WO2001007577A2 - Protease conjugates having sterically protected epitope regions - Google Patents

Abstract

The present disclosure relates to subtilisin protease conjugate comprising a protease moiety and one or more addition moieties. Each addition moiety is covalently attached to an epitope protection position of the protease moiety. The protease conjugates have decreased immunogenicity relative to a parent protease. The present disclosure further relates to cleaning and personal care compositions comprising the protease conjugates.

Description

PROTEASE CONJUGATES HAVING STERICALLY PROTECTED EPITOPE REGIONS

FIELD OF THE INVENTION

The present invention relates to chemically modified subtilisin proteases which are useful in compositions such as, for example, personal care compositions, laundry compositions, hard surface cleansing compositions, and light duty cleaning compositions

BACKGROUND OF THE INVENTION Enzymes make up the largest class of naturally occurring proteins One class of enzyme includes proteases which catalyze the hydrolysis of other proteins This ability to hydrolyze proteins has typically been exploited by incorporating naturally occurring and genetically engineered proteases into cleaning compositions, particularly those relevant to laundn applications.

In the cleaning arts, the mostly widely utilized of these proteases are the seπne proteases Most of these seπne proteases are produced by bacteπal organisms while some are produced by other organisms, such as fungi See Siezen et al.. "Homology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtihsin-Like Seπne Proteases", Protein Engineering, Vol 4, No 7, pp. 719 - 737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment is frequently not optimized for the artificial environment of a cleaning composition. Specifically, protease characteπstics such as, for example, thermal stability, pH stability, oxidative stability, and substrate specificity are not necessaπly optimized for utilization outside the natural environment of the protease.

Several approaches have been employed to alter the wild-type ammo acid sequence of senne proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign and / or chemical modification of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions

However, because such modified proteases are foreign to mammals, they are potential antigens As antigens, these proteases cause an lmmunogenic and / or allergenic response (herein collectively described as lmmunogenic response) in mammals.

Furthermore, while genetic redesign and chemical modification of proteases has been prominent m the continuing search for more highly effective proteases for laundry applications, such proteases have not been commercially utilized m personal care compositions and light dutj detergents. A pπmary reason for the absence of these proteases m products such as, for example, soaps, gels, body washes, shampoos, and light duty dish detergents is due to the problem of human sensitization leading to undesirable lmmunogenic responses. It would therefore be highly advantageous to provide a personal care composition or light duty detergent which provides the cleansing properties of proteases without the provocation of an lmmunogenic response.

Presently, lmmunogenic response to proteases may be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne proteases, still present the πsks associated with extended tissue contact with the finished composition and worker exposure to protease-contaimng dust or aerosol during manufacturing.

It has also been proposed that reduction in immunogenicity of a protease may be achieved by attaching polymers to the protease. See, e.g., U.S. Patent No. 4,179,337, Davis et al.. issued December 18, 1979; U.S. Patent No. 5,856,451, Olsen et al.. assigned to Novo Nordisk, issued January 5, 1999; WO 99/00489, Olsen et al.. assigned to Novo Nordisk, published January 7, 1999; WO 98/30682, Olsen et al.. assigned to Novo Nordisk, published July 16, 1998; and WO 98/35026, Von Per Osten et al. published August 13, 1998. However, such proposals have not suggested the importance of attaching polymers to the ammo acid regions of the protease which are responsible for the immune response (i.e , epitopes).

It has recently been discovered that the subtilisin protease compπses three epitope regions and that conjugation of one or more polymers, polypeptides. or other groups should be attached at one or more of these regions to effect significant reduction in immunogenicity of the protease. See, e.g., U.S. Patent Application Seπal No. 09/088,912, Weisgerber et al.. assigned to The Procter & Gamble Co., filed June 2, 1998.

The present inventors have discovered that steπc protection near one or more of the epitope regions of the protease is an alternative mechanism to prevent or impede presentation of an epitope and decrease the immunogenicity of the protease. Accordingly, the present inventors herein provide modified subtihsms wherein the modification is at a region in steπc proximity to one or more of the epitope regions. The present inventors have therefore discovered subtilisin proteases which evoke a decreased lmmunogenic response yet maintain their activity as an efficient and active protease. Accordingly, the present protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.

