Asn + Va1197His + Asn198Ser +G1n200Asn + Asn212Asp Asp 58G1u + Leu 99A1a + Leu124Pro +A1a156G1y + Leu211G1n I1e105Thr + G1y157Pro + Phe183Pro +Va1197Asp + Tyr208Va1 Gly 95G1u + G1yl00Gln + Tyr102G1y +Tyr161His + T'yr208Cys Thr 64G1y + Val 93Asp + Gly 95Pro +G1yl00Pro + Asn198G1n Gln 57Asn + Asn 60G1n + SerlOlGlu +G1y155Ser + Pro204Ser G1y125Ser + G1n185Asn + Asn198G1n +Ser206G1u + A1a209His Gly 98Ser + Pro129Asn + Va1197G1u +G1n200Asn + Tyr208Asn Gly 98Asp + Asn153G1n + G1n200Asn +Pro204Ser + Leu211Ser Ala 96Thr + A1a181Thr + Va1197Asn +G1n200Asn + Thr207Pro Gly 95G1n + Pro129Asn + Tyr161A1a +G1n185Asp + Thr207Asn Gly 95Asn + G1y152G1n + Ser160G1u +A1a194Asn + Tyr208His Gln 57Asp + Phe183Leu + Thr207Ser +A1a209G1n + Leu211Met I1e105His + G1y125Asp + G1y152Asn +Pro204Asn + Tyr208Leu Gln 57Asn + Leu 94G1u + Ala 96G1n +Pro127G1y + Leu211Pro Asn153Ser + Tyr161Met + A1a181G1n +Asn198Ser + Ser210Asp Gly 95Asn + Gly 98G1u + Pro129Asr.+A1a209Pro + Thr219Pro Gly 6lAsn + Asn198Ser + A1a209G1t:+Leu211Ser + G1y213Pro Ser 99Asp + Pro127Asn + Pro195Asn +Tyr208His + Leu211Cys Asn 60G1n + Thr 64Asn + Glyl00Asn.+I1e105Leu + Pro127Asn Pro127G1u + A1a194G1n + G1n200Asn.+Thr207Ser + Leu211Cys Thr 64G1y + Leu 94G1y + Va1197Asn.+G1n200Ser + A1a209Pro Gly 59G1n + Leu 94Ser + Ala 96Ser +Leu211Thr + Thr214G1u Pro129G1y + A1a194Asn + G1n200Ser +Leu211G1n + Asn212Asp A1a131G1y + Asn153G1u + Thr207Asn +Leu211Thr + G1y213G1n Gly 59Pro + Thr 64Asn + Asn198G1n +A1a209G1u + Ser210Asp G1y155Asn + Ser160Asp + Tyr161G1u +Asn198Ser + Leu211Ser Ser126G1u + Pro127G1u + G1n200Ser +Thr207Ser + Leu211Pro A1a131G1y + A1a209Thr + Ser210Asp +Leu211G1u + Thr214G1y Gly 6lAsn + I1e159Ser + Ser206G1u +Thr207Asp + Leu211A1a Gly 98G1u + Ser 99G1u + Tyr161Leu +Phe183G1n + Asn198G1n Gln 57Ser + Val 93Asn + G1y155Asn +G1n200G1u + Ser210G1u Thr 64Asn + Gly 95G1u + Ala 96Asp +Gly 98Asp + G1n200Ser I1e105Ser + Ser210G1u + Leu211Asp +Asn212Asp + G1y213Ser Ala 96Asn + Glyl00Ser + I1e105Cys +Asn198Asp + Ser210G1u Ala 96G1n + Gly 98Asn + Asn198Asp +Pro209Asn + Ser210Asp Gly 98G1n + Asn198G1u + G1n200G1u +Tyr208Asn + Ser210Asp I1e105Pro + Leu129Asn + I1e159Ser +Asn198Asp + Leu211Asp Leu 94G1n + Tyr161Pro + Asn198G1u +Thr207G1y + Leu211G1u Tyr161G1y + Va1197A1a + Asn198G1n +Pro209G1u + Ser206Asp Leu124A1a + Asn198Asp + Tyr208G1y +Ser210G1u + Asn212Asp G1n185Asn + Asn198Asp + G1n200G1u +Thr207G1n + Leu211Asp Pro127G1y + Tyr161A1a + Pro209G1u +Thr207Asp + Thr214Ser Gln 57Asn + Leu124Ser + Va1197G1u +Asn198G1u + G:Ln200G1u Asp 58G1u + Ala 96Asp + Va1197A1a +G1y205Ser + Thr207G1y Ser158Asp + Ser160Asp + Pro195Ser +Thr207Ser + Leu211Met Asp 58G1u + Asn 60G1u + Thr 64G1u +G1y157Asn + Leu211A1a Leu124G1u + Ser126Asp + I1e159Met +Phe183Va1 + Va1193His Leu 94I1e + Pro127G1u + Pro129Asp +Ser130Asp + Tyr203His Leu 99Cys + Ser 99Asp + Ser101G1u +Asn198G1n + Asn212G1n Gly 6lAsn + Ser128G1u + Ser160G1u +A1a181Asn + Thr207G1n Tyr161Met + Va1197Asp + G1n200Asp +Ser210G1u + Asn212G1n Val 93A1a + G1y125Pro + Ser159G1u +G1y157Asp + Ser158Asp Leu 94G1u + SerlOlAsp + Phe183A1a +G1n200Asn + Leu211Met WO 95!30011 :. PCTlUS95104760 Leu 94Asp + Ser103G1u Ser104Asp + Asn198Ser + Tyr208G1y +

G1y125Asp + Asn153G1u + Ser160G1u + Tyr161Asn + Asn198Ser Leu 94Asn + Pro127Asn Ser154Asp + G1n185G1u + Thr214Asp +