SUMMARY OF THE INVENTION The present invention relates to protease conjugates comprising a protease moiety and one or more addition moieties, wherein each addition moiety is covalently attached to an epitope protection position of the protease moiety, wherein:

(a) the epitope protection positions for the first epitope region are selected from 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215, and 216 corresponding to subtilisin BPN';

(b) the epitope protection positions for the second epitope region are selected from 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145 corresponding to subtilisin BPN¹; and

(c) the epitope protection positions for the third epitope region are selected from 9, 10, 22, 23. 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203. 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275 corresponding to subtilisin BPN', and wherein the addition moieties each, independently, have the structure:

wherein X is selected from nil and a linking moiety; R_! is selected from nil, a first polypeptide, and a first polymer; and R₂ is selected from nil, a second polypeptide, and a second polymer; wherein at least one of X, R_u and R is not nil.

The protease conjugates of the present invention have decreased immunogenicity relative to the parent protease. Accordingly, such protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.

DETAILED DESCRIPTION OF THE INVENTION

The essential components of the present invention are herein described below. Also included are non-limiting descπptions of vaπous optional and preferred components useful m embodiments of the present invention.

The present invention can compπse, consist of, or consist essentially of any of the required or optional components and / or limitations described herein.

All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated.

All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impuπties, for example, residual solvents or byproducts, which may be present in commercially available sources. All documents referred to herein, including all patents, patent applications, and publications, are hereby incorporated by reference m their entirety.

Referred to herein are trade names for materials including, but not limited to, enzymes The inventors herein do not intend to be limited by mateπals under a certain frade name. Equivalent mateπals (e g , those obtained from a different source under a different name or catalog (reference) number) to those referenced by frade name may be substituted and utilized in the protease conjugates and compositions herein

As used herein, abbreviations will be used to describe ammo acids. Table I provides a list of abbreviations used herein-

Table I

Definitions

As used herein, the term "mutation" refers to an alteration in a gene sequence and / or an am o acid sequence produced by those gene sequences Mutations include deletions, substitutions, and additions of ammo acid residues to the wild-type protein sequence

As used herein, the term "parent" refers to a protein (wild-type or vaπant) which is utilized for further modification to form a protease conjugate herein

As used herein, the term "wild-type" refers to a protein, for example a protease or other enzyme, produced by unmutated organisms

As used herein, the term "variant" means a protein having an amino acid sequence which differs from that of the corresponding wild-type protein

As used herein, all polymer molecular weights are expressed as weight average molecular weights

As referred to herein, while the conjugates of the present invention are not limited to those compπsmg subtilisin BPN' and variants thereof, all ammo acid numbeπng is with reference to the ammo acid sequence for subtilisin BPN' which is represented by SEQ ID NO 1 The ammo acid sequence for subtilisin BPN' is further described by Wells et al . Nucleic Acids Research, Vol π, pp 7911 - 7925 (1983)

Protease Comugates of the Present Invention

The protease conjugates of the present invention are compounds which compπse a protease moiety and one or more addition moieties, wherein the protease moiety and the addition moieties are connected via covalent attachment (i e , covalent bonding) Protease Moieties

The protease moieties herein are subtilism-like proteases, either wild-type or vaπants thereof As used herein, the term "subtilism-like protease" means a protease which has at least 50%, and preferably 80%, ammo acid sequence identity with the sequences of subtilisin BPN' Wild-type subtilism-like proteases are produced by, for example, Bacillus alcalophilus, Bacillus amylohquefaciens, Bacillus amylosaccharicus , Bacillus hcheniformis, Bacillus lentus, and Bacillus subtilis microorganisms A discussion relating to subtilism-like seπne proteases and their homologies may be found in Siezen et al . "Homology Modelling and Protein Engmeeπng Strategy of Subtilases, the Family of Subtihsin-Like Seπne Proteases", Protein Engineering Vol 4, No 7, pp 719 - 737 (1991)

Preferred protease moieties for use herein include, for example, those obtained from Bacillus amylohquefaciens, Bacillus hcheniformis, and Bacillus subtilis, subtilisin BPN, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, protemase K, and thermitase, including A/S Alcalase® (commercially available from Novo Industries, Copenhagen, Denmark),

Esperase® (Novo Industπes), Savmase® (Novo Industπes), Maxatase® (commercially available from Genencor International Inc.), Maxacal® (Genencor International Inc.), Maxapem 15® (Genencor International Inc.), and variants of the foregoing. Especially preferred protease moieties for use herein include those obtained from Bacillus amylohquefaciens and variants thereof. The most preferred protease moieties herein are subtilisin BPN' and vaπants thereof

Especially preferred vaπants of subtilisin BPN', hereinafter referred to as "Protease A", for use as parent ammo acid sequences herein are disclosed U.S. Patent No. 5,030,378, Venegas, issued July 9, 1991, as characteπzed by the subtilisin BPN' amino acid sequence with the following mutations

(a) Gly at position 166 is substituted with an ammo acid residue selected from Asn, Ser, Lys, Arg, His, Gin, Ala and Glu; Gly at position 169 is substituted with Ser; and Met at position 222 is substituted with an ammo acid residue selected from Gin, Phe, His, Asn, Glu, Ala and Thr; or

(b) Gly at position 160 is substituted with Ala, and Met at position 222 is substituted

Additionally preferred vaπants of subtilisin BPN', hereinafter referred to as "Protease B", for use as parent ammo acid sequences herein are disclosed in EP 251 ,446, assigned to Genencor International. Inc., published January 7, 1988, as characteπzed by the wild-type subtilisin BPN^" ammo acid sequence with mutations at one or more of the following positions: Tyr21, Thr22. Ser24, Asp36, Ala45, Ala48, Ser49, Met50, Hιs67, Ser87, Lys94, Val95, Gly97, Ser 101, Gly 102, Gly 103, Ilel07, Gly 110, Met 124, Glyl27, Glyl28, Prol29, Leul35, Lysl70, Tyrl71, Prol72. Aspl97, Metl99, Ser204, Lys213, Tyr214, Gly215, and Ser221; or two or more of the positions listed above combined with one or more mutations at positions selected from Asp32, Ser33, Tyrl04, Alal52, Asnl55, Glul56, Gly 166, Glyl69, Phel89, Tyr217, and Met222.

Other preferred subtilisin BPN' vaπants for use herein are hereinafter referred to as

"Protease C", and are descπbed in WO 95/10615, assigned to Genencor International Inc., published Apπl 20, 1995, as characteπzed by the wild-type subtilisin BPN' ammo acid sequence with a mutation to position Asn76, in combination with mutations in one or more other positions selected from Asp99, SerlOl, Gin 103, Tyrl04, Serl05, Ilel07, Asnl09, Asnl23, Leul26,

Glyl27, Glyl28, Leul35, Glul56, Gly 166, Glul95, Aspl97, Ser204, Gln206, Pro210. Ala216,

Tyr217, Asn218, Met222, Ser260, Lys265, and Ala274. Other preferred subtilisin BPN' vaπants for use herein, hereinafter referred to as "Protease D", are descπbed m U.S. Patent No 4,760,025, Estell et al . issued July 26, 1988, as characteπzed by the wild-type subtilisin BPN' ammo acid sequence with mutations to one or more ammo acid positions selected from the group consisting of Asp32, Ser33, Hιs64, Tyrl04, Asnl55, Glul56, Glyl66, Glyl69, Phel89, Tyr217, and Met222.

The more preferred protease moieties herein are selected from the group consisting of subtilisin BPN', Protease A, Protease B, Protease C, and Protease D, with Protease D being the most preferred

Without intending to be limited by theory, the protease moieties herein have three epitope regions: a first epitope region, a second epitope region, and a third epitope region. The first epitope region occurs at positions 70 - 84 corresponding to subtilisin BPN'; the second epitope region occurs at positions 103 - 126 corresponding to subtilisin BPN', and the third epitope region occurs at positions 220 - 246 corresponding to subtilisin BPN' See, e.g.. U.S Patent Application Serial No. 09/088,912, Weisgerber et al.. assigned to The Procter & Gamble Co., filed June 2, 1998; copendmg U.S. Provisional Patent Application Seπal No. 60/144,991, Rubmgh et al . "Seπne Protease Variants Having Ammo Acid Substitutions and Deletions m Epitope Regions" filed July 22, 1999; and copendmg U.S. Provisional Patent Application Seπal No 60/144,980, Sikorski et al.. "Serme Protease Vaπants Having Ammo Acid Substitutions in Epitope Regions" filed July 22, 1999.

The present inventors have surprisingly discovered epitope protection positions which are in steπc proximity to at least one of the foregoing epitope regions It has further been discovered that these epitopes are protected from hydrolysis, and thus exposure of epitopes, by covalently attaching one or more addition moieties to an ammo acid of the protease moiety at an epitope protection position.