Gly 6lGln + Thr 64Asn + Asn198G1u + Ser210Asp + G1y213Asp Leu124G1n + Asn198G1u + G1n200Asp + Thr207G1y + Tyr208G1u Leu 94G1u + Leu124Asp + Pro129Asn + A1a156Ser + Thr207G1y Gly 98Asn + A1a156Pro + Asn198G1u + G1n200Asn + G1y213Asp Gly 95G1u + Gly 98G1n + Ser 99G1u + Tyr102Cys + Ser104G1u G1y125Asn + G1n200Asp + Thr207G1u + Ser210Asp + Leu211His Gly 59Asp + Ser 97Asp + Ser 99Asp + A1a131Pro + Pro204G1y Gln 57Ser + Tyr102G1n + Asn198G1u + A1a209G1u + Leu211Ser Asn153Asp + I1e159Asp + Tyr161His + Phe183His + G1y213Pro G1yl00Ser + G1y155Asp + I1e159G1u + Va1197Met + Thr207Pro Pro127Ser + G1n200Asp + Thr207G1y + Ser210Asp + G1y213G1u Ala 96Thr + Ser126G1u + G1y152G1u + Ser158G1u + Thr207Asn Leu 94A1a + Gly 95Asp + Ser 97Asp + Ser101Asp + Thr202G1y G1y152Ser + G1y205Ser + Thr207G1u + Ser210G1u + Leu211Asp Tyr161Pro + Pro195Ser + Thr207Asp + Ser210G1u + Leu211Asp Asp 58G1u + Ser 97G1u + Ser 99Asp + G1n185Asn + G1y196Ser Thr 64G1y + SerlOlGlu + I1e105G1u + Ser126Asp + Tyr161Leu Thr 69G1y + Ser 99Asp + I1e105G1u + G1y205Pro + Leu211A1a Asn153G1u + A1a199Thr + Asn198G1u + Tyr208I1e + G1y213G1u G1y152G1n + G1n200Asn + Ser206G1u + A1a209Asp + Ser210G1u Ser103G1u + G1y125Pro + A1a131G1u + Tyr161Cys + Leu211Thr G1yl00Ser + G1n200Asn + Thr207G1u + Ser210Asp + Leu211G1n G1yl00Pro + Asn198Asp + G1n200G1u + G1y205G1n + Thr207G1u Ala 96Asp + Ser 99G1u + Ser109Asp + Va1197Met + G1y213Ser Gly 98Asp + Glyl00Pro + Ser101Asp + G1y125Ser + A1a156Ser Gly 98Asp + G1y125Asp + Ser160G1u + Tyr161I1e + Thr214G1y Gly 6lGlu + G1y155G1n + Asn198G1n + Ser206G1u + Tyr208His Asp 58G1u + Asn 60G1u + Leu129Asn + Ser206Asp + Thr207Asn Gly 6lAsp + Thr202Pro + Thr207G1n + Ser210G1u + Leu211Pro Gly 6lGlu + Tyr102His + Phe183His + G1n185Asn + Ser210Asp Ser 99Asp + Ser126Asp + Pro129Asp + G1y152Ser + Leu211His Leu 94I1e + G1y125G1n + Ser128Asp + Pro129G1u + Ser154Asp Asn198Asp + G1n200Ser + G1y205Pro + G1y213Ser + Thr214Asp Gly 6lAsn + Ser182Asp + Va1193G1n + Asn198Asp + Ser210G1u Gly 6lGlu + Leu 94Cys + G1n200Ser + G1y205G1u + Ser210G1u Asp 58G1u + Phe183Ser + Pro204G1u + Thr207Ser + Ser210G1u Leu 94Met + A1a156Asp + Tyr161Cys + Asn212G1u + Thr214G1u Asp 58G1u + Leu124Pro + I1e159Thr + Leu211Asp + Asn212G1u Gln 57G1u + Leu 94Thr + Va1197Ser + A1a209G1u + Ser210Asp Ser104G1u + Pro129Ser + A1a156G1y + A1a209G1u + Ser210G1u Asn153Asp + Ser159G1u + Asn198Asp + Thr207G1n + Leu211G1n Gly 59Asp + Asn 60G1u + SerlOlGlu + Tyr102His + Thr207Asn Gly 6lGln + Ser104Asp + I1e105Asp + Asn153Ser + Thr207Asp Ser 97G1u + Va1197G1n + Asn198G1i;+ Asn212Asp + G1y213Asp Tyr102Va1 + Ser154G1u + G1y205G1u + Ser206Asp + Leu211Cys Asn153G1u + Tyr161Thr + Pro195G1y + Ser206Asp + Thr207G1u Gly 59G1n + Thr 64Pro + G1y 98G1u + Ser130Asp + A1a131G1u G1y125Pro + G1y152Asn + Ser158G1u + I1e159G1u + Ser210G1u Ser 97Asp + Gly 98Asp + I1e105Va1 + Leu124Thr + G1n200Asp G1yl00Glu + SerlOlAsp + Ser160Asp + Va1197A1a + Leu211G1n Ser103Asp + + A1a181Ser + Asn198Asp + Tyr208I1e Ser104Asp WO 95130011 ~ PCT/US95104760 Ser 99G1u + Glyl00Glu + G1y125Asn + Ser182Asp + Thr207G1y Leu 94G1y + G1yl00Asp + A1a131G1y + G1n200G1u + Ser210Asp Ser 97G1u + A1a131G1n + G1n200Asp + Thr207G1y + Ser210G1u G1y125Asn + Ser130G1u + Phe183Asn + G1n200Asp + Ser210G1u Gly 95G1n + Tyr102Leu + Pro127G1u + G1n200Asp + Ser210Asp G1y125G1u + G1y155Asn + G1n200G1u + Ser210Asp + Thr219G1y Gln 57Ser + Asp 58G1u + G1n200Asp + Thr207G1y + Ser210G1u Pro127Asp + Tyr161Cys + G1n185Asn + G1n200Asp + Ser210Asp G1yl00Glu + G1n200Asp + Thr207Pro + Ser210Asp + Leu211A1a Gly 59Pro + Ser160Asp + Asn198G1n + G1n200Asp + Ser210G1u Thr 64G1u + I1e105Cys + Leu124Ser + Va1197G1u + Ser210Asp Gln 57G1u + Val 93G1u + Gly 95Ser + Ser103G1u + Asn198G1n Asn 60G1u + Ala 96Asp + Tyr102G1u + G1n200Ser + Thr207G1y Gln 57Ser + Asn 60Ser + Leu 94G1y + G1n200G1u + G1y213Asp Asn198Asp + Pro204G1n + Ser206G1u + Ser210G1u + Leu211Thr Val 93G1u + Ser126G1u + Ser160Asp + Va1197G1n + Leu211Ser Gly 98Asp + Ser125G1u + Pro129Asp + A1a181Ser + G1y213Ser Ala 96G1u + Gly 98G1u + Ser10.3Asp+ Prol?7Ser + I1e159Pro Gly 63G1n + Ser128G1u + G1y155Ser + Asn198G1u + Ser210G1u Gln 57Asp + Ala 96Thr + G1n185Asn + Asn198Asp + Ser210Asp Asn 60G1n + Gly 95Asp + Asn198G1u + G1n200Asn + Ser210Asp Ser104Asp + I1e105Met + Asn198Asp + Ser210Asp + Leu211Asn Tyr102G1u + Asn198Asp + G1n200Asn + Ser210Asp + Leu211Va1 Gln 57Asp + G1y125Ser + Asn198Asp + G1y205Asn + Ser210Asp Tyr102Leu + G1n185G1u + Asn198G1u + G1n200Ser + Ser210G1u Gly 59Asp + Tyr102I1e + Asn198G1u + Ser210Asp + Thr219Ser Val 93Thr + Leu124Va1 + I1e159G1u + Asn198Asp + Ser210G1u Ser101Asp + Pro129G1y + Asn198G1u + Thr207Ser + Leu211G1u G1y152G1n + G1y155G1n + Asn198Asp + G1y205Asp + Leu211Asp Gly 59G1n + Thr 64Ser + A1a156Asp + Ser158G1u + G1y205G1u Pro127G1u + Asn198Ser + Pro204G1u + Ser206Asp + Thr207Pro Asp 58G1u + I1e159G1y + Asn198Asp + Leu211I1e + Asn212G1u Ser130Asp + I1e159G1y + Asn198G1u + Leu211Met + Asn212G1u Asp 58G1u + Asn 60G1u + I1e159Met + Tyr161G1n + G1n200Asp Ser 97Asp + Ser103G1u + I1e105G1u + Va1197Cys + Leu211Va1 Ser103G1u + I1e105G1u + Asn153Ser + G1n200Ser + Ser210Asp Ala 96Pro + G1y152Asp + Ser160Asp + Thr207G1u + Leu211G1n Gly 95Asp + Ser 99G1u + I1e105G1y + G1y157Pro + Leu211G1u Ser 97Asp + Ser 99G1u + Va1197G1u + Asn198Ser + Thr219G1y Gln 57Asn + Ser 97G1u + Ser 99G1u + Asn153G1u + Va1193His Thr 64G1u + A1a131Asn + Tyr208Leu + Ser210Asp + G1y213Ser Gln 57G1u + A1a156Pro + Ser158Asp + G1n185G1u + Thr207G1n Ser 97Asp + Pro129Asn + Ser158G1u + G1n185G1u + Leu211Ser Ala 96Thr + A1a156His + Ser158G1u + G1n185Asp + Leu211Asp Leu 94A1a + SerlOlAsp + Ser126G1u + A1a131G1n + Ser154Asp G1y157G1u + I1e159Leu + G1n200Asn + Pro204Asp + Thr207G1u Leu 94Thr + Ser126G1u + Pro204G1u + Thr207Asp + G1y213Asn G1yl00Gln + Ser154G1u + Asn198G1u + G1n200Asp + Leu211Ser Gln 57G1u + Asn198G1u + G1n200G1u + Tyr203Va1 + G1y213Pro I1e105Met + Ser158G1u + I1e159Ser + Asn198G1u + G1n200Asp Ser109Asp + A1a181G1n + Asn198G1u + G1n200Asp + Thr207G1y G1yl00Asn + G1y152Pro + Ser182Asp + G1n200Asp + G1y213Asp Tyr102Met + Ser128G1u + Ser130Asp + Asn198Asp + Leu211Met Ser128G1u + Ser130G1u + A1a131Thr + Asn198G1u + Asn212G1n l22 Val 93Thr + Leu129G1u + I1e159Asn + G1n200G1u + Leu211Asp G1y157Ser + Ser182Asp + G1n200G1u + Thr207Pro + Leu211G1u Ser130G1u + G1n200Asp + G1y205G1n + Thr207G1n + Leu211G1u Gln 57Asn + Gly 59Asn + Asn 60G1u + Gly 98G1u + Ser130Asp Gly 6lAsp + Tyr102Pro + Ser206G1u + Thr207Asn + Ser210G1u Thr 64Asn + Ser 97Asp + Pro129Asn + Va1197Asp + Leu211G1u Ser 99G1u + A1a181Thr + Va1197G1u + A1a209Asn + Leu211Asp Gly 59Asp + Val 93Ser + Asn153Ser + Va1197G1u + Leu211Asp Ser101G1u + Asn153Ser + Tyr208Met + Leu211G1u + G1y213Asp G1y157Asp + G1n185Asn + Thr207G1u + A1a209Asp + Asn212G1n G1yl00Gln + G1y125Ser + Phe183Asp + Ser210Asp + Thr214G1u Asn198G1n + Ser206G1u + Thr207Pro + Ser210Asp + Asn212Asp A1a181Thr + G1n200G1u + Thr207Asp + Leu211A1a + G1y213G1u Asn 60Asp + Gly 98Pro + Ser126G1u + Pro129G1u + G1n200Ser Asp 58G1u + I1e159Pro + Ser210G1u + Asn212Asp + Thr214G1n Asp 58G1u + G1y125Ser + A1a131Ser + Ser210Asp + Asn212G1u Gly 59Ser + SerlOlGlu + A1a131His + Ser210G1u + Asn212G1u Asn 60G1n + Val 93A1a + Ser103G1u + Ser210G1u + Asn212G1u Ser158G1u + Asn198Ser + Tyr208G1u + Ser210Asp + Leu211Ser 2o Ala 96Ser + Ser160Asp + Asn198G1u + Asn212Ser + Thr214G1u Leu 94Asn + Gly 98Asp + Glyl00Glu + Pro127G1u + Va1197Met Asn 60Ser + Gly 98Asp + Glyl00Asp + Tyr102Asn + G1n200G1u Leu 99Ser + Ala 96Thr + I1e105Asp + Tyr161G1u + Leu211I1e Gly 95Ser + Ser 99Asp + SerlOlAsp + Leu211Met + G1y213G1u Gly 6lGlu + G1y125Ser + Ser128G1u + Ser160Asp + A1a181Pro Ser182G1u + Va1193Met + Asn198G1u + G1y205G1n + A1a209Asp Leu 94G1u + Glyl00Asn + Ser104Asp + I1e105Va1 + Ser206G1u Thr 64Asp + Ser126Asp + G1y152Asp + Pro204G1y + Leu211Asn Gly 59Asp + Val 93G1u + Leu 94Pro + I1e105Met + Leu211Ser Pro129G1u + Ser160Asp + G1n200Ser + G1y205G1n + Ser210G1u I1e159Va1 + Ser160G1u + Phe183G1u + Pro204G1n + A1a209Asn Leu 94Asp + Va1197Asp + Thr207G1y + Ser210Asp + Leu211Met Leu129G1u + Phe183Pro + Va1197G1u + Tyr208Leu + Ser210G1u Leu 94Met + Leu129Asp + Asn153Asp + Tyr161A1a + Leu211G1u Gly 98Asp + G1y152G1u + A1a156Asp + Tyr208Met + Asn212Ser Val 93Asp + Gly 98G1n + Ser126Asp + Ser130G1u + A1a209Pro Asp 58G1u + Tyr102A1a + Ser126Asp + Ser130G1u + I1e159Asn Ser126Asp + Ser130G1u + Va1197G1y + Leu211G1u + Asn212Ser Ser104G1u + I1e105Pro + Asn198Asp + Leu211His + G1y213Asp Ser 97Asp + G1y100G1u + G1y152Pro + Tyr161G1u + A1a181G1n Ser128G1u + A1a131G1u + Ser154Asp + Asn198G1n + A1a209His Ser 99Asp + Pro129Ser + Ser154G1u + Ser160Asp + Pro204G1n Asp 58G1u + I1e105Cys + Leu129Ser + Ser154G1u + Ser158G1u A1a131Asp + Va1197G1u + G1n200G1u + Thr207Pro + Thr214G1y Ser 99Asp + Va1197Asp + Asn198G1n + G1n200G1u + Thr207G1y Ser101G1u + Va1197Asp + G1n200G1u + Pro204G1y + Thr207Ser A1a131Asp + Va1197Asp + Asn198G1n + G1n200G1u + A1a209Asn Gly 95Pro + Gly 98Asp + Leu129G1u T Thr207Asn + Asn212G1u Gln 57G1u + Asn 60G1u + Val 93Thr + Gly 95Asn + Ser182Asp Asn 60Asp + I1e105G1n + A1a131Thr + Thr207Ser + A1a209G1u Ser103G1u + G1n200G1u + Thr207Pro + A1a209G1n + Asn212Asp Gly 6lPro + Leu 94G1u + G1n200Asp + Leu211Asn + Asn212G1u Gly 59Pro + + G1n200Asp + Leu211Va1 + Asn212Asp Val 93G1u Ser 97G1u + + A1a181Pro + G1n200G1u + Asn212G1u G1y157Ser "'"' WO 95/30011 PCT/US95104760 t23 Val 93Thr +Gly 98G1n + Tyr102G1u + A1a131G1u + Ser210Asp Gly 6lGlu +Ser104Asp + Phe183A1a + Thr207G1y + A1a209G1u A1a156Asp +Ser160Asp + A1a181G1y + Asn198Ser + G1n200Asp Gln 57G1u +Gly 6lAsp + Asn153G1n + Asn198Asp + G1y205Ser G1yl00Glu +I1e105Ser + Leu129G1u + Va1197Met + Ser210Asp Tyr102G1u +Ser126G1u + Va1193Pro + Pro204Asn + Ser210Asp Gly 59Pro +A1a131Asp + Asn153Ser + Ser154Asp + Thr214Asp Ser103G1u +Ser130G1u + G1n200Ser + G1y205G1u + Thr214G1y Asp 58G1u +A1a156Pro + G1y157Asp + Ser160Asp + G1n200Ser Thr 64G1u +Leu 94Asp + Ala 96G1n + G1n185Ser + Ser210Asp Ser130Asp +G1y157G1u + Phe183G1u + Asn198G1n + Leu211Met Asp 58G1u +Pra129G1u + A1a156His + Pro204Ser + G:Ly205G1u Ser126G1u +I1e159Leu + A1a209Asp + Leu211A1a + Asn212G1u Gly 59Asp +Ser126Asp + Ser154G1u + Phe183Leu + Leu211Va1 G1y155Asn +G1y157Asp + G1n200G1u + Thr207G1u + Leu211Asn Gly 59G1u +Gly 95Pro + A1a181His + G1n200G1u + Thr207Asp Gln 57G1u +Pro127G1y + G1y152Ser + G1n20UAsp + Thr207Asp Ala 96G1u +Asn198Ser + G1n200G1u + Thr207G1u + Thr214Ser Thr 64Asp +Leu129Ser + Pro129Asp + Tyr161Asn + Thr207Asp Ser 97Asp +Tyr161G1y + Ser210G1u + Leu211Pro + G1y213Asp Pro127Ser +Pro129Asn + G1y157Asp + Ser210Asp + G1y213G1u Tyr102Cys +Ser130Asp + Pro209G1n + Ser210G1u + G1y213Asp Ser 99G1u +A1a156Ser + A1a209Thr + Ser210Asp + G1y213Asp G1y196Pro +G1n200Asp + Pro204Asp + Thr207G1n + Leu211Asn Asp 58G1u +Ser 97G1u + Ser104Asp + Pro129G1y + Tyr161Thr Asp 58G1u +Leu 94Ser + Ser 97Asp + Gly 98Pro + G1n200Asp Gly 6lGlu +SerlOlAsp + Thr207Asp + Leu211I1e + Thr214Ser Gly 6lAsp +SerlOlGlu + Tyr102Va1 + Thr207G1u + Thr219Ser Gly 6lAsp +Asn153G1n + I1e159G1y + Asn198Asp + Thr207G1u Gly 6lGlu +I1e159Ser + Va1197Met + Asn198G1u + Thr207Asp Thr 69G1n +G1yl00Glu + G1n200Asn + Thr207G1u + Ser210Asp Ser126G1u +G1y155Pro + Tyr161Thr + Thr207G1u + Ser210Asp Tyr102G1u +G1y125G1n + A1a181Pro + Thr207Asp + Ser210G1u G1yl00Glu +G1y205Ser + Thr207G1u + Ser210G1u + Asn212G1n Gly 59Ser +Gly 95G1u + Thr207G1u + Ser210Asp + Leu211Va1 Tyr161Asp +Asn198Ser + Thr207G1u + Ser210G1u + G1y213Pro Pro129G1n +Ser130G1u + Asn198G1n + Thr207Asp + Ser210G1u Gln 57Asp +Phe183G1y + Pro204G1n + Thr207G1u + Ser210Asp Asn 60G1n +G1y157G1u + Pro204G1y + Thr207Asp + Ser210Asp Pro129Asp +G1y152Pro + Thr207Asp + Ser210G1u + Leu211Asn Gly 6lGlu +Ser 99Asp + G1y155Asn + Pro195G1n + G1n200Asn Gly 98G1u +Ser101G1u + A1a181G1y + G1n185G1u + Thr207Pro Gly 59Pro +Thr 64Asp + Pro129G1n + A1a181G1u + Asn198Asp Gly 98Asp +Tyr161Met + A1a181G1u + Asn198Asp + Leu211Va1 I1e159G1u +Va1197Thr + Tyr208G1n + A1a209G1u + Thr214Asp Ser126G1u +G1y157G1u + Ser182G1u + Asn198G1n + Leu211Va1 Gly 6lAsp +Ser128G1u + Ser206Asp + Tyr208G1y + Leu211Met Gly 98Pro +Ser130G1u + Ser160G1u + Thr207G1y + Ser210Asp Gln 57Asp +G1y125G1n + Ser130Asp + Asn153Ser + Ser160Asp Tyr161Met +G1n200G1u + Ser206G1u + A1a209His + G1y213Asp Gly 6lAsp +A1a156G1u + G1y2U5Asn + Thr207G1n + Ser210G1u I1e105G1u +Ser130Asp + G1n200Asn + G1y205G1u + Thr207Asn Gln 57Ser +Asn 60Asp + G1n200Asp + G1y205G1u + Thr207Ser Ser101G1u +Ser126G1u + Va1197Asp + Thr207G1y + Leu211Cys s SerlOlAsp +Ser126Asp + G1y152Ser Asn198G1u + G1n200Asn Gln 57Asp +G1y125Asp + G1y205Asp + Thr207Ser + Tyr208Thr Leu 99Met +Ser128Asp + Ser158G1u + Asn198G1n + Ser210Asp Gly 59Asp +Gly 95Pro + A1a199G1n + Asn198Asp + Thr214Asp Gln 57G1u +Gly 6lAsn + Gly 98Asp + G1yl00Pro + Va1193Cys Thr 64G1n +Val 93G1u + Ser 97G1u + Pro129Ser + G1n200G1u Gln 57G1u +Thr 59Asn + Val 93Asp + G1n200G1u + Thr207Asn G1yl00Asp +G1y125Ser + Ser126G1u + G1y196G1n + Va1197G1u Val 93Met +Glyl00Asp + Ser126G1u + Tyr161A1a + Asn198Asp G1yl00Glu +Ser126Asp + A1a181His + Va1197Cys + G1n200G1u Asn 60G1n +Ser 99Asp + Ser103G1u + Ser210Asp + Leu211A1a Gln 57Ser +Ser 99G1u + Ser103G1u + G1n200Asp + Thr207Asn Gly 63Ser +Ala 96Ser + Ser210G1u + Leu211Pro + Thr214Asp Pro129G1y +Va1197G1n + Ser210G1u + Leu211His + Thr214Asp Pro127Asp +G1y155G1u + Tyr161Ser + Thr207Ser + Ser210G1u Asp 58G1u +Val 93His + G1y155Pro + G1n200Asn + Thr207G1u Thr 69Asn +Asn198Ser + G1n200G1u + Ser206G1u + Thr214G1n Gly 59Pro +Gly 6lGlu + Ser154Asp + G1n200Asp + Thr214Asn Ser126Asp +Pro129Ser + A1a156Asp + Ser210Asp + Leu211A1a Ser103G1u +I1e159Leu + Pro204G1u + G1y205Asn + Ser210Asp A1a131Thr +Asn153G1u + Tyr203Asn + Pro209G1u + Ser210G1u G1y157Asn +Ser160G1u + Pro204G1u + G1y205Ser + Ser210G1u Gly 95G1n +Gly 98Pro + G1y152G1u + G1y157Pro + Va1197Asp Gln 57G1u +Leu 94Pro + G1y125Ser + Asn153G1u + Leu211G1u Gly 59Ser +Ser109Asp + Ser130G1u + Ser210G1u + Leu211Ser Ser104G1u +Ser130Asp + G1y155Asn + Leu211G1y + G1y213Asp Ser104G1u +Ser130G1u + Asn198Asp + G1n200Asn + Thr207G1n Ser158G1u +Thr202Ser + Thr207Pro + Leu211Thr + Thr214Asp Gly 59G1u +Leu 94Va1 + SerlOlAsp + A1a131G1u + Tyr161Met Asp 58G1u +Ser 99G1u + Tyr102Asn + A1a209Asn + Ser210G1u Ser101Asp +Phe183Ser + G1n200Asp + Pro204G1u + Thr214G1n Gly 6lAsp +Gly 98Asn + Ser 99G1u + I1e105Ser + Va1197G1u Asn 60Ser +G1y125Pro + A1a156Asp + Tyr161Asp + Ser210Asp Gly 59Pro +Ser128G1u + Ser159Asp + Thr207G1u + Leu211I1e Ser103Asp +A1a156G1y + Ser182G1u + Asn198G1u + Tyr203His Val 93A1a +G1y125Pro + Ser160G1u + Thr207Asp + Leu211G1u Ser 97Asp +G1y152Pro + Thr207G1u + Leu211G1u + G1y213G1n Gln 57G1u +Ser103Asp + Pro129G1u + A1a181G1y + Thr207Pro Ser158Asp +Tyr161Pro + Ser182Asp + Thr207G1n + Ser210Asp Multi-loop Sextuple Mutation Variants Tyr102Cys + I1e105Va1 + Leu124I1e + Ser154G1u + Asn198G1n + Thr207G1y Ser160G1u + Tyr203Cys + Thr207Pro + Leu211Met + Asn212G1n + G1y213G1n Val 93Ser + Ser103G1u + G1y125Ser + G1n185Asn + A1a194G1n + Asn198G1n G1yl00Ser + I1e105Met + G1y157Pro + G1n200Asn + Thr207Asn + Leu211Va1 Thr 64G1u + Leu 94Asn + Va1197Cys + Thr202G1n + Thr207G1n + Leu211Va1 "~" WO 95/30011 PCT/US95/04760 t25 G1y152Pro + G1y157G1n + A1a181Asp + Asn198G1n + G1n200Asn + Thr207Asn Gly 95G1n + Ala 96Asn + Leu124Thr + Asn153Ser + Ser154G1u + Va1197His Val 93Met + Pro127Ser + G1y155Pro + Ser182Asp + G1n200Ser + G1y213Ser Gly 6lPro + Gly 95Pro + Ser154G1u + A1a181His + Leu211Asn + G1y213Pro Tyr161Thr + G1n200Asn + Thr207G1y + Tyr208Leu +
Pro195Asn + Leu211Pro Leu 94Pro + Ala 96Ser + Pro127Asp + I1e159A1a + Thr207Pro + G1y213G1n G1yl00Asn + G1y125G1n + Pro129G1y + G1n185G1u +
I1e105G1n + Thr207G1n Gly 98G1u + Pro129G1y + G1y155Pro + Thr207G1y + A1a209His + Thr214G1y I1e105G1n + Phe183His + A1a194G1n + G1y205Ser +
G1y152Asp + Asn212G1n A1a194G1n + G1n200Asn + Ser2C>6Asp+ Leu211His +
Asn198Ser + Thr219G1n Ser 99G1u + Pro129Asn + A1a131Pro + G1n185Asn + Thr207Pro + Leu211Va1 Leu 94Va1 + Gly 98G1n + Leu129G1y + Va1197G1u + G1n200Asn + Thr207G1y Gln 57Ser + Gly 98Asn + Tyr102Asp + Tyr161Asn + G1n200Ser + Thr207G1n Asn 60G1n + Gly 6lSer + Tyr102Cys + G1y152Asp + Va1199Ser + Pro204Asn Tyr1 02Thr + G1y155G1u + A1a156Asn + Tyr161Pro + G1n200Asn + Leu211Thr Tyr1 02G1u + G1y157Pro + Phe183His + Asn198G1n + Thr207Pro + Leu211Cys Leu1 24Cys + Pro127Asn + Tyr208Met + Ser210Asp + Leu211His + G1y213G1n Gly 98Ser + Leu124A1a + Ser126Asp -+A1a181Asn + A1a209G1y + G1y213G1n Ala 96His + Tyr102Ser + I1e105Ser -+G1y157Ser + I1e159G1n + Leu211Asp Thr 64Pro + Ala 96Pro + Ser 97Asp ~+Pro127Ser + G1y205Asn + Leu211Ser Val 93G1y + Gly 98Ser + Tyr102Asn + Va1197Met + Thr207G1n + Ser210Asp Asn 60G1n + Ala 96G1n + Asn153G1u -~Thr202G1n + G1y205Asn + Leu211Met Gly 59Asn + Leu 94Va1 + Pro127Ser -~I1e159A1a + G1n200Asn + Leu211Met I1e1 05G1n + G1y157G1n + Va1193Asn ,-Va1197Asp + Thr207Pro + Tyr208Cys Asn 60G1n + Gly 63G1n + I1e105G1u -~Asn198G1n + Leu211I1e + Thr214G1y Gly 6lGln + G1y125G1u + Tyr161G1y ~-G1y205G1n + Thr207Ser + A1a209G1n Gly 98Ser + A1a156G1n + Tyr161A1a + Ser182G1u + G1n200Ser + Thr207G1n ~~~9~2?