The epitope protection positions which are appropπate for covalent modification with an addition moiety depend upon which epitope one desires to protect. Most preferably, at least one addition moiety is covalently attached to an epitope protection position for the first epitope region.

It has been discovered that the epitope protection positions for the first epitope region are 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215, and 216 corresponding to subtilisin BPN'. Preferably, the epitope protection positions for the first epitope region are 1, 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86, 87, 89, 206, 209, 214, and 215 corresponding to subtilisin BPN' Most preferably, the epitope protection positions for the first epitope region are 1, 2, 3, 4, 5, 17, 40, 41, 43, 67, 86, 87, and 214 corresponding to subtilisin BPN'.

It has further been discovered that the epitope protection positions for the second epitope region are 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145 corresponding to subtilisin BPN. Preferably, the epitope protection positions for the second epitope region are 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138, and 141 corresponding to subtilisin BPN'.

It has further been discovered that the epitope protection positions for the third epitope region are selected from the group consisting of 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275 corresponding to subtilisin BPN' Preferably, the epitope protection positions for the second epitope region are 22, 23, 24, 143, 146, 155, 173, 189, 197, 203. 204, 253, 254, 265, and 275 corresponding to subtilisin BPN'.

In a preferred embodiment of the present invention, the protease moiety compπses a modified sequence of a parent am o acid sequence. The parent ammo acid sequence may be any of the above proteases descπbed above, with the same preferred limitations as descπbed above. In this embodiment, the parent ammo acid sequence is substituted at one or more of the parent ammo acid residues with a substituting ammo acid to produce a protease moiety suitable for attachment with one or more of the present addition moieties. In accordance with the present invention, the substitution should be made at one or more of the epitope protection positions The epitope protection positions, and preferred limitations thereof, are descπbed above

In order to best achieve selective attachment at one or more of the epitope protection positions of one or more addition moieties to the protease moiety, the substitution should be with a substituting ammo acid which does not occur in (is unique to) the parent ammo acid sequence. In this respect, any substituting ammo acid which is unique to the parent ammo acid sequence may be utilized. For example, because a cysteme residue does not occur in the wild-type ammo acid sequence for subtilisin BPN', a substitution of subtilisin BPN' with one or more cysteme residues at one or more of the epitope protection positions is suitable for the present invention Wherein a cysteme residue occurs at a position other than an epitope protection position of the parent ammo acid sequence, it is preferable to substitute another ammo acid residue for in each of those positions to enable selective coupling with one or more addition moieties at an epitope protection position. Cysteme is the most preferred substituting ammo acid for substitution at one or more of the epitope protection positions. Other preferred substituting ammo acids include lysme. Wherein the substituting ammo acid is lysine, it is preferred to mutate lysine residues which occur at positions other than an epitope protection position of the parent amino acid sequence to another ammo acid residue such that functionahzation of one or more of the lysine residues at an epitope protection position is selective. For example, a lysine residue occurs at position 43 of subtilisin BPN' which is an epitope protection position as defined herein. Site-selective mutation of all other lysme residues occurring in the subtilisin BPN' sequence may be performed followed by selective functionahzation of the lysine residue at position 43 with an addition moiety Alternatively, ammo acid residues at any of the epitope protection positions may be mutated to lysine (for example) followed by selective functionahzation at those positions by an addition moiety. Addition Moieties

The protease conjugates of the present invention comprise one or more addition moieties wherein each of the addition moieties is covalently attached to one of the ammo acid residues at an epitope protection position as descπbed herein The addition moiety may be any chemical structure Preferably, the addition moiety steπcally hinders the epitope protection position to which it is attached, or any other epitope protection position as defined herein. Non-limitmg examples of addition moieties include organic molecules including, but not limited to, molecules having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300; polypeptides, and polymers. As used herein, the term "polypeptide" means a molecule comprising two or more ammo acid residues. As used herein, the term "polymer" means any molecule which comprises two or more identical (preferably five or more identical) monomer units

Preferably, the addition moiety has the structure.

^R-x-

wherein X is selected from nil and a linking moiety; R_l is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R₂ is selected from the group consisting of nil, a second polypeptide, and a second polymer, wherein at least one of X, Ri, and R₂ is not nil

Preferably, the protease conjugate comprises from 1 to about 15, more preferably from about 2 to about 10, and most preferably from about 1 to about 5 addition moieties.

Wherein Ri