Gly + Leu124G1y + A1a131Thr +Va1197G1u 6lPro +
Val 93A1a + Asn198G1n Val 93Asp + Ala 96Asn+ Va1197His + Asn198Ser +Va1199Ser + Thr207G1n Tyr102I1e + A1a181Asn + G1n200Ser +Pro209G1n +
I1e159Asp + Leu211His G1yl00Pro + G1n200Ser + Thr207Pro +A1a209G1u +
Pro129Ser + Ser210G1u Va11 97Pro + Asn198G1n+ G1n200G1u + Pro204G1y +Thr207G1n l0+ Ser210Asp Ala 96G1n + Leu129Met+ I1e159Ser + G1n200G1u +Pro204G1y + Ser210Asp Asn 60G1n + I1e159Thr+ Asnl98ser + G1n200G1u +Ser210G1u + Leu211Asp 15Gly 6lSer + Va1197G1n+ Asn198G1u + G1n200Ser +Ser210Asp + Leu211His G1yl 00Asn + A1a181Pro+ Asn198Asp + Thr207Ser +Ser210G1u + G1y213G1n Gly 98Asn + Pro129Ser+ Asn198Asp + G1n200Asp +Ser210G1u 20+ Leu211Pro Gly 63Ser + Leu 99G1n+ I1e159A1a + G1y205G1u +Thr207G1u + Thr214Asn Asp 58G1u + Gly 6lGlu+ Gly 98Asn + Asn153Ser +Tyr161Asn + Leu211Cys 25Leu1 24Ser + I1e159Thr+ Asn198G1u + G1n200G1u +Pro204G1y + Thr207G1n Gly 6lGln + Gly 95Asn+ A1a199G1y + Asn198Asp +G1n200Asp + Leu211Va1 Gln 57Ser + Tyr102G1n+ Asn198G1u + A1a209G1u +Ser210G1u 30+ Leu211Ser Gly 59G1n + G1y125Pro+ Ser159G1u + G1y155Asp +Tyr161A1a + Ser182Asp Gln 57G1u + Gly 59Asp+ Thr 64G1n + Tyr208Pro +A1a209G1n + Leu211Asn 35Asn 60Asp + Gly 61G1n+ Gly 95G1u + Gly 98G1u +Asn153Ser + I1e159Leu I1e105Thr + Ser158Asp + Ser160Asp +Thr207G1y +
G1y152Ser + Leu211A1a Gly 6lGln + Thr 64Asn+ Asn198G1u + Ser210Asp +Leu211Asp 40+ G1y213Asp Leu 94Pro + Ala 96G1y+ Thr207Ser + Ser210Asp +Leu211Cys + Asn212G1u Gly 98G1u + Glyl00Asp+ A1a181Pro + Tyr203Va1 +Pro204Asn + Thr207Ser 45Asp 58G1u + G1y157Ser+ G1n185Asn + Va1197G1n +Pro204G1u + Asn212G1n Gly 6lAsn + Leu124Thr+ Ser128G1u + Ser160G1u +A1a181Asn + Thr207G1n I1e105Leu + G1y125Asp + G1y152G1u +Asn153G1u +
Leu129Ser 50+ G1y213G1u Leu129G1n + G1n200Asp + Thr207G1y +Tyr208G1u +
Asn198G1u + Leu211Asp Leu 94I1e + G1y152Ser+ G1y155G1u + Ser158Asp +A1a181G1y + Va1197His w° WO 95130011 PCTIUS95104760 Asp 58G1u + Tyr161G1y + Va1197A1a + Asn198G1n + Pro204G1u + Ser206Asp Gln 57Ser + I1e159Ser + Asn198Asp + G1n200Asn + Tyr208Asn + G1y213G1u G1y152Ser + Asn198G1u + Va1199Ser + Leu211Cys +
Phe183A1a + G1y213G1u Leu 94I1e + Ser154Asp + I1e159G1u + Asn198Ser + G1n200Asn + Leu211Met Ala 96Pro + Ser159Asp + A1a156His + G1y157Asn + Ser158G1u l0 + Pro204Ser Gln 57Asp + Gly 59Pro + Asn 60Asp + Gly 95Pro + Thr207Ser + A1a209G1n Thr 64Ser + G1y152G1n + A1a181His + G1n200G1u + Leu211His + Asn212G1u Gln 57Asn + Gly 98G1n + G1n200Asp + Leu211Cys + Asn212Asp + G1y213Ser Ala 96Asn + G1yl00Asp + Leu129Asp + Va1197G1y + Thr207Pro + G1y213Ser Ala 96Ser + Ser154Asp + Phe183G1n + Thr207Ser + Leu211Thr + G1y213Asp Gly 95Asp + Gly 98Pro + Ser 99Asp + G1y100Ser + Tyr102G1u + Ser103G1u Gln 57Asp + Asp 58G1u + Thr 69Asp + Ser 97G1u + G1n200Ser + Tyr208Thr G1y1 25Asp + Ser130Asp + Asn153G1u + A1a156Thr + Ser160G1u + Asn198Ser Gly 59Asn + Leu1.29Thr+ I1e159Met + A1a209Asp + Leu211Cys + Asn212Asp Gly 95G1n + G1y1.00Asn+ I1e105Met + A1a181G1n + G1n200G1u + Thr207G1u I1e1 05Met + Ser126Asp + Asn153G1u + G1y205G1n + Thr207Pro + Leu211Met Pro1 29G1y + A1a131His + Va1197Thr + Thr207Asn + Ser210G1u + G1y213G1u Gly 98Asp + G1y152G1u + Asn153G1u + Ser154Asp + Va:1197Ser + G1n200Asn Asp 58G1u + Gly 6lPro + Val 93G1y + Ser 97G1u + Pro129Asn + Tyr161I1e Ser1 30Asp + G1y152Ser + Va1197G1y + Asn198G1u + Ser210G1u + Leu211Asp Tyr1 02Va1 + G1y155Asn + Ser182Asp + Va1197Asp + G1n200Asp + Ser210Asp Thr 64G1y + Ser128G1u + G1n200G1u + Thr207Ser + Ser210Asp + Leu211Asp Gln 57G1u + Tyr102Ser + Thr207Pro + Ser210Asp + Leu211Asp + Asn212Asp Pro1 27Asn + G1y152G1u + I1e159Asn ~~Asn198Asp + G1n200Asn + G1y213G1u Asn1 53Ser + G1y155Ser + I1e159Thr -~Asn198Asp + Ser210G1u + Thr214Asp Val 93Thr + Ser154Asp + Ser158G1u -+Ser182G1u + Asn198Ser + Thr207Ser Ser1 01Asp + G1y155Pro + Asn198G1u -~G1n200G1u + A1a209Asn + Ser210Asp , i?s Ser 99G1u + Ser126Asp + Pro127Asp + I1e159Ser + G1n200Ser + Leu211Met Pro129Asn + Ser158Asp + G1n200Ser + Pro204Ser +
Asn153Asp + G1y205Ser Va11 97Cys + G1y205Asp + Thr207Asp + Ser210Asp + Leu211I1e + Asn212Ser Gly 6lAsn + Asn198Asp + G1n200G1u + Thr207G1u + Tyr208Cys + Leu211His Asn 60Asp + Pro127G1n + Asn153G1u + Ser154G1u + A1a156G1u + Thr207Pro Ser 99G1u + Asn153Asp + Ser154Asp + A1a156G1u + G1n185Asn + Pro195Ser Thr 64Asn + A1a156Asn + Va1197Asp + G1n200G1u + Leu211Pro + Thr219G1u Ser1 26Asp + Pro127G1n + 5er128Asp + Ser160Asp + G1n200G1u + Thr219Ser Asn1 53Asp + G1n185G1u + Asn198Ser + G1n200G1u + Leu211Cys + G1y213Asp I1e1 05G1n + Leu129G1u + G1y125G1u + Ser160Asp + A1a199G1n + Pro204G1u Asp 58G1u + Leu 99Pro + Ser154G1u + G1y155Asp + Ser182Asp + Asn198G1n Ser 99G1u + Va1197G1u + Thr207G1y + A1a209Asn + Ser210G1u + Leu211Asp Gln 57Asn + G1y125G1u + Asn153G1u + G1n200Asn + Leu211Ser + Asn212Asp Leu 99Ser + G1y125G1n + Ser126G1u + Va1197Asp + Ser210Asp + Asn212G1u Thr 64G1u + I1e159His + Asn198G1u + G1n200Asp + Leu211Cys + Thr214G1y Gly 98Asp + G1y152G1u + G1y155G1u + A1a156Asp + Tyr208Met + Asn212Ser Gly 98Pro + Ser104G1u + Asn198G1u + G1n200Asp + G1y205G1n + Asn212Asp Asp 58G1u + Thr 64G1u + Ala 96Ser + Tyr102His + G1n200G1u + Leu211G1y Leu 94A1a + G1yl00Gln + Phe183Asp + Va1193G1n + Va1197Asp + G1n200G1u Asn 60G1n + Thr 69G1y + G1y125G1u + Ser159G1u + G1y157Asp + Thr207G1y Val 93Met + G1yl00Asp + Ser126G1u + Pro129Asp + A1a181Asn + Thr214Ser G1y125Asn + Phe183Tyr + G1n185Asp + Asn198Asp +
Ser182G1u + Asn212G1u Leu 94G1n + G1y125Asp + Ser128G1u + Pro129G1u + Asn198G1n + Ser210G1u Gly 59G1n + Thr 69Asp + Asn198G1n + G1n200Asn + Ser210Asp +
Asn212Asp Gly 59G1n + G1y125G1u + Asn198Asp + G1n200Asp + Leu211I1e SO +
G1y213Asp Thr 64Asn + G1y125G1u + Asn153Asp + G1n185G1u + Thr207Ser +
Leu211Asp Asp 58G1u + Gly 95G1u + Gly 98G1u + G1n185Asn + Pro204Ser +
Ser210Asp Gln 57G1u + Gly 59G1u + Thr 69Asp + G1y152Ser + A1a156G1u + Leu211Ser Asn 60Ser + A1a156G1n + Asn198G1u + G1y205G1n + Thr207Asp + Leu211Asp Leu 94A1a + Ser101Asp + Ser154G1u + Ser158Asp + Ser160Asp + Leu211Thr Asp 58G1u + G1n200G1u + Thr207G1u + Ser210G1u + Leu211Thr + G1y213Pro Gly 6lGln + G1y152Asp + G1n200G1u + Ser210G1u + Leu211G1y + Thr214Asp Asn 60G1u + Tyr102I1e + G1n200G1u + Thr207Asp + Ser210Asp + Asn212Ser Gly 59Ser + Val 93Asp + I1e159Asp + Ser160G1u + Thr207Asn + G1y213Ser Thr 69Asn + Ser104Asp + I1e105Cys + A1a156G1y + Ser210Asp + Leu211Asp Gln 57Asn + Gly 98Asp + Glyl00Gln + Ser206G1u + Thr207G1u + Tyr208Met Gln 57Asn + Ser158Asp + I1e159G1u + Phe183Met + Asn198Asp + Thr207Asn Gly 95Asp + Ala 96G1y + A1a131G1n + Thr207G1u + Tyr208G1u + Leu211Thr Ser1 03Asp + Ser104Asp + G1n200Asn + Leu211Asn + Asn212Asp + G1y213Asp Gly 6lGln + Gly 98G1u + G1y155Asp + Phe183Asp + Thr207G1n + Leu211Pro Leu 94Ser + Gly 95Ser + Ser 97G1u + Gly 98G1u + A1a131G1u + Asn212Ser Thr 64Asn + Tyr102I1e + Ser103Asp + Ser104Asp + A1a181Asn + Tyr208Asp Thr 64G1y + Tyr102Asp + G1n200G1u + Thr207Asn + Tyr208G1n + Ser210G1u Ser 99G1u + Tyr102His + I1e159A1a + G1n200Asp + Thr207G1n + Ser210G1u Thr 64G1u + Leu129Cys + Va1197G1u + G1n200Asn + Ser210Asp + Leu211G1y Thr 64G1y + Ser154Asp + A1a181Asn + G1n200G1u + Thr207G1u + Ser210Asp Tyr1 02A1a + A1a181Asn + Ser182G1u + G1n200Asp + Thr207Asp + Ser210Asp G1y1 25Pro + G1y152Asn + Ser158G1u + I1e159G1u + Asn198Asp + Ser210G1u Gln 57Ser + Asn 60Ser + Val 93G1n + Leu 99G1y + G1n200G1u + G1y213Asp Gly 6lGlu + Thr 64Ser + Val 93Asp + Asn198Ser + Pro204G1u + Tyr208G1y Ser1 01Asp + Ser103G1u + Leu129Met + G1y155Ser + Ser210Asp + Leu211G1n Ser 99G1u + Pro127Asp + Pro129Ser + Ser159G1u + Ser158G1u + Thr207Ser Gly 6lGlu + Ser101G1u + Ser104G1u -+I1e159Leu + G1y196Asn + Asn198G1n Gly 59Asp + Asn 60G1u + Ser 99G1u + Va1197Thr + Thr207Asp + G1y213G1n Gly 6lAsn + Ala 96G1u + Gly 98G1u + Leu124G1n + Pro127Ser + G1y152G1u G1y125Ser + Phe183Asp + G1n185G1u + A1a209G1u + Ser210Asp + Leu211His Ser130Asp + I1e159G1y + Asn198G1u + Thr207G1n + Ser210Asp + Leu211Met Pro127G1y + Ser130Asp + Tyr161Thr + Asn198G1u + Ser210G1u + Thr214G1n Gly 98Pro + Tyr102G1u + Phe183Met + Asn198Asp + G1n200Asn + Ser210Asp Ser 99G1u + Pro127Ser + G1y155Pro + Va1197Cys + Asn198G1u + Leu211G1u Leu 94Ser + Ser101G1u + I1e105G1u + Pro195G1n + Va1197G1u + Asn198G1u IS Pro127G1u + Asn198Ser + Pro204G1u + G1y205Ser + Ser206Asp + Thr207Pro Gln 57Ser + Asn 60Asp + G1n185Asn + Asn198G1u + Leu211Thr + Asn212Asp Asp 58G1u + Asn 60Asp + Tyr102A1a + I1e159Ser + Ser160G1u + G1y205Pro Ala 96G1y + A1a181Asp + Asn198Asp + Thr207G1u + A1a209His + Ser210G1u Gln 57Asn + Asn 60G1u + Gly 95G1u + G1yl00Glu + G1y125Ser + Ser210G1u Asp 58G1u + Phe183Ser + Pro209G1u + Thr207Ser + Ser210G1u + Asn212G1u Leu 94Asp + Pro129G1u + A1a156Ser + Tyr161Asp + G1n185Ser + Asn212Ser Gly 95G1u + Ser 99Asp + A1a156His + Va1197G1u + Asn198G1n + Thr207Asn Thr 64G1u + Pro127Asn + A1a156G1u + A1a209Asp + Ser210Asp + Leu211Asn Gly 59Ser + Gly 63Pro + Pro129Ser + G1y157G1u + G1n185Asp + Ser210G1u Gln 57Ser + Gly 59Asp + Leu 94G1u + G1y125Asp + Ser160G1u + G1n185Asn I1e105G1n + Ser126G1u + Ser128Asp + Leu211G1u + G1y213Asp + Thr214G1y Pro127Ser + Ser182G1u + G1n185G1u + Asn198Asp + Thr207Pro + Ser210G1u Ala 96Asn + A1a131Pro + A1a181Asp + Thr207Asp + Ser210Asp + Leu211Asp Asp 58G1u + Gly 59Pro + Ser126Asp + G1y152Asp + Ser158G1u + Leu211I1e G1y155Asp + Ser182Asp + Va1197G1y + G1n200Asp + Thr202Asn + Leu211G1u Ser126Asp + I1e159Ser + Asn198Ser + G1n200Ser + Pro204G1u + Thr207Asp Asn 60Ser + Gly 63Asn + G1n185G1u + Pro195Ser + Asn198Asp + G1n200G1u Gly 59Ser + I1e105Asp + A1a181Asn + Asn198Asp + G1n200Asp + Leu211Thr Leu 94G1u + Ser 99Asp + G1yl00Gln + Ser103Asp + G1n200G1u + Tyr208Met "~" WO 95/30011 PCT/US95/04760 Asp 58G1u + Leu 99Asp + Ser 99G1u + G1yl00Pro + G1y157Asp + Tyr203Va1 Asp 58G1u + Thr 64G1n + Gly 98G1u + Ser 99Asp + Pro129G1u + G1y155Ser Val 93G1n + SerlOlAsp + I1e105Asp + Asn198Asp + G1y205Ser + Leu211Asn Ser 99G1u + I1e105A1a + Ser210G1u + Leu211His + Asn212G1u + Thr214Asp Val 93A1a + Leu124His + A1a131G1y + G1y157Asp + Va1197Asp + G1y213Asp I1e105His + A1a156Asn + Ser158G1u + Va1197Asp +
G1y152Ser + G1y213Asp Gly 6lAsp + Leu 99Thr + Va1197Met + Asn198G1n + G1n200G1u + G1y205G1u Tyr102Pro + Ser130G1u + A1a131Thr + G1n200Asp +
Ser128Asp + Leu211Asp Gly 98Ser + Ser126Asp + Ser128G1u + G1y152G1n + Ser158Asp + G1y205G1u Ser130Asp + Thr207G1y + Tyr208Asp + Ser210G1u +
Phe183I1e + Asn212Asp Asp 58G1u + Leu 94Thr + G1yl00Gln + Tyr102His + Ser206Asp + Thr214Pro Asp 58G1u + Thr 64G1u + Ser126G1u + Pro129Ser + Alal8lGly + Asn198G1n Asn 60Ser + Leu 99Asp + Ala 96G1u + A1a181G1u + G1n185Asn + Thr207G1y Pro127Asp + A1a181G1n + Pro195Ser + Ser206Asp +
Pro129G1u + Thr214G1n G1y152G1n + Thr207Asp + A1a209G1u + Leu211Pro +
A1a156Asp + G1y213Pro Ser103G1u + Ser158G1u + Ser160G1u + Tyr161His +
G1y157Ser + Pro204Ser Leu 94Asp + Gly 95G1n + Gly 98Asp + Ser126G1u + Ser154Asp + Thr207Ser Ser 97G1u + I1e105G1u + Leu124Asp + Phe183G1y + Thr207G1y + Leu211G1n G1yl 00Glu + Pro129Ser + A1a131Ser + Tyr161Va1 + Ser210Asp + Asn212G1u Gly 59Ser + SerlOlGlu + I1e105G1n + A1a131His + Ser210G1u + Asn212G1u Gln 57Asn + Pro127G1n + A1a156Asp + I1e159Asp + Pro195G1y + Ser206Asp Val 93His + G1yl00Glu + Ser126Asp ~+-Ser128G1u + G1n185Asp + Pro204G1y Gly 95Ser + Ser 99Asp + SerlOlAsp ~~Va1197Asn + Leu211Met + G1y213G1u Gln 57Ser + Gly 6lGlu + Ala 96G1u ~ Asn153Asp + G1y213Pro + Thr219G1u Gln 57Ser + A1a156Asp + Ser160Asp -~Va1197G1y + Leu211Asp + G1y213Asp Gly 98Pro + Ser104Asp + A1a181G1u -~Ser182G1u + Asn198G1u + Asn212G1n I1e1 05G1u + Leu124Thr + Va1197Ser -hPro204G1u + Thr207Asp + Ser210Asp Asn 60Ser + Gly 6lPro + Ser160Asp + G1n185Asp + G1n200Asn + Ser210G1u Gly 6lSer + Leu 94Asp + Ser104Asp + Leu124Asn + Asn198Ser + G1y213Asp Asn 60G1u + Val 93Met + Gly 95Asp + Gly 98Asn + Asn198G1u + Leu211Cys Gly 95Ser + Gly 98Asp + Ser 99G1u + Ser126G1u + Ser182G1u + Thr207G1y Asp 58G1u + Gly 98Asp + Leu129Cys + G1n200Asp + Ser210Asp 10+ Asn212Ser Leu 94G1u + Ser 97Asp + Ser158G1u + Va1197Thr + Tyr208Ser + Leu211Ser Ser101G1u + I1e105G1u + Ser154G1u + G1y157G1u + Asn198Ser + Leu211A1a 15Gly 63Ser + Gly 98G1u + G1yl00Pro + G1y152Asp + G1n200Asn + G1y213Asp Leu 94G1u + SerlOlAsp + Phe183A1a + G1n200Asn + Ser206G1u + Leu211Met Gln 57G1u + Asp 58G1u + Leu129Pro + Asn198Ser + Ser206Asp 20+ G1y213Asp G1y125G1u + A1a131His + G1y205Asp + Ala2U9Glu + Ser210G1u + Thr214Ser Gly 59Ser + G1yl00Glu + Ser103Asp + Asn198Ser + Tyr208Leu + Ser210G1u 25Asp 58G1u + Asn 60Asp + G1y155Asp + Va1197Asn + Thr207Ser + Thr214Asp Ser 99Asp + Ser128G1u + Pro129Asp + G1y152G1u + G1n200Asn + Thr207Asn Pro127G1n + Asn153G1u + Ser182Asp + Va1197Cys + Ser210G1u 30+ Leu211Pro Ser104G1u + Leu124Pro + Asn153G1u + A1a156Thr + I1e159Leu + Ser182G1u Thr 64Asn + Tyr102Cys + Ser128Asp + Ser158Asp + Phe183G1u + Asn198Ser 35Ala 96G1n + Tyr161Asn + G1n185Asn + Va1197G1u + G1y205G1u + Ser210Asp Gly 98Ser + Ser101G1u + Pro127G1y + G1n200Asp + Leu211Asp + Thr214G1u Gly 59G1n + Thr 69G1u + Gly 95Ser + G1y155G1u + Ser158Asp 40+ Va1197Cys Tyr102Va1 + Ser103G1u + Asn198Asp + G1n200G1u + Thr207G1u + A1a209Asn Asn 60G1u + Gly 98G1n + Asn153G1u + Ser210Asp + Leu211A1a + G1y213G1u 45Asp 58G1u + Gly 6lGlu + SerlOlGlu + Leu129Asn + Pro129G1y + Ser130Asp Ser126G1u + Ser130Asp + A1a131Thr + G1n200Asn + Thr202G1n + Pro204Asp Gly 63G1n + Ser 97G1u + Ser126Asp + Ser130Asp + Asn198Ser SO+ G1n200Ser 99C + Ser 97G1u + Ser101G1u + Asn212G1n ys Val 93G1n + Leu + G1y213Ser Asp 58G1u + Gly 95G1u + Ser159Asp + Ser158Asp + Thr207Pro + Tyr208Va1 ~. r W O 95/30011 ~ ~ ~ ~ ~ ~,~ ~ PCTIUS95104760 Asn 60G1n + Thr 64Ser + Pro129Asn + Pro204Asp + Ser210Asp + Leu211I1e Ser 99Asp + I1e159G1n + Va1197G1y + Asn198G1u + Leu211Va1 + G1y213Asp Ser 97G1u + G1yl00Glu + G1y152G1u + G1y155G1n + A1a181Ser + A1a209His Asp 58G1u + Ala 96G1u + Ser126G1u + Asn153Asp + Phe183Leu + G1y205Asn Gly 98Asp + Pro127G1u + G1y152Pro + Ser158G1u + G1y205Pro + Leu211G1n Ser154G1u + I1e159Asn + Asn198G1u + G1n200Asn +
G1y157Asp + Tyr203Asn Gly 63Ser + Phe:183G1u+ Thr207G1u + Ser210G1u + Leu211Pro + Thr214Asp Glyl00Glu + Ser154Asp + Ser160G1u + Asn198Ser +
Asn153Ser + G1y213G1u Gln 57G1u + Ala 96His + Gly 98Pro + Ser160Asp + Pro204Asp + Tyr208G1y Gly 59Asp + Gly 95Pro + A1a199G1n + Asn198Asp + G1n200Asp + Thr214Asp Gly 95G1u + Va1197G1u + Asn198G1n + G1n200G1u + Thr207G1n + Leu211A1a Gly 98Asp + Ser128Asp + G1y155Pro + Ser158Asp + G1n185G1u + G1n200Ser Gly 98G1u + Leu124Asp + G1y125Asn + I1e159G1y + Va1197Asp + Leu211A1a Thr 64Asp + G1y152Asn + Ser160G1u + G1n185Asn + Thr207Asp + Ser210Asp Thr 64Asp + G1y155Asp + A1a156Ser + Va1197Ser + Asn198G1u + Leu211Asp Gln 57Asn + Thr 64Pro + I1e105G1u + Ser126G1u + Ser160Asp + Ser206G1u Gly 98Asn + G1y157Asp + Phe183G1u + Va1197Asn + Ser206Asp + Thr214G1u Ser 97Asp + Ser126G1u + Ser128Asp + Pro129Asn + A1a156Asp + Leu211G1n Gln 57G1u + Leu1.24Pro+ Phe183Asn + Va1197G1u + G1y205G1u + G1y213G1u Ser1 26Asp + Ser158G1u + G1n200Asp + Thr207Pro + Leu211G1u + Asn212Ser Gly 98Ser + G1yl00Pro + Pro129G1u + G1y157G1u + Ser158G1u + Asn198Asp Gly 6lAsp + G1n185Asp + Asn198G1n + G1n200Asp + Thr207Asp + Leu211Ser Asp 58G1u + Asn198G1n + G1n200G1u + Thr207G1u + Leu211A1a + Asn212G1u Gly 59Asp + Ser 97G1u + Pro129Asn + G1y152Pro + Ser182G1u + Thr214Asp Gly 59G1u + Thr 69G1u + Pro127G1n + Pro129Asn + A1a131G1y SO + Asn153G1u Pro1 27G1u + Ser130Asp + Ser154G1u + I1e159Cys + Ser210G1u + Leu211Ser Asp 58G1u + Val 93Asn + Ser 97Asp + G1yl00Glu + G1y152Pro + Asn198G1u G1y125G1u + G1y155Asp + G1y157Asn + G1n200G1u + G1y205Asn + Ser210Asp Ala 96Ser + Ser103G1u + Leu129Asp + G1n200G1u + Ser210G1u + Leu211Cys Ser101Asp + Pro129Asp + G1y155Asn + A1a181G1u + Ser182Asp + G1y205Pro Asp 58G1u + Gly 6lGlu + Ala 96His + Asn153Asp + Va1197G1n + G1n200G1u Asn 60Asp + Ser 99G1u + G1y155Pro + A1a181G1u + Va1197Asn + Asn212Asp Asn 60G1n + Gly 98Asn + Ser130Asp + A1a181Asp + A1a209Asp + Asn212Asp Gly 6lAsp + A1a131Asp + Va1197A1a + Asn198Asp + G1n200Asp + Thr214Asn Thr 64Asp + Ser 99Asp + SerlOlGlu + A1a181G1n + G1n200Ser + Ser210G1u Leu124Ser + Tyr161G1u + Ser182Asp + Va1197G1u + Thr207G1n + Leu211Asn Gly 95G1u + Gly 98Asp + Ser103Asp + A1a181G1n + Thr207G1n + Asn212Asp Asn 60G1u + Ser 97G1u + SerlOlGlu + Leu124A1a + Pro127G1u + Va1197G1n Val 93Asp + Ala 96G1u + A1a131Asp + G1y152G1n + G1y196Ser + Va1197His Val 93G1y + A1a131Asp + I1e159G1y + Ser182G1u + Asn198G1n + G1y213Asp Ser130Asp + Ser182Asp + G1n185Ser + Va1197Asn + Asn198G1u + Ser210Asp I1e159Thr + Ser182Asp + G1n200Asp + G1y205Asp + Thr207Asn + Asn212G1u Val 93Thr + Ser104G1u + Pro127Ser + Ser128Asp + Ser130G1u + Asn153G1u Asn 60G1u + Leu 94Asp + G1n200Ser + G1y205Asp + Ser210Asp + Leu211G1n Gly 95Asp + SerlOlAsp + Ser130G1u + Asn198Asp + G1n200Ser + Leu211A1a Ser104Asp + Ser154G1u + Thr207Asn + A1a209Asp + Ser210G1u + Leu211Asn Asp 58G1u + Gly 59G1u + I1e105Leu + Ser126Asp + Phe183Asn + Ser210Asp Gly 59G1u + Asn 60Asp + Gly 95Asn + Pro129G1u + G1y152Ser + G1n185G1u Gly 59Asp + Ser160G1u + G1y196Ser + G1y205Ser + Ser210Asp + Leu211Asp Asn 60G1u + Ser 99Asp + G1y157G1u + Phe183Leu + Va1197Asn + Thr207G1n Ser 99G1u + Asn198Asp + Ser206G1u + Thr207Asp + Tyr208Asn + Leu211His Gly 98G1u + Ser 99Asp + A1a131Pro + Asn153G1u + Tyr161Pro + Tyr208G1u Ser 97G1u + Gly 98G1u + Ser182Asp + Thr207G1n + Ser210Asp + G1y213Ser Ser 97Asp + Gly 98Asp + I1e105Va1 + Pro129G1y + G1n200Asp + G1y205G1u '' W0 95130011 ~ 'PCTIUS95/04760 13~
Asp 58G1u + Ser103Asp + Ser109Asp + Phe183G1y + Va1197Pro + Asn198Asp Asn 60Ser + Ser 99G1u + Glyl00Asp + Pro129G1y + G1y205Asp + Ser210G1u Ser 99G1u + Ser154Asp + Tyr161Cys + Va1197Ser + G1n200Asp + Ser210Asp Ser101G1u + G1n185Ser + Asn198G1n + G1n200Asp +
G1y155Asp + Ser210G1u Gly 59Asp + Tyr102Asn + Ser158Asp + G1n200Asp + Tyr208Va1 + Ser210G1u Gly 59Asp + A1a156G1n + I1e159Asp + Phe183G1y + G1n200Asp + Ser210Asp Pro127Ser + Ser182Asp + Va1197G1n + G1n200G1u +
Ser160Asp + Ser210Asp Gln 57Asn + Val 93Asp + Pro127G1u + Asn198G1n + G1n200G1u + Ser210G1u Leu 94G1u + Pro129G1n + Ser159Asp + Va1197His + G1n200Asp + Ser210G1u Ala 96Ser + Gly 98G1n + Ser126Asp + Ser154Asp + Va1197Thr + Thr207Asp Gln 57Ser + Serl04Asp + G1y125Asp + Ser160G1u + G1y205Pro + Ser210G1u Leu 94G1n + Ser 99Asp + G1y155Pro + Va1193Asn + G1n200Asp + Thr207Asp Gly 95G1u + G1n200G1u + Thr202Ser + Thr207Asp + Leu211G1n + Thr214G1n Thr 69G1n + Ala 96Thr + Ser104Asp + G1n200Asp + Thr207Asp + Thr219Pro Thr 64G1u + Tyr102Cys + A1a156ser + Phe183G1u + Pro195G1n + Thr207G1u Gly 98G1u + Va1197His + Thr207G1n + A1a209Ser + Ser210Asp + G1y213Asp Gly 95Ser + G1y157Asp + Phe183G1u + Thr207Asp + A1a209G1n + Ser210Asp Asp 58G1u + Val 93Asp + Ser128G1u + A1a131G1u + G1y196G1n + Pro204Asn Gln 57Ser + Pro127G1u + G1n185Asn + Asn198Asp + G1n200G1u + Ser206G1u Val 93G1u + G1y152Asn + Thr207G1u + Ser210Asp + Leu211Ser + G1y213G1u Thr 64G1y + Ser 99G1u + Ser103G1u + Pro127Asn + G1y152G1u + Ser158G1u G1y1 57Asn + Ser158G1u + Asn198Asp -rSer206Asp + Thr207G1n + Ser210Asp Ala 96Asp + Ser126G1u + Ser160G1u -+Tyr161Leu + Ser210G1u + G1y213Ser Asp 58G1u + Ala 96Ser + SerlOlAsp -+Ser104G1u + A1a209G1n + Ser210Asp Gly 6lAsp + Thr 69G1n + Val 93Cys -+I1e105Ser + Leu211Va1 + Asn212G1u Gly 6lGlu + Phe183Ser + Tyr203Thr -~Thr207Asn + Tyr208G1y + Asn212G1u Ser 97G1u + I1e105Cys + Pro127Ser -~Ser160Asp + Asn198G1u + Ser210Asp W O 95!30011 Leu 94Asp + Ser130G1u + Asn198Asp + Pro204Asn + Thr207G1n + Ser210Asp I1e105Asp + Leu124Pro + I1e159Leu + Asn198G1u + Ser206Asp + Leu211G1u Tyr102G1y + A1a131Ser + Ser182G1u + Phe183Pro + Thr207G1u + Ser210Asp Asn 60G1n + Gly 6lGlu + Ser 97Asp + G1y125G1u + Tyr208Pro + Ser210Asp Gly 98G1n + Ser160G1u + A1a181Thr + Asn198Asp + Ser206Asp + Asn212G1u Leu124A1a + Ser128G1u + Asn153G1n + G1y205Asp + Thr207G1u + Asn212G1u Gly 59G1u + G1yl00Pro + Ser103G1u + IlelUSGlu + Va1197G1y + Ser210G1u Asn 60G1u + Ser 97G1u + Pro127Asp + Va1193Met + Thr207Asp + A1a209Asn Thr 64G1n + Ala 96Asn + SerlOlGlu + Ser130Asp + Asn198Ser + Leu211Asp Gly 6lPro + Leu 94G1u + Ser103Asp + I1e159Pro + Leu211Asn + Asn212G1u Val 93Cys + Leu 94Asp + Glyl00Ser + Ser103G1u + Ser206Asp + Tyr208G1y Ser130Asp + G1y152G1u + Ser154G1u + G1y157Ser + A1a181Pro + Asn198G1u Gly 59G1u + Ser130Asp + Ser154Asp + G1n185Asp + Thr207G1y + Leu211Pro Gln 57G1u + Leu 94A1a + Ser101Asp + Ser126G1u + A1a131G1n + Ser154Asp Ser103Asp + I1e105Met + G1y125Asn + Ser160Asp + Va1197Ser + Thr214Asp Asp 58G1u + Val 93Asp + G1y125Ser + G1y152Ser + Va1193His + G1n200G1u Asp 58G1u + Gly 98Asp + Leu124Ser + G1y125G1u + Tyr161Met + G1n200G1u Gly 6lAsp + Leu 94I1e + Asn153Asp + Ser158Asp + Va1197A1a + Ser210G1u Asn 60Ser + Val 93Ser + Ser103G1u + Ser160G1u + Asn198Asp + G1n200Asp Asn 60G1n + Ser 97Asp + Ser128Asp + Asn198G1u + G1n200Asp + A1a209G1y Val 93Ser + Tyr102Asn + Ser104Asp + G1y152Asp + Asn198G1u + G1n200G1u Gly 59Asp + I1e105Met + Ser158G1u + I1e159Ser + Asn198G1u + G1n200Asp Gly 59G1u + Ser101G1u + I1e159Cys + Asn198G1u + Va1199G1n + G1n200Asp Gly 6lAsp + Pro127Asp + G1n185Asp + Asn198Ser + G1n200Ser + Ser210Asp G1yl00Glu + Ser104G1u + Ser182G1u + Thr207G1y + Tyr208Met + Ser210G1u Thr 69G1y + G1y157Asp + Ser182G1u + Va1197G1n + Asn198Ser + Ser210Asp Ser128G1u + Ser130Asp + Tyr161Va1 + Asn198G1u + Ser206G1u + G1y213Asn 'I~94~7 °'""" WO 95/30011 . PCT/US95I04760 Gly 59G1n + Thr 64Asp + Ser 99G1u + Asn198Asp + Pro204G1u + Thr207Asn Gly 95G1u + Ser126Asp + G1n185Ser +Va1197Cys + G1n200G1u + Leu211G1u Gly 98Pro + Ser 99G1u + Ser158Asp +G1n200Asp + A1a209Ser + Leu211Asp Ser101G1u + Asn153Ser +Ser160Asp + Thr207G1u +
Pro129G1u + Leu211Cys Asn 60G1u + Gly 98Asp + Pro127Asp +A1a131G1n + A1a181Asp l0 + Asn198Ser Asp 58G1u + Ser126Asp + G1y157Asn +Thr207Ser + Leu211Asp + G1y213Asp Ser104Asp + Asn153Asp +Ser182G1u + Asn198G1u +
Leu124G1y + Thr207Asn Gly 95Ser + Ser103G1u + Pro129G1u +Ser160Asp + G1n200Asp + Leu211Pro Gly 59G1u + G1y125Asp + Pro127G1u +A1a131His + A1a209Asp + Leu211I1e Pro127G1u + Pro204G1y +Thr207Asp + A1a209Asp +
G1y157Asn + Thr214Asp Gln 57G1u + Leu 94G1u + Ser103Asp +G1y152Asp + G1y205Ser + Leu211A1a Ala 96G1u + Ser103Asp + G1y152G1n +Ser158G1u + Ser160Asp + Thr219Asn Gly 6lGlu + Tyr102His + Asn153Asp +Phe183His + G1n185Asn + Ser210Asp Ser 97Asp + Ser126Asp + Pro129G1u +Va1197His + Asn198Asp + G1n200Asn Asn 60G1u + Ala 96Pro + I1e105Asp +Leu129A1a + Ser130G1u + I1e159Ser Gln 57G1u + Pro1.27G1y+ Ser128G1u +A1a131Ser + Ser210Asp + Asn212G1u Ala 96G1u + Tyr102Asp + Ser128G1u +A1a156Pro + Tyr161Va1 + Leu211Thr Gln 57Ser + Asp 58G1u + Ser 99Asp +G1n200G1u + Tyr208Ser + Asn212G1u Gly 59Asp + Leu 94Met + I1e105A1a +G1y152G1u + Ser182G1u + G1n185Ser Asn 60G1u + A1a156G1y + Va1197Asp +Asn198Ser + G1n200G1u + Ser206Asp G1yl 00Ser + Ser101Asp + G1y125G1n +Ser126Asp + Pro127Asn + Asn198G1u Ala 96G1u + Ser 99Asp + Ser160Asp +A1a181G1y + G1y205Ser + Ser210Asp Leu1 24Asp + G1y125Pro + Ser130G1u +Ser154Asp + G1y205G1n + A1a209Thr Ser1 01G1u + G1y152G1u + G1y155G1u ~+Asn198G1n + G1n200Asp + Asn212Ser Gly 95Asn + Ser103Asp + G1y152G1u +G1y155Asp + Tyr208Va1 + Ser210G1u Ser 99G1u + G1yl00Ser + G1y125Asn ~+G1y152Asp + G1y155Asp + G1y205Asp Ala 96G1u + Ser160Asp + Tyr161Met +Phe183Leu + G1n185Asp + G1y205Asp WO 95130011 ~ ~ ~ ' PCTIUS95/04760 Leu 94G1u + Ser104Asp + G1y157G1u + Asn198G1n + Thr207Asn + A1a209Asp Thr 64G1n + SerlOiGlu + I1e105Pro + G1y125Ser + Pro129G1u + Thr219Asn S Gly 98G1n + Tyr102G1n + G1y125Asn + Ser126G1u + Ser158G1u + Asn198G1u Leu 94Asp + Tyr161I1e + A1a181G1y + Phe183Asp + G1n200G1u + Thr214Asp to TABLE 38 Preferred Subtilisin 309 Variants --Single Mutation--Thr207G1u 15 Ser210G1u Ser210Asp Ser210G1y Va1197G1u 20 --Double Mutation--G1n200G1u + Ser210G1u Va1199Leu + Ser210G1u Va1199Leu + Ser210Asp Pro204A1a + A1a209Thr 25 Thr207G1u + Ser210G1u Tyr208Phe + Leu211Asn A1a194G1u + Ser210G1u G1n200G1u + Tyr211Asn G1n206G1u + Thr207G1u --Triple Mutation--G1n200Pro + G1y205A1a + Ser210G1u Thr207G1u + Ser210G1u + 1eu211Asn Va1199I1e + Pro209Asn + Thr207G1u G1n200G1u + Ser210G1u + Leu211Asp G1n200G1u + Thr207G1u + Leu211G1u G1n200G1u + Thr207G1n + Ser210G1u --Quadruple Mutation--Pro204A1a + Thr207G1u + Ser210G1u + Leu211Asn G1n206G1u + Thr207G1u + Ser210G1u + Leu211G1y --Quintuple Mutation--Va1197Leu + Pro204A1a + Thr207G1u + Ser210G1u + Leu217Asp Asn198G1u + G1n200G1u + Thr207G1u + Ser210G1u + Leu211Asp II. Cleaning Compositions In another embodiment of the present invention, an effective amount of one or more enzyme variants of the present invention are included in so compositions useful for cleaning a variety of surfaces in need of proteinaceous ""'~' W O 95/30011 PCTlUS95104760 l39 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 s 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). As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity to 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 is cleaning compositions of the present invention 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 of the present invention may be employed are 2o 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 surtace cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions.
2s A. Cleaning Compositions for Hard SurfacE~s Dishes and Fabrics The enzymes of the present invention can be used in any detergent composition where high sudsing and good insoluble substrate removal are desired. Thus the enzyme variants of the preaent invention can be used with various conventional ingredients to provide fully-formulated hard-surface 3o 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 modern "concentrated"
detergents which contain as much as 30%-60°/~ by weight of surfactants.
The cleaning compositions herein can optionally, and preferably, 3s contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.
Nonlimiting examples of surfactants useful herein include the »o conventional C11-C1g alkyl benzene sulfonates and primary and random alkyl sulfates, the C10-C18 secondary (2,3) alkyl sulfates of the formulas CH3(CH2)x(CHOS03)-M+)CH3 and CH3(CH~)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-C1g alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C10-C1g alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C1p-C1g alkyl polyglycosides, and their corresponding sulfated polyglycosides, C12-C18 alpha-sulfonated fatty acid esters, C 12-C 1 g alkyl and alkyl phenol alkoxylates io (especially ethoxylates and mixed ethoxylpropoxy), C12-C1g 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 andlor betaine or sultaine surfactants is also preferred, depending on the is desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C 1 p-C 1 g 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 2o 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 C10-C16 alkoiamides can be incorporated into the compositions, typically at about 1% to about 10% levels. The C10-25 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%, 3o to provide additionally sudsing.
The liquid detergent compositions herein can contain water and other solvents as carriers. Low molecular weight prirnary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable.
Monohydric alcohols are preferred for solubilizing surfactants, but polyols such 3s 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.

"'"" WD 95/30011 ? PCTJUS95I04760 ia~
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 s 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.
to 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 15 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 20 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, 2s 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 3o 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 of 3s the present invention. 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 %).

PCTlUS9~104760 1. _Hard surface cleaning compositions As used herein "hard surface 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 s 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 enzyme variants of the present invention, such hard to surface cleaning compositions typically comprise a surfactant 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 filmylstreaky residue on the glass surface.
The surfactant component, when present, may comprise as little as 15 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 2o 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, 25 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 3o 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 3s typically present at levels of from about 2% to about 12% in the compositions.
The hard surface cleaning composition embodiment of the present invention is illustrated by the following examples.

"' WO 95130011 ~ ~ PCTIUS95104760 14:, ...
Examales 7-1:?
Liquid Hard Surface Cleaning Compositions Example No.

Component 7 8 9 ~ 0 11 12 s Thr207G1u ~ 0.05 0.50 0.02 0.03 0.10 0.03 Va1199Leu + Ser210Asp - - - - 0.20 0.02 Na2DIDA*

EDTA** - - 2.90 2.90 - -Na Citrate - - - - 2.90 2.90 ~oNaCl2 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 15C12 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%

20*Disodium N-diethyleneglycol-N,N-iminodiacetate **Na4 ethylenediamine diacetic acid *''*Diethyleneglycol monohexyl ether ****All formulas adjusted to pH 7 In Examples 7-10, the subtilisin 309 variants recitedTables 3-38, in 2samong others, are substituted for Thr207G1u, with milar results.
substantially si In Examples 11-12, any combination of the subtilisin309 variants recited in Tables 3-38, among others, are substitutedVa1199Leu for +

Ser210Asp, with substantially similar results.

2189427 »=
Ekamples 13-18 Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew Example No.
s Component 13 14 15 16 17 18 GIn200G1u + Thr207G1u 0.50 0.05 0.60 0.30 0.20 0.30 Ser210G1u - - - - 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 to 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.
is In Examples 13-16, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for GIn200G1u + Thr207G1u, with substantially similar results.
In Examples 17-18, any combination of the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for GIn200G1u +
2o Thr207G1u and Ser210G1u, with substantially similar results.
2. Dishwashing 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 2s 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.

""' WO 95/30011 PCT/US95I04760 1~5 Examples 19-24 Dishwashing Composition Example No.

Component 19 20 21 ~ 22 23 24 s GIn200Pro + GIy205A1a 0.05 0.50 0.0:2 0.40 0.10 0.03 + Ser210Asp Va1199Leu + Ser210Asp - - - - 0.40 0.02 C 12-C 14 N-methyl- 0.90 0.90 0.90 0. 90 0.90 0.90 glucamide to C12 ethoxy (1 ) sulfate 12.0012.00 12.00 12.00 12.0012.00 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 is 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%

2o Product pH is adjusted to 7.

In Examples 19-22, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for GIn200Pro + GIy205A1a + Ser210Asp, with substantially similar results.
In Examples 23-24, any combination of the subtilisin 309 variants 2s recited in Tables 3-38, among others, are substituted for GIn200Pro +
GIy205A1a + Ser210Asp and Va1199Leu + Ser210Asp, with substantially similar results.
3. Fabric cleaninq compositions In another embodiment of the present invention, fabric cleaning so 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.
3s a. Granular fabric cleaning compositions The granular fabric cleaning compo:>itions 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 surfactant, one or more builders, and,. in some cases, a bleaching s agent.
The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples.
Examples 25-28 Granular Fabric Cleaning Composition to Example No.

Component 25 26 27 28 Ser99Asp 0.10 0.20 0.03 0.05 Ser99Gly - - 0.02 0.05 C13 linear alkyl benzene sulfonate22.00 22.00 22.00 22.00 15 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 2o 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 subtilisin 309 variants recited in Tables 3-38, 25 among others, are substituted for Ser99Asp, with substantially similar results.
In Examples 27-28, any combination of the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Ser99Asp and Ser99Gly, with substantially similar results.

~"~" WO 95130011 ~ PCT/US95/04760 Examples 29-32 Granular Fabric Cleaning Composition Example No.
Component 29 ~ 30 31 32 s GIn200G1u + Thr207G1u + Ser21 0.10 0.20 0.03 0.05 OGIu Asn74Asp + Pro204A1a + Thr207G1u - - 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 to 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 is Water and minors balance to 100%

In Examples 29-30, the subtilisin ariants recitedTables 309 v in 3-38, among others, are substituted for r210G1u, GIn200G1u + Thr207G1u + Se with substantially similar results.

In Examples 31-32, any combination of the subtilisin309 variants 2o recited in Tables 3-38, among others,substituted GIn200G1u arf~ for +

Thr207G1u + Ser210G1u and Asn74Asp + Pro204A1a + Thr207G1u, with substantially similar results.

Examples 33-36 Granular Fabric Cleaning (:omposition 2s Example No.

Component 33 34 35 36 Leu 94GIy + GIn200G1u 0.10 0.20 0.03 0.05 Gln 57Ser + Leu 94GIy + GIn200G1u - 0.02 0.05 -C13 linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 so 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 3s 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%

14s In Examples 33-34, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Leu 94GIy + GIn200G1u, with substantially similar results.
In Examples 35-36, any combination of the subtilisin 309 variants s recited in Tables 3-38, among others, are substituted for Leu 94GIy +
GIn200G1u and Gln 57Ser + Leu 94GIy + GIn200G1u, with substantially similar results.
Examples 37-40 Granular Fabric Cleaning Composition to Example No.
Component 37 38 39 40 Asn74His + Gln 57Ser + Asn 60Ser 0.10 0.20 0.03 0.05 + Leu 94GIy + GIn200G1u Val 93GIn + Tyr102Cys + Ser154G1u - - 0.02 0.05 1s + Asn198G1n + Thr207G1y 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 2o 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%
2s In Examples 37-38, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Asn74His + Gln 57Ser + Asn 60Ser + Leu 94GIy + GIn200G1u, with substantially similar results.
In Examples 39-40, any combination of the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Asn74His + Gln 30 57Ser + Asn 60Ser + Leu 94GIy + GIn200G1u and Val 93GIn + Tyr102Cys +
Ser154G1u + Asn198G1n + Thr207G1y, with substantially similar results.

"~ WO 95/30011 ~ PCTJUS95104760 !49 Examples 41-42 Granular Fabric Cleanincl Composition Example No .

Component 41 42 s 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 toDispersant 0.07 0.1 Silicone fluid 0.80 0.80 Trisodium citrate 14.00 15.00 Citric acid 3.00 2.50 Zeolite 32.50 32.10 isMalefic acid acrylic acid copolymer5.00 5.00 Diethylene triamine penta methylene1.00 0.20 phosphonic acid Ser21 OAsp 0.30 0.30 Lipase 0.36 0.40 2oAmylase 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 2sPhenol sulphonate 0.1 0.2 Peroxidase 0.1 0.1 Minors Up to 100 Up to 100 21~94~"~
Examples 43-44 Granular Fabric Cleaning Composition Example No.
Component 43 44 s Sodium linear C12 alkyl benzene-sulfonate 8.0 6.5 Sodium sulfate 15.0 18.0 Zeolite A 26.0 22.0 Sodium nitrilotriacetate 5.0 5.0 Polyvinyl pyrrolidone 0.5 0.7 to Tetraacetylethylene diamine 3.0 3.0 Boric acid 4.0 -Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Va1199Leu + Ser210G1u 0.4 0.4 is Fillers (e.g., silicates; carbonates; Up to 100 Up to 100 perfumes; water) Example 45 Compact Granular Fabric Cleaning Composition Component Weight Alkyl Sulphate 8.0 Alkyl Ethoxy Sulphate 2.0 Mixture of C25 and C45 alcohol 3 and 7 times ethoxylated 6.0 Polyhydroxy fatty acid amide 2.5 Zeolite 17.0 Layered silicatelcitrate 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 vinylpyrrolidone0.1 PEG2000 0.2 Asn74Asp + Va1197G1u + GIn200G1u + Ser210G1u0.5 Lipase 0.2 Cellulase 0.2 Tetracetylethylene diamine 6.0 Percarbonate 22.0 Ethylene diamine disuccinic acid 0.3 "'' WO 95/30011 ~ ~ PCTlUS95104760 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) biphenyl 0.05 Water, Perfume and Minors Up to 100 x 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 ammonium chloride 1.4 Dispersant 0.07 Silicone fluid 0.8 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 Tyr208Phe + Leu211 Asn 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 WO 95130011 ~ ~ PCT/US95104760 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 Zeolite 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 Asn74His + Va1197G1u + Pro204A1a + AIa209Thr0.2 + Ser210G1u Lipase 0.36 Cellulase 0.13 Perborate tetrahydrate 11.64 Perborate monohydrate 8.7 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 b. Liquid fabric cleaning compositions Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants 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 compositions typically additionally comprise an anionic surfactant, a to fatty acid, a water-soluble detergency builder 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.

Component 48 49 50 51 52 s Pro204A1a + AIa209Thr 0.05 0.03 0.30 0.03 0.10 GIn200G1u + Thr207G1u - - - 0.01 0.20 + Ser210G1u 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 to Sodium citrate 1.00 1.00 1.00 1.00 1.00 C1 p alcohol ethoxylate 13.00 13.00 13.00 13.00 13.00 (3) Monethanolamine 2.50 2.50 2.50 2.50 2.50 Waterlpropylene glycollethanol balance to (100:1:1 ) 100%

In Examples 48-50 the subtilisin309 Tables variants 3-38, recited in is among others, are substitutedfor AIa209Thr, substantially Pro204A1a with +

similar results.

In Examples 51-52, any combination of the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Pro204A1a +
AIa209Thr and GIn200G1u + Thr207G1u + Ser210G1u, with substantially similar 2o results.
Examples 53-~57 Liquid Fabric Cleaning Compositions Example No.
Component 53 54 55 56 57 2s Tyr102Cys + IIe105Va1 + Leu12411e0.050.03 0.30 0.03 0.10 + Ser154G1u + Asn198G1n + Thr207G1y Asn74Asp + Ser97Asp + Gln 57Ser- - - 0.01 0.20 + Asn 60Ser 3o C12- C14 alkyl sulfate, Na 20.0020.0020.00 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 C~ p alcohol ethoxylate (3) 13.00 13.0013.00 13.00 13.00 Monethanolamine 2.50 2.50 2.50 2.50 2.50 ss Waterlpropylene glycollethanol balance (100:1:1 ) to 100%

In Examples 53-55 the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Tyr102C;ys + IIe105Va1 + Leu12411e +
Ser154G1u + Asn198G1n + Thr207G1y, with substantially similar results.

WO 95!30011 21 g g 4 2 ~ PCTIUS95I04760 I s=1 In Examples 56-57, any combination of the subtilisin 309 variants recited in Tables 212, among others, are substituted for Tyr102Cys +
IIe105Va1 + Leu12411e + Ser154G1u + Asn198G1n + Thr207G1y and Asn74Asp + Ser97Asp + Gln 57Ser + Asn 60Ser, with substantially similar results.
s Examples 58-59 Granular Fabric Cleaning Composition Example No.
Component 58 59 C12-14 alkenyl succinic acid 3.0 8.0 to Citric acid monohydrate 10.0 15.0 Sodium C12-15 alkyl sulphate 8.0 8.0 Sodium sulfate of C12-15 alcohol 2 times - 3.0 ethoxylated C12-15 alcohol 7 times ethoxylated - 8.0 C12-15 alcohol 5 times ethoxylated 8.0 -ls Diethylene triamine penta (methylene phosphonicd)0.2 -aci Oleic acid 1.8 -Ethanol 4.0 4.0 Propanediol 2.0 2.0 Asn74Asp + Ser210G1u 0.2 0.2 2o 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 2s Peroxidase 0.4 0.1 Waters and minors up to parts In each of Examples 58 and 59 herein, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Asn74Asp +
Ser210G1u, with substantially simila«esults.

°
'~"'' WO 95/30011 '~ ~ PCT/US95/04760 I~5 Examples 60-Ei2 Liquid Fabric Cleaning Composition Example 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 l0 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 is Ser210G1u 0.0145 --Asn74Asp + Ser97Asp + Ser210G1u - 0.0145 -GIn200G1u + Ser210G1u - - 0.0145 Lipase 0.200 0.200 0.200 Cellulase - 7.50 7.50 2o 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 so Bar fabric cleaning compositions n suitable of the present inventio for hand-washing soiled fabrics containeffiectiveamount one or an of more enzyme variants of the present preferably invention, from about 0.001 % to about 10%, more preferably from 01 % about 1 weight about 0. to % by of the composition.

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

WO 95130011 PCTIUS95I04'760 2489 42' Examples 63-66 Bar Fabric Cleaning Compositions Example No.

Component 63 64 65 66 Va1197G1u 0.3 - 0.1 0.02 Tyr208Phe + Leu211 Asn - - 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, 10.0 10.0 10.0 10.00 Na to 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 2o Water 4.0 4.0 4.0 4.00 Filler* balan ce to 100%

*Can be selected from convenient materials such as CaC03, talc, clay, silicates, and the like.
In Examples 63-64 the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Va1197G1u, with substantially similar results.
In Examples 65-fib, any combination of the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Va1197G1u and Tyr208Phe + Leu211Asn, with substantially similar results.

A""' WO 95130011 PCT/US9i104760 2189~~7 ~5' Examples 67-TO
Bar Fabric Cleaning Compositions Example No.
Component 67 68 69 70 s Asn 60Ser + Val 93GIn + GIy213Asp 0.3 - 0.1 0.02 Val 93GIn + Tyr102Cys + Thr207G1y - 0.3 0.4 0.03 + GIy213Asp 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 to 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-.10t~) 5.0 5.0 5.0 5.00 1s 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 2o 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.
2s In Example 67, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Asn 60Ser + Val 93GIn + GIy213Asp, with substantially similar results.
In Example 68, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Val 93GIn + Tyr10:2Cys + Thr207G1y + GIy213Asp, 3o with substantially similar results.
In Examples 69-70, any combination of the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Asn 60Ser + Val 93GIn + GIy213Asp and Val 93GIn + Tyr102Cys + Thr207G1y + GIy213Asp, with substantially similar results.
3s B. Additional Cleaning Compositions In addition to the hard surface cleaning, dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated inl:o a variety of other cleaning iss compositions where hydrolysis of an insoluble substrate ~s desired. Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions.
1. Oral cleaninct 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 io 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 i5 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, 2o instability, irritation, allergic response, and the like, commensurate with a reasonable benefitlrisk 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 25 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 3o 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.
s5 The oral cleaning composition embodiment of the present invention is illustrated by the following examples.

~ 18942 1~9 Examples 71-74 Dentifrice Composition Example No.

Component 71 72 73 74 s Va1199Leu + Pro204A1a + Thr207G1u 2.000 3.500 1.5002 +Ser21 OGIu .

Sorbitol (70% aqueous solution) 35.000 35.000 35.00035.000 PEG-6* 1.000 1.000 1.000 1.000 Silica dental abrasive** 20.000 2'0.000 20.000 20.000 ~o 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) is 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 gl col havin l l y g a mo ecu ar weight of 600.
20 '*PrecipitateM silica identified as Zeodent 119 offered by J.M
Huber . .
***Ca b opol offered by B.F. Goodrich Chemical Company.

~
*'**lota Carrageenan offered by Hercules Chemical Company.

In Examples 71-74 the subtilisin 309 variants recitedTables in 3-38, among others, are substituted for Va1199Leu + Pro204A1aThr207G1u + +

a Ser210G1u, with substantially similar results.

WO 95!30011 PCTIUS95I04760 '""'°
,w ., a Examples 75-78 Mouthwash Comp osition Example No.

Component 75 76 77 78 s Ser210Asp 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 1o Glycerin 10.00 10.00 10.00 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 is Water bal ance 100%
to In Examples 75-78, the subtilisin Tables 3-38, 309 variants recited in among others, are substituted th substantially for Ser210Asp, wi similar results.

Examples 79-82 Lozenge Composition 2o Example No.

Component 79 80 81 82 Tyr208Phe + Leu211 Asn 0.0'1 0.03 0.10 0.02 Sorbitol 17.5() 17.50 17.50 17.50 Mannitol 17.5() 17.50 17.50 17.50 2s Starch 13.6() 13.60 13.60 13.60 Sweetener 1.20 1.20 1.20 1.20 Flavor 11.7() 11.70 11.70 11.70 Color 0.10 0.10 0.10 0.10 Corn Syrup balance to 100%

3o In Examples 79-82, the subtilisin Tables 3-38, 309 variants recited in among others, are substituted Leu211 Asn, substantially for Tyr208Phe + with similar results.

2 ~ 89427 IGI
Examples 83-86 Chewing Gum Composition Example No.
Component 83 84 85 86 s Va1199Met + Pro204A1a + Thr207G1u 0.03 0.02 0.10 0.05 Sorbitol crystals 38.44 38.40 38.40 38.40 Paloja-TTgum 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 ~o 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 subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Va1199Met + Pro204A1a + Thr207G1u, with is substantially similar results.
2. Denture cleaning 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 2o 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 25 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 3o is illustrated by the following examples.
a~.

WO 95130011 PCTIUS95104760 "w Examples 87-90 Two-layer Effervescent Denture Cleansing Tablet Example No.

Component 8T 88 89 90 s Acidic Layer Ser210G1u 1.0 1.5 0.01 0.05 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 to 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 is 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 monohydrate32.0 32.0 32.00 32.00 2o 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 25 Sodium carbonate 2.0 2.0 2.00 2.00 Pyrogenic silica 2.0 2.0 2.00 2.00 Dyelflavor 2.0 2.0 2.00 2.00 *Tetraacetylethylene diamine In Examples 87-90, the sub tilisinvariants Tables 3-38, 309 recited in 3o among others, are substituted milar results.
for Ser210G1u, with substantially si 3. Contact Lens Cleaning positions r Com In another embodiment of invention,contactlens cleaning the present compositions comprise one the ent invention.
or more enzyme variants pres of Such contact lens cleaning prise ffective compositions com an e amount of one 3s or more of the enzyme variants, about 50% of preferably from about 0.01 % to one or more of the enzyme preferablyfrom out 0.01 %
variants, more ab to about 20%, more preferably % to weight of the still from about ~ about 5%, by composition, and a contact cleaningcarrier.Variouscontact lens 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 s 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 to lens.
The contact lens cleaning composition embodiment the present of invention is illustrated by the following examples.

Examples 91-94 Enzymatic Contact Lens Cleaning Solution 15 ' Example No.

Component 91 92 93 94 Va1199Leu + Ser210Asp 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 2o 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 25 Water balance to 100%

In Examples 91-94, the subtilisin 309 variants recited in Tables 3-38, among others, are substituted for Va1199Leu + Ser210Asp, with substantially similar results.
While particular embodiments of the subject invention have been 3o 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.
~.

WO 95/30011 PCT/US9sI04760 '''"
1 G.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: PHILIP F. HRODE, III
BOBBY L. BARNETT
DONN N. RUBINGH
CHANCHAL K. GHOSH
(ii) TITLE OF INVENTION: SUBTILISIN 309 VARIANTS
WITH

DECREASED ADSORPTION AND

INCREASED HYDROLYSIS

IS (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY

(B) STREET: 11810 EAST MIAMI RIVER ROAD

(C) CITY: ROSS

(D) STATE: OH

(E) COUNTRY: USA

(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 #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: CORSTANJE, BRAHM J.

(B) REGISTRATION NUMBER: 34,809 (C) ATTORNEY DOCKET NO.: 52338 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 513-627-2858 (B) TELEFAX: 513-627-0260 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser "'""'" ~ PCTIUS95104760 ~

IGs Phe ValProGly GluPro SerThr G1nAspGlyAsn GlyHisGly Thr His ValAlaGly ThrIle AlaAla LeuAsnAsnSer IleGlyVal Leu Gly ValAlaPro SerAla GluLeu TyrAlaValLys ValLeuGly Ala Ser GlySerGly SerVal SerSer IleAlaGlnGly LeuGluTrp Ala Gly AsnAsnGly MetHis ValAla AsnLeuSerLeu GlySerPro Ser Pro SerAlaThr LeuGlu GlnAla ValAsnSerAla ThrSerArg Gly Val LeuValVal AlaAla SerGly AsnSerGlyAla G1ySerIle Ser Tyr ProAlaArg TyrAla AsnAla MetAlaValGly AlaThrAsp Gln Asn AsnAsnArg AlaSer PheSer GlnTyrGlyAla GlyLeuAsp Ile Val AlaProGly ValAsn ValGln SerThrTyrPro GlySerThr Tyr Ala SerLeuAsn GlyThr SerMet RlaThrProHis ValAlaGly Ala Ala AlaLeuVal LysGln LysAsn ProSerTrpSer AsnValGln Ile Arg AsnHisLeu LysAsn ThrAla ThrSerLeuGly SerThrAsn Leu Tyr GlySerGly LeuVal AsnAla GluAlaAlaThr Arg (2) INFORMATION FOR SEQID
N0:2:

(i) SEQUENC E ARACTERISTIC S:
CH

(A)LE NGTH: 5 inoacids 27 am (B)TY PE: amino id ac (D)TO POLOGY: linear (ii)MOLECUL E PE:protein TY

(xi) SEQUENCE DESCRIPTION: N0:2:
SEQ
ID

Ala GlnSer ValProTyrGly ValSerG1n IleLysAla ProAlaLeu S

His SerGln GlyTyrThrGly SerAsnVal LysValAla ValIleAsp Ser GlyIle AspSerSerHis ProAspLeu LysValAla GlyGlyAla Ser MetVal ProSerGluThr AsnProPhe GlnAspAsn AsnSerHis Gly ThrHis ValAlaGlyThr ValAlaAla LeuAsnAsn SerIleGly Val LeuGly ValAlaProSer AlaSerLeu TyrAlaVal LysValLeu Gly AlaAsp GlySerGlyGln TyrSerTrp IleIleAsn GlyIleGlu Trp AlaIle AlaAsnAsnMet AspValIle AsnMet5er LeuGlyGly 2$ 115 120 125 Pro SerGly SerAlaAlaLeu LysAlaAla ValAspLys AlaValAla Ser GlyVal ValValValAla AlaAlaGly AsnGluGly ThrSerGly Ser SerSer ThrValGlyTyr ProGlyLys TyrProSer ValIleAla Val GlyAla ValAspSerSer AsnGlnArg AlaSerPhe SerSerVal Gly ProGlu LeuAspValMet AlaProGly ValSerIle GlnSerThr Leu ProGly AsnLysTyrGly AlaTyrAsn GlyThrSer MetAlaSer Pro HisVal AlaGlyAlaAla AlaLeuIle LeuSerLys HisProAsn Trp ThrAsn ThrGlnValArg SerSerLeu GluAsnThr ThrThrLys Leu GlyAsp SerPheTyrTyr GlyLysGly LeuIleAsn ValGlnAla Ala Ala Gln

Claims

Claims:

1. A subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution in at least one of positions 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 205, 207, 208, 209, 211, or 214, wherein a. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln or Ser;
e. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln, Asn or Ser and the single amino acid substitution at position 199 is combined with at least one further substitution selected from the group consisting of positions: 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 205, 207, 208, 209, 211 and 214;
f. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
g. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
h. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
i. when a substitution occurs at position 203, the substituting amino acid is Ile, Met, Ala, His, Pro, Gln, Asn or Ser;
j. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
k. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser;

l. when a substitution occurs at position 207, the substituting amino acid is Pro, Gly, Gln, Asn or Ser; but when position 207 is substituted the variant is not a double mutation variant having a substitution at position 211; and wherein when the variant is a single mutation variant the substitution does not occur at position 207;
m. when a substitution occurs at position 208, the substituting amino acid is Leu, Ile, Met, Cys, Ala, His, Pro, Gly, Gln or Asn; but when position 208 is substituted the variant is not a double mutation variant having a substitution at position 211; and wherein when the variant is a single mutation variant the substitution does not occur at position 208;
n. when a substitution occurs at position 209, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn or Ser; but where position 209 is substituted the variant is not a double mutation variant having a substitution at position 211;
and wherein when the variant is a single mutation variant the substitution does not occur at position 209;
o. when a substitution occurs at position 211, the substituting amino acid is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn or Ser; wherein when the variant is a single mutation variant, the substitution does not occur at position 211; and p. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

2. The subtilisin 309 variant of Claim 1 wherein a. when a substitution occurs at position 200, the substituting amino acid is Ser;
b. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser;
c. when a substitution occurs at position 208, the substituting amino acid is Leu, Ile, Met, Cys, Ala, His, Pro, Gly, Gln or Asn; and d. when a substitution occurs at position 209, the substituting amino acid is Pro, Gln, Asn or Ser.

3. The subtilisin 309 variant of Claim 2 wherein the modified amino acid sequence further comprises His or Asp substituted for Asn at position 74.

4. The subtilisin 309 variant of Claim 2, wherein when a substitution occurs in at least one of positions 194, 195, 196, 197, 199, 201, 202, 203, 204, 205, 207, 208, 209, 211 or 214, the substituting amino acid for any of positions 194, 195, 196, 201, 202 or 214 is Asp or Glu.

5. The subtilisin 309 variant of Claim 2 wherein a substitution occurs in at least one of positions of 194, 195, 196, 197, 201, 202, 203, 204, 205 or 209.

6. The subtilisin 309 variant of Claim 5 wherein the substitution occurs in at least one of positions 194, 195, 196, 197 or 201.

7. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution in at least two of positions 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 207, 208, 209, 211, 212 or 214, wherein a. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Pro, Thr, Gly, Gln, Asn or Ser;

e. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
f. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln, Asn or Ser;
g. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
h. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
i. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
j. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser or Glu;
k. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
l. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn or Ser;
m. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser or Asp; but when position 207 is substituted the variant is not a double mutation variant having a substitution at position 211;
n. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Met, Cys, Thr, Ala, His, Pro, Gly, Gln or Asn; but when position 208 is substituted the variant is not a double mutation variant having a substitution at position 211;
o. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn or Ser; but when position 209 is substituted the variant is not a double mutation variant having a substitution at position 211;
p. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu;

q. when a substitution occurs at position 212, the substituting amino acid Gln, Ser or Asp; [provided that if position 211 is substituted with Glu, Asn or Val, then position 212 is not substituted with Asp]but when position 212 is substituted the variant is not a double mutation variant having a substitution at position 211; and r. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

8. The subtilisin 309 variant of Claim 7 comprising two amino acid substitutions, wherein the substitutions are Ala for Pro at position 204 and Thr for Ala at position 209.

9. The subtilisin 309 variant of Claim 7 wherein a. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
b. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
c. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser;
d. when a substitution occurs at position 208, the substituting amino acid is Leu, Ile, Met, Cys, Ala, His, Pro, Gly, Gln or Asn; and e. when a substitution occurs at position 209, the substituting amino acid is Pro, Gln, Asn or Ser.

10. The subtilisin 309 variant of Claim 9, wherein the modified amino acid sequence further comprises His or Asp substituted for Asn at position 74.

11. The subtilisin 309 variant of Claim 7 wherein a substitution occurs in at least two of positions 193, 194, 195, 196, 197, 201, 202, 203, 204, 205 or 209.

12. The subtilisin 309 variant of Claim 11 wherein a substitution occurs in at least two of positions 194, 195, 196, 197 or 201.

13. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution in at least three of positions 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 207, 208, 209, 211, 212 or 214 is substituted, wherein a. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Pro, Thr, Gly, Gln, Asn or Ser;
e. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
f. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln, Asn or Ser;
g. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
h. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
i. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
j. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser or Glu;
k. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
l. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser;

m. when a substitution occurs at position 207, the substituting amino acid is Pro, Gly, Gln, Asn, Ser or Asp;
n. when a substitution occurs at position 208, the substituting amino acid is Leu, Ile, Met, Cys, Thr, Ala, His, Pro, Gly, Gln or Asn;
o. when a substitution occurs at position 209, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn or Ser;
p. when a substitution occurs at position 211, the substituting amino acid is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser or Glu;
q. when a substitution occurs at position 212, the substituting amino acid is Gln, Ser or Asp; and r. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

14. The subtilisin 309 variant of Claim 13 wherein a. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
b. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
c. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser;
d. when a substitution occurs at position 208, the substituting amino acid is Leu, Ile, Met, Cys, Ala, His, Pro, Gly, Gln or Asn; and e. when a substitution occurs at position 209, the substituting amino acid is Pro, Gln, Asn or Ser.

15. The subtilisin 309 variant of Claim 14 wherein a substitution occurs in at least three of positions 194, 195, 196, 197, 201, 202, 203, 204, 205 or 209.

16. The subtilisin 309 variant of Claim 14, wherein the modified amino acid sequence further comprises Asp or His substituted for Asn at position 74.

17. The subtilisin 309 variant of Claim 15, wherein a substitution occurs in at least three of positions 194, 195, 196, 197 or 201.

18. The subtilisin 309 variant of Claim 17, wherein a substitution occurs in at least four of positions 194, 195, 196, 197 or 201.

19. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a detergent composition for cleaning fabrics, a dishwashing composition, an oral cleaning composition, a denture cleansing composition and a contact lens cleaning composition, wherein the cleaning composition comprises the subtilisin 309 variant of Claim 1 and a cleaning composition carrier.

20. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a detergent composition for cleaning fabrics, a dishwashing composition, an oral cleaning composition, a denture cleansing composition and a contact lens cleaning composition, wherein the cleaning composition comprises the subtilisin 309 variant of Claim 7 and a cleaning composition carrier.

21. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a detergent composition for cleaning fabrics, a dishwashing composition, an oral cleaning composition, a denture cleansing composition and a contact lens cleaning composition, wherein the cleaning composition comprises the subtilisin 309 variant of Claim 13 and a cleaning composition carrier.

22. A hard surface cleaning composition comprising the subtilisin 309 variant of Claim 1 and a hard surface cleaning carrier.

23. A hard surface cleaning composition comprising the subtilisin 309 variant of Claim 7 and a hard surface cleaning carrier.

24. A hard surface cleaning composition comprising the subtilisin 309 variant of Claim 13 and a hard surface cleaning carrier.

25. A detergent composition for cleaning fabrics comprising the subtilisin 309 variant of Claim 1 and at least one cleaning composition material compatable with the subtilisin 309 variant.

26. A detergent composition for cleaning fabrics comprising the subtilisin 309 variant of Claim 7 at least one cleaning composition material compatable with the subtilisin 309 variant.

27. A detergent composition for cleaning fabrics comprising the subtilisin 309 variant of Claim 13 and at least one cleaning composition material compatable with the subtilisin 309 variant.

28. An isolated DNA sequence encoding the subtilisin 309 variant of Claim 1.

29. An isolated DNA sequence encoding the subtilisin 309 variant of Claim 7.

30. An isolated DNA sequence encoding the subtilisin 309 variant of Claim 13.

31. A substilisin 309 variant having a modified amino acid sequence of substilisin 309 wild-type amino acid sequence, wherein the modified amino acid sequence comprises Glu substituted for Gln at position 200, Glu substituted for Thr at position 207 and Glu substituted for Ser at position 210.

32. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 wild-type amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, as fourth loop region, a fifth loop region, and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at one position in one of the loop regions; wherein A. when the substitution occurs in the first loop region, the substitution occurs at one of positions 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 59, the substituting amino acid is Asn, Gln, Pro or Ser;
b. when a substitution occurs at position 60, the substituting amino acid is Gln or Ser;

c. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and e. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
B. when the substitution occurs in the second loop region, the substitution occurs at one of positions 93 or 94; wherein a. when a substitution occurs at position 93, the substituting amino acid is Ala, Asn, Gln, Gly, His, Met, Pro, Ser or Thr; and b. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
C. when the substitution occurs in the third loop region, the substitution occurs at position 131; wherein the substituting amino acid is Asn, Gln, Gly, His, Pro, Ser or Thr;
D. when the substitution occurs in the fifth loop region, the substitution occurs at one of positions 181, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu or Ser;
b. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and c. when a substitution occurs at position 185, the substituting amino acid is Asn or Ser; and E. when the substitution occurs in the sixth loop region, the substitution occurs at one of positions 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 205 or 214; wherein a. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

b. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln or Ser;
e. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln, Asn or Ser and the single amino acid substitution at position 199 is combined with at least one further substitution selected from the group consisting of positions: 194, 195, 196, 197, 198, 200, 201, 202, 203, 204, 205, 207, 208, 209, 211 and 214;
f. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
g. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
h. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
i. when a substitution occurs at position 203, the substituting amino acid is Ile, Met, Ala, His, Pro, Gln, Asn or Ser;
j. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
k. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser; and l. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

33. The subtilisin 309 variant of Claim 32, wherein the substitution occurs in the sixth loop region.

34. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when a substitution occurs in the first loop region, the substitution occurs in at least one of positions 57, 58, 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser;
b. when a substitution occurs at position 58, the substituting amino acid is Glu;
c. when a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser;
e. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
f. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

B. when a substitution occurs in the second loop region, the substitution occurs in at least one of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 105; wherein a. when a substitution occurs at position 93, 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 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; provided that if position 96 or position 207 is substituted with Asp, and the variant is a double mutation variant, then position 95 is not substituted with Asp; and when position 95 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183 and 211, and with the proviso that when position 95 is substituted with Asn, then position 96 is not substituted with Pro;
d. when a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; provided that if position 207 is substituted with Asp, and the variant is a double mutation variant, then position 96 is not substituted with Asp; and when position 96 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211, and with the proviso that when position 96 is substituted with Pro, then position 95 is not substituted with Asn;
e. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
f. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
g. when a substitution occurs at position 99, the substituting amino acid is Asp or Glu; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;

179 a h. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 101, the substituting amino acid is Asp or Glu; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
j. when a substitution occurs at position 102, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;
k. when a substitution occurs at position 103, the substituting amino acid is Asp or Glu;
I. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; and m. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125, 126, 127, 128, 129, 130 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
but when position 124 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
c. when a substitution occurs at position 126, the substituting amino acid is Asp or Glu; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
d. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
e. when a substitution occurs at position 128, the substituting amino acid is Asp or Glu;
f. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;
g. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 131, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
D. when a substitution occurs in the fourth loop region, the substitution occurs in at least one of positions 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161;
wherein a. when a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 152 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
b. when a substitution occurs at position 153, the substituting amino acid is Asp, Gln, Glu, Pro or Ser;

c. when a substitution occurs at position 154, the substituting amino acid is Asp or Glu; but when position 154. is substituted the variant is not a double, triple, quadruple, or quintuple mutation variant having substitutions at positions selected from the group consisting of 102, 160, 183, and 211;
d. when a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 155 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
g. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
h, when a substitution occurs at position 159, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; wherein when position 159 is substituted with Leu, the variant is not a double mutation variant having a substitution at position 158 with Glu; and when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double mutation variant having a substitution at position 60 with Asp or Glu; and j. when a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
E. when a substitution occurs in the fifth loop region, the substitution occurs in at least one of positions 181, 182, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
b. when a substitution occurs at position 182, the substituting amino acid is Asp or Glu;
c. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;
d. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 185, the substituting amino acid is Asn, Asp, Glu or Ser; and F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 193, 194, 195, 193, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; wherein a. when a substitution occurs at position 193, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

b. b.when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
e. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
f. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
g. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
h. when a substitution occurs at position 200, the substituting amino acid is Asn, Ser, Glu or Asp;
i. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
j. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
k. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
I. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
m. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn, Ser, Asp or Glu;
n. when a substitution occurs at position 206, the substituting amino acid Asp or Glu;
o. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 207 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
p. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Val, Met, Cys, Thr, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
but when position 208 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
q. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 209 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
r. when a substitution occurs at position 210, the substituting amino acid Asp or Glu;
s. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu or Asp;
wherein when position 211 is substituted with Glu, the variant is not a double mutation variant having a substitution of Asn at position 102;
t. when a substitution occurs at position 212, the substituting amino acid Gln, Ser, Asp, or Glu; wherein the variant is not a double mutation variant having substitutions at positions 211 and 212 and wherein when position 212 is substituted with Asp, the variant is not a double mutation variant having a substitution at position 160 with Asp;
u. when a substitution occurs at position 213, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;

whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

35. 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 subtilisin 309 variant of Claim 34 and a cleaning composition carrier.

36. The cleaning composition of Claim 35, wherein the cleaning composition is a hard surface cleaning composition.

37. The cleaning composition of Claim 35, wherein the cleaning composition is a fabric cleaning composition.

38. The fabric cleaning composition of Claim 37, wherein the composition is in the form of a liquid.

39. The fabric cleaning composition of Claim 38, wherein the composition comprises at least about 5% surfactant and at least about 5% builder, by weight of the composition.

40. The fabric cleaning composition of Claim 39 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.

41. The fabric cleaning composition of Claim 39 further comprising at least one bleaching agent.

42. An isolated DNA sequence encoding the subtilisin 309 variant of Claim 34.

43. The subtilisin 309 variant of Claim 34, wherein at least two substitutions occur in the sixth loop region.

44. An isolated subtilisin 309 variant having a modified .amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when the substitution occurs in the first loop region, the substitution occurs in at least one of positions 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 59, the substituting amino acid is Asn, Gln, Pro or Ser;
b. when a substitution occurs at position 60, the substituting amino acid is Gln or Ser;
c. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and e. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
B. when a substitution occurs in the second loop region, the substitution occurs in at least of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or 105; wherein a. when a substitution occurs at position 93, 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 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; provided that if position 96 or position 207 is substituted with Asp, and the variant is a double mutation variant, then position 95 is not substituted with Asp; and when position 95 is substituted the variant. is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183 and 211, and with the proviso that when position 95 is substituted with Asn, then position 96 is not substituted with Pro;
d. when a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; provided that if position 207 is substituted with Asp, and the variant is a double mutation variant, then position 96 is not substituted with Asp; and when position 96 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211, and with the proviso that when position 96 is substituted with Pro, then position 95 is not substituted with Asn;
e. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
f. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 54, 160, 183 and 211;
g. when a substitution occurs at position 99, the substituting amino acid is Asp or Glu; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
h. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted 188 a the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
when a substitution occurs at position 101, the substituting amino acid is Asp or Glu; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
j. when a substitution occurs at position 102, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;
k. when a substitution occurs at position 103, the substituting amino acid is Asp or Glu;
when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; and m. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125, 126, 127, 128, 129, 130 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
but when position 124 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; burr when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
c. when a substitution occurs at position 126, the substituting amino acid is Asp or Glu; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 18:1, and 211;
d. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
e. when a substitution occurs at position 128, the substituting amino acid is Asp or Glu;
f. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;
g. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 131, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
D. when a substitution occurs in the fourth loop region, the substitution occurs in at least one of positions 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161;
Wherein a. when a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; bul: when position 152 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
b. when a substitution occurs at position 153, the substituting amino acid is Asp, Gln, Glu, Pro or Ser;
c. when a substitution occurs at position 154, the substituting amino acid is Asp or Glu;_but when position 154 is substituted the variant is not a double, triple, quadruple, or quintuple mutation variant having substitutions at positions selected from the group consisting of 102, 160, 183, and 211;

d. when a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 155 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
g. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
h. when a substitution occurs at position 159, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; wherein when position 159 is substituted with Leu, the variant is not a double mutation variant having a substitution at position 158 with Glu; and when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; and j. when a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
E. when a substitution occurs in the fifth loop region, the substitution occurs in at least one of positions 181, 182, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
b. when a substitution occurs at position 182, the substituting amino acid is Asp or Glu;
c. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;
d. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 185, the substituting amino acid is Asn, Asp, Glu or Ser; and F. when a substitution occurs in the sixth loop region, the substitution occurs in at least one of positions 193, 194, 195, 193, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; wherein a. when a substitution occurs at positions 193, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;

e. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
f. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
g. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
h. when a substitution occurs at position 200, the substituting amino acid is Asn, Ser, Glu or Asp;
i. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
j. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
k. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
l. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
m. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn, Ser, Asp or Glu;
n. when a substitution occurs at positions 206, the substituting amino acid Asp or Glu;
o. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 207 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
p. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Val, Met, Cys, Thr, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
but When position 208 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

q. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 209 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

r. when a substitution occurs at position 210, the substituting amino acid Asp or Glu;

s. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu or Asp;
but when position 211 is substituted with Glu, the variant is not a double mutation variant having a substitution of Glu at position 127 or a substitution of Asn at position 102;

t. when a substitution occurs at position 212, the substituting amino acid Gln, Ser, Asp, or Glu; but when position 212 is substituted the variant is not a double mutation variant having a substitution at position 211;
and wherein when position 212 is substituted with Asp, the variant is not a double mutation variant having a substitution at position 160 with Asp;

u. when a substitution occurs at position 213, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

45. The subtilisin 309 variant of Claim 44, wherein at least two substitutions occur in the sixth loop region.

46. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when a substitution occurs in the first loop region, the substitution occurs in at least one of positions 57, 58, 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser;
b. when a substitution occurs at position 58, the substituting amino acid is Glu;
c. when a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser;
e. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
f. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
B. when a substitution occurs in the second loop region, the substitution occurs in at least one of positions 93, 94, 95, 96, 98, 100, 102 or 105; wherein a. when a substitution occurs at position 93, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Met, Pro, Ser or Thr;
b. when a substitution occurs at position 94, 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 95, the substituting amino acid is Gln or Ser; but when position 95 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
d. when a substitution occurs at position 96, the substituting amino acid is Asn, Gly, His, Ser or Thr; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 98, the substituting amino acid is Asn, Gln, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
g. when a substitution occurs at position 102, the substituting amino acid is Cys or Met; and h. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125, 126, 127, 128, 129, 130 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
but when position 124 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
c. when a substitution occurs at position 126, the substituting amino acid is Asp or Glu; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
d. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
e. when a substitution occurs at position 128, the substituting amino acid is Asp or Glu;
f. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;
g. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 131, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
D. when a substitution occurs in the fourth loop region, the substitution occurs in at least one of positions 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161;
wherein a. when a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 152 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
b. when a substitution occurs at position 153, the substituting amino acid is Asp, Gln, Glu, Pro or Ser;
c. when a substitution occurs at position 154, the substituting amino acid is Asp or Glu; but when position 154 is substituted the variant is not a double, triple, quadruple, or quintuple mutation variant having substitutions at positions selected from the group consisting of 102, 160, 183, and 211;
d. when a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 155 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but: when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
g. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
h. when a substitution occurs at position 159, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 159 is substituted with Leu, the variant is not a double mutation variant having a substitution at position 158 with Glu;
and when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double mutation variant having a substitution at position 60 with Asp or Glu; and j. when a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
E. when a substitution occurs in the fifth loop region, the substitution occurs in at least one of positions 181, 182, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
b. when a substitution occurs at position 182, the substituting amino acid is Asp or Glu;
c. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;

d. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 185, the substituting amino acid is Asn, Asp, Glu or Ser; and F. when a substitution occurs in the sixth loop region, the substitution occurs in at least one of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; wherein a. when a substitution occurs at position 193, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
e. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
f. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
g. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
h. when a substitution occurs at position 200, the substituting amino acid is Asn, Ser, Glu or Asp;
i. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
j. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;

k. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
I. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
m. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn, Ser, Asp or Glu;
n. when a substitution occurs at position 206, the substituting amino acid Asp or Glu;
o. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 207 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
p. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Val, Met, Cys, Thr, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
but when position 208 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
q. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 209 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
r. when a substitution occurs at position 210, the substituting amino acid Asp or Glu;

s. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu or Asp;
wherein when position 211 is substituted with Glu, the variant is not a double mutation variant having a substitution at position 127 with Glu;
t. when a substitution occurs at position 212, the substituting amino acid Gln, Ser, Asp, or Glu; but when position 212 is substituted the variant is not a double mutation variant having a substitution at position 211;
and wherein when position 212 is substituted with Asp, the variant is not a double mutation variant having a substitution at position 160 with Asp;
u. when a substitution occurs at position 213, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

47. The subtilisin 309 variant of Claim 46, wherein at least two substitutions occur in the sixth loop region.

48. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when a substitution occurs in the first loop region, the substitution occurs in at least one of positions 57, 58, 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser;
b. when a substitution occurs at position 58, the substituting amino acid is Glu;

c. when a substitution occurs a position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs a position 60, the substituting amino acid is Asp, Gln, Glu or Ser;
e. when a substitution occurs a position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
f. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
B. when a substitution occurs in the second loop region, the substitution occurs in at least one of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 105; wherein a. when a substitution occurs at position 93, 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 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; provided that if position 96 or position 207 is substituted with Asp, and the variant is a double mutation variant, then position 95 is not substituted with Asp; and when position 95 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183 and 211, and with the proviso that when position 95 is substituted with Asn, then position 96 is not substituted with Pro;

d. when a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; provided that if position 207 is substituted with Asp, and the variant is a double mutation variant, then position 96 is not substituted with Asp; and when position 96 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211, and with the proviso that when position 96 is substituted with Pro, then position 95 is not substituted with Asn;
e. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
f. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
g. when a substitution occurs at position 99, the substituting amino acid is Asp or Glu; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
h. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple 204 a mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
i.~when a substitution occurs at position 101, the substituting amino acid is Asp or Glu; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
j.~when a substitution occurs at position 102, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;

k. when a substitution occurs at position 103, the substituting amino acid is Asp or Glu;
l. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; and m. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from,the group consisting of 102, 153, 154, 160, 183, and 211;
C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Met, Pro, Ser, Thr or Val; but when position 124 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Glu, Pro or Ser; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211; and c. when a substitution occurs at position 131, the substituting amino acid is Asn, Gln, Gly, His, Pro, Ser or Thr;
D. when a substitution occurs in the fourth loop region, the substitution occurs in at least one of positions 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161;
wherein a. when a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 152 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
b. when a substitution occurs at position 153, the substituting amino acid is Asp, Gln, Glu, Pro or Ser;
c. when a substitution occurs at position 154, the substituting amino acid is Asp or Glu; but when position 154 is substituted the variant is not a double, triple, quadruple, or quintuple mutation variant having substitutions at positions selected from the group consisting of 102, 160, 183, and 211;
d. when a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 155 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
g. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;

h. when a substitution occurs at position 159, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 159 is substituted with Leu, the variant is not a double mutation variant having a substitution at position 158 with Glu;
and when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double mutation variant having a substitution at position 60 with Asp or Glu; and j. when a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
E. when a substitution occurs in the fifth loop region, the substitution occurs in at least one of positions 181, 182, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
b. when a substitution occurs at position 182, the substituting amino acid is Asp or Glu;
c. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;
d. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 185, the substituting amino acid is Asn, Asp, Glu or Ser; and F. when a substitution occurs in the sixth loop region, the substitution occurs in at least one of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; wherein a. when a substitution occurs at position 193, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
e. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
f. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
g. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
h. when a substitution occurs at position 200, the substituting amino acid is Asn, Ser, Glu or Asp;
i. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
j. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
k. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

l. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
m. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn, Ser, Asp or Glu;
n. when a substitution occurs at position 206, the substituting amino acid Asp or Glu;
o. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 207 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
p. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Val, Met, Cys, Thr, Ala, His., Pro, Gly, Gln, Asn, Asp or Glu;
but when position 208 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
q. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 209 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
r. when a substitution occurs at position 210, the substituting amino acid Asp or Glu;
s. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu or Asp;
wherein when position 211 is substituted with Glu, the variant is not a double mutation variant having a substitution at position 102 with Asn;

t. when a substitution occurs at position 212, the substituting amino acid Gln, Ser, Asp, or Glu; but when position 212 is substituted the variant is not a double mutation variant having a substitution at position 211;
and wherein when position 212 is substituted with Asp, the variant is not a double mutation variant having a substitution at position 160 with Asp;
u. when a substitution occurs at position 213, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

49. The subtilisin 309 variant of Claim 48, wherein in at least two substitutions occur in the sixth loop region.

50. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when a substitution occurs in the first loop region, the substitution occurs in at least one of positions 57, 58, 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser;
b. when a substitution occurs at position 58, the substituting amino acid is Glu;
c. when a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser;

d. when a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser;
e. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
f. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
B. when a substitution occurs in the second loop region, the substitution occurs in at least one of positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 105; wherein a. when a substitution occurs at position 93, 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 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; provided that if position 96 or position 207 is substituted with Asp, and the variant is a double mutation variant, then position 95 is not substituted with Asp; and when position 95 is substituted the variant is not a double, triple or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183 and 211; and with the proviso that when position 95 is substituted with Asn, then position 96 is not substituted with Pro; ~
d. when a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; provided that if position 207 is substituted with Asp, and the variant is a double 211 a mutation variant, then position 96 is not substituted with Asp; and when position 96 is substituted the variant is not a double, triple or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183 and 211; and with the proviso that when position 96 is substituted with Pro, then position 95 is not substituted with Asn.

e. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

f. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

g. when a substitution occurs at position 99, the substituting amino acid is Asp or Glu; but when position 99 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

h. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

i. when a substitution occurs at position 101, the substituting amino acid is Asp or Glu; but when position 101 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

j. when a substitution occurs at position 102, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;

k. when a substitution occurs at position 103, the substituting amino acid is Asp or Glu;

l. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; and m. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125, 126, 127, 128, 129, 130 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
but when position 124 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 125 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

c. when a substitution occurs at position 126, the substituting amino acid is Asp or Glu; but when position 126 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

d. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; but when position 127 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

e. when a substitution occurs at position 128, the substituting amino acid is Asp or Glu;

f. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;

g. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 131, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;

D. when a substitution occurs in the fifth loop region, the substitution occurs in at least one of positions 181, 182, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;

b. when a substitution occurs at position 182, the substituting amino acid is Asp or Glu;

c. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;

d. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 185, the substituting amino acid is Asn, Asp, Glu or Ser; and E. when a substitution occurs in the sixth loop region, the substitution occurs in at least one of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; wherein a. when a substitution occurs at position 193, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

b. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

c. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;

d. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;

e. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

f. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;

g. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

h. when a substitution occurs at position 200, the substituting amino acid is Asn, Ser, Glu or Asp;

i. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;

j. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;

k. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;

l. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;

m. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn, Ser, Asp or Glu;

n. when a substitution occurs at position 206, the substituting amino acid Asp or Glu;

o. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 207 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

p. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Val, Met, Cys, Thr, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
but when position 208 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

q. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 209 is substituted the variant is not a double, triple, or quadruple mutation variant having substitutions at positions selected from the group consisting of 102, 183, and 211;

r. when a substitution occurs at position 210, the substituting amino acid Asp or Glu;

s. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu or Asp;
but when position 211 is substituted with Glu, the variant is not a double mutation variant having a substitution of Glu at position 127 or a substitution of Asn at position 102;

t. when a substitution occurs at position 212, the substituting amino acid Gln, Ser, Asp, or Glu; but when a substitution occurs at position 212 the variant is not a double mutation variant having a substitution at position 211;

u. when a substitution occurs at position 213, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;

whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

51. The subtilisin 309 variant of Claim 50, wherein at least two substitutions occur in the sixth loop region.

52. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when a substitution occurs in the first loop region, the substitution occurs in at least one of positions 57, 58, 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser;

b. when a substitution occurs at position 58, the substituting amino acid is Glu;

c. when a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

d. when a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser;

e. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

f. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

B. when a substitution occurs in the second loop region, the substitution occurs in at least one of positions 93, 94, 95, 96, 9T, 98, 99, 100, 101, 102, 103, or 105; wherein a. when a substitution occurs at position 93, 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 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; provided that if position 96 or position 207 is substituted with Asp, and the variant is a double mutation variant, then position 95 is not substituted with Asp; and when position 95 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183 and 211, and with the proviso that when position 95 is substituted with Asn, then position 96 is riot substituted with Pro;

d. when a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; provided that if position 207 is substituted with Asp, and the variant is a double mutation variant, then position 98 is not substituted with Asp; and when position 96 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211, and with the proviso that when position 96 is substituted with Pro, then position 95 is not substituted with Asn;

e. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;

f. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;

g. when a substitution occurs at position 99, the substituting amino acid is Asp or Glu; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;

218 a h. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154,160, 183 and 211;

i. when a substitution occurs at position 101, the substituting amino acid is Asp or Glu; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;

j. when a substitution occurs at position 102, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;

k. when a substitution occurs at position 103, the substituting amino acid is Asp or Glu;

l. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; and m. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125, 126, 127, 128, 129, 130 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
but when position 124 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

c. when a substitution occurs at position 126, the substituting amino acid is Asp or Glu; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple. sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

d. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

e. when a substitution occurs at position 128, the substituting amino acid is Asp or Glu;

f. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;

g. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 131, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;

D. when a substitution occurs in the fourth loop region, the substitution occurs in at least one of positions 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161;
wherein a. when a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 152 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;

b. when a substitution occurs at position 153, the substituting amino acid is Asp, Gln, Glu, Pro or Ser;

c. when a substitution occurs at position 154, the substituting amino acid is Asp or Glu; but when position 154 is substituted the variant is not a double, triple, quadruple, or quintuple mutation variant having substitutions at positions selected from the group consisting of 102, 160, 183, and 211;
d. when a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 155 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; burr when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 18.3, and 211;
g. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
h. when a substitution occurs at position 159, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 159 is substituted with Leu, the variant is not a double mutation variant having a substitution at position 158 with Glu;
and when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;

i. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double mutation variant having a substitution at position 60 with Asp or Glu; and j. when a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;

E. when the substitution occurs in the fifth loop region, the substitution occurs at one of positions 181, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Ser or Thr;
b. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Ser, Thr, Tyr or Val;
c. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and d. when a substitution occurs at position 185, the substituting amino acid is Asn or Ser; and F. when a substitution occurs in the sixth loop region, the substitution occurs in at least one of positions 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 or 214; wherein a. when a substitution occurs at position 193, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;

d. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
e. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
f. when a substitution occurs at position 198, the substituting amino acid is Glu, Gln, Asp or Ser;
g. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
h. when a substitution occurs at position 200, the substituting amino acid is Asn, Ser, Glu or Asp;
i. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
j. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
k. when a substitution occurs at position 203, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
I. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
m. when a substitution occurs at position 205, the substituting amino acid Pro, Gln, Asn, Ser, Asp or Glu;
n. when a substitution occurs at position 206, the substituting amino acid Asp or Glu;
o. when a substitution occurs at position 207, the substituting amino acid Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 207 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

p. when a substitution occurs at position 208, the substituting amino acid Leu, Ile, Val, Met, Cys, Thr, Ala, His, Pro, Gly, Gln, Asn, Asp or Glu;
but when position 208 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
q. when a substitution occurs at position 209, the substituting amino acid His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu; but when position 209 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
r. when a substitution occurs at position 210, the substituting amino acid Asp or Glu;
s. when a substitution occurs at position 211, the substituting amino acid Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Glu or Asp;
but when position 211 is substituted with Glu, the variant is not a double mutation variant having a substitution of Glu at position 127 or a substituion of Asn at position 102;
t. when a substitution occurs at position 212, the substituting amino acid Gln, Ser, Asp, or Glu; but when a substitution occurs at position 212 the variant is not a double mutation variant having a substitution at position 211; and wherein when position 212 is substituted with Asp, the variant is not a double mutation variant having a substitution at position 160 with Asp;
u. when a substitution occurs at position 213, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

53. The subtilisin 309 variant of Claim 52, wherein at least two substitutions occur in the sixth loop region.

54. An isolated subtilisin 309 variant having a modified amino acid sequence of subtilisin 309 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 in at least two positions in at least one of the loop regions;
wherein A. when a substitution occurs in the first loop region, the substitution occurs in at least one of positions 57, 58, 59, 60, 61, 63, or 64; wherein a. when a substitution occurs at position 57, the substituting amino acid is Asn, Asp, Glu or Ser;
b. when a substitution occurs at position 58, the substituting amino acid is Glu;
c. when a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
d. when a substitution occurs at position 60, the substituting amino acid is Asp, Gln, Glu or Ser;
e. when a substitution occurs at position 61, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;
f. when a substitution occurs at position 63, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and g. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;
B. when a substitution occurs in the second loop region, the substitution occurs in at least one of positions 93, 94, 95, 96, 9T, 98, 99, 100, 101, 102, 103, or 105; wherein a. when a substitution occurs at position 93, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, this, Met, Pro, Ser or Thr;

b. when a substitution occurs at position 94, 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 95, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; provided that if position 96 is substituted with Asp, and the variant is a double mutation variant, then position 95 is not substituted with Asp; and when position 95 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183 and 211, and with the proviso that when position 95 is substituted with Asn, then position 96 is not substituted with Pro;

d. when a substitution occurs at position 96, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 96 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211, and with the proviso that when position 96 is substituted with Pro, then position 95 is not substituted with Asn;

e. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu; but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;
f. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;

226 a g. when a substitution occurs at position 99, the substituting amino acid is Asp or Glu; but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183 and 211;

h. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 101, the substituting amino acid is Asp or Glu; but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
j. when a substitution occurs at position 102, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;
k. when a substitution occurs at position 103, the substituting amino acid is Asp or Glu;
l. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu; and m. when a substitution occurs at position 105, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 105 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

C. when a substitution occurs in the third loop region, the substitution occurs in at least one of positions 124, 125, 126, 127, 128, 129, 130 or 131; wherein a. when a substitution occurs at position 124, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;
but when position 124 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

b. when a substitution occurs at position 125, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 125 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

c. when a substitution occurs at position 126, the substituting amino acid is Asp or Glu; but when position 126 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

d. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; but when position 127 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;

e. when a substitution occurs at position 128, the substituting amino acid is Asp or Glu;

f. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser;

g. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 131, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;

D. when a substitution occurs in the fourth loop region, the substitution occurs in at least one of positions 152, 153, 154, 155 156, 157, 158, 159, 160 or 161;
wherein a. when a substitution occurs at position 152, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 152 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
b. when a substitution occurs at position 153, the substituting amino acid is Asp, Gln, Glu, Pro or Ser;
c. when a substitution occurs at position 154, the substituting amino acid is Asp or Glu; but when position 154 is substituted the variant is not a double, triple, quadruple, or quintuple mutation variant having substitutions at positions selected from the group consisting of 102, 160, 183, and 211;
d. when a substitution occurs at position 155, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 155 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
e. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
f. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
g. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu; but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
h. when a substitution occurs at position 159, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; but when position 159 is substituted with Leu, the variant is not a double mutation variant having a substitution at position 158 with Glu;
and when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
i. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu; but when position 160 is substituted the variant is not a double mutation variant having a substitution at position 60 with Asp or Glu; and j. when a substitution occurs at position 161, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val; but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 102, 154, 160, 183, and 211;
E. when a substitution occurs in the fifth loop region, the substitution occurs in at least one of positions 181, 182, 183, 184 or 185; wherein a. when a substitution occurs at position 181, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;
b. when a substitution occurs at position 182, the substituting amino acid is Asp or Glu;
c. when a substitution occurs at position 183, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;
d. when a substitution occurs at position 184, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 185, the substituting amino acid is Asn, Asp, Glu or Ser; and F. when the substitution occurs in the sixth loop region, the substitution occurs at one of positions 194, 195, 196, 197, 199, 200, 201, 202, 203, 204, 205, 207, 208, 209, 211 or 214; wherein a. when a substitution occurs at position 194, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
b. when a substitution occurs at position 195, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
c. when a substitution occurs at position 196, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
d. when a substitution occurs at position 197, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln or Ser;
e. when a substitution occurs at position 199, the substituting amino acid is Met, Cys, Ala, His, Pro, Gly, Gln, Asn or Ser;
f. when a substitution occurs at position 200, the substituting amino acid is Asn or Ser;
g. when a substitution occurs at position 201, the substituting amino acid is Asp or Glu;
h. when a substitution occurs at position 202, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
i. when a substitution occurs at position 203, the substituting amino acid is Ile, Met, Ala, His, Pro, Gln, Asn or Ser;
j. when a substitution occurs at position 204, the substituting amino acid is Gly, Gln, Asn or Ser;
k. when a substitution occurs at position 205, the substituting amino acid is Pro, Gln, Asn or Ser;
l. when a substitution occurs at position 207, the substituting amino acid is Pro, Gly, Gln, Asn or Ser; but when position 207 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
m. when a substitution occurs at position 208, the substituting amino acid is Leu, Ile, Met, Cys, Ala, His, Pro, Gly, Gln or Asn; but when position 208 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
n. when a substitution occurs at position 209, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn or Ser; but when position 209 is substituted the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 102, 153, 154, 160, 183, and 211;
o. when a substitution occurs at position 211, the substituting amino acid is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn or Ser; and p. when a substitution occurs at position 214, the substituting amino acid is Pro, Gly, Gln, Asn, Asp, Ser or Glu;
whereby the subtilisin 309 variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin 309.

55. The subtilisin 309 variant of Claim 54, wherein at least two substitutions occur in the sixth loop region.

56. 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 subtilisin 309 variant of Claim 32 and a cleaning composition carrier.

57. 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 subtilisin 309 variant of Claim 31 and a cleaning composition carrier.

58. 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 subtilisin 309 variant of Claim 44 and a cleaning composition carrier.

59. 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 subtilisin 309 variant of Claim 46 and a cleaning composition carrier.

60. 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 subtilisin 309 variant of Claim 48 and a cleaning composition carrier.

61. 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 subtilisin 309 variant of Claim 50 and a cleaning composition carrier.

62. 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 subtilisin 309 variant of Claim 52 and a cleaning composition carrier.

63. 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 subtilisin 309 variant of Claim 54 and a cleaning composition carrier.

64. The subtilisin 309 variant of Claim 32, wherein the substitution occurs in the first loop region.

65. The subtilisin 309 variant of Claim 32, wherein the substitution occurs in the second loop region.

66. The subtilisin 309 variant of Claim 32, wherein the substitution occurs in the third loop region.

67. The subtilisin 309 variant of Claim 32, wherein the substitution occurs in the fifth loop region.

68. The subtilisin 309 variant of Claim 34, wherein at least two substitutions occur in the first loop region.

69. The subtilisin 309 variant of Claim 34, wherein at least two substitutions occur in the second loop region.

70. The subtilisin 309 variant of Claim 34, wherein at least two substitutions occur in the third loop region.

71. The subtilisin 309 variant of Claim 34, wherein at least two substitutions occur in the fourth loop region.

72. The subtilisin 309 variant of Claim 34, wherein at least two substitutions occur in the fifth loop region.

73. The subtilisin 309 variant of Claim 44, wherein at least two substitutions occur in the first loop region.

74. The subtilisin 309 variant of Claim 44, wherein at least two substitutions occur in the second loop region.

75. The subtilisin 309 variant of Claim 44, wherein at least two substitutions occur in the third loop region.

76. The subtilisin 309 variant of Claim 44, wherein at least two substitutions occur in the fourth loop region.

77. The subtilisin 309 variant of Claim 44, wherein at least two substitutions occur in the fifth loop region.

78. The subtilisin 309 variant of Claim 46, wherein at least two substitutions occur in the first loop region.

79. The subtilisin 309 variant of Claim 46, wherein at least two substitutions occur in the second loop region.

80. The subtilisin 309 variant of Claim 46, wherein at least two substitutions occur in the third loop region.

81. The subtilisin 309 variant of Claim 46, wherein at least two substitutions occur in the fourth loop region.

82. The subtilisin 309 variant of Claim 46, wherein at least two substitutions occur in the fifth loop region.

83. The subtilisin 309 variant of Claim 48, wherein at least two substitutions occur in the first loop region.

84. The subtilisin 309 variant of Claim 48, wherein at least two substitutions occur in the second loop region.

85. The subtilisin 309 variant of Claim 48, wherein at least two substitutions occur in the third loop region.

86. The subtilisin 309 variant of Claim 48, wherein at least two substitutions occur in the fourth loop region.

87. The subtilisin 309 variant of Claim 48, wherein at least two substitutions occur in the fifth loop region.

88. The subtilisin 309 variant of Claim 50, wherein at least two substitutions occur in the first loop region.

89. The subtilisin 309 variant of Claim 50, wherein at least two substitutions occur in the second loop region.

90. The subtilisin 309 variant of Claim 50, wherein at least two substitutions occur in the third loop region.

91. The subtilisin 309 variant of Claim 50, wherein at least two substitutions occur in the fifth loop region.

92. The subtilisin 309 variant of Claim 52, wherein at least two substitutions occur in the first loop region.

93. The subtilisin 309 variant of Claim 52, wherein at least two substitutions occur in the second loop region.

94. The subtilisin 309 variant of Claim 52, wherein at least two substitutions occur in the third loop region.

95. The subtilisin 309 variant of Claim 52, wherein at least two substitutions occur in the fourth loop region.

96. The subtilisin 309 variant of Claim 52, wherein at least two substitutions occur in the fifth loop region.

97. The subtilisin 309 variant of Claim 54, wherein at least two substitutions occur in the first loop region.

98. The subtilisin 309 variant of Claim 54, wherein at least two substitutions occur in the second loop region.

99. The subtilisin 309 variant of Claim 54, wherein at least two substitutions occur in the third loop region.

100. The subtilisin 309 variant of Claim 54, wherein at least two substitutions occur in the fourth loop region.

101. The subtilisin 309 variant of Claim 54, wherein at least two substitutions occur in the fifth loop region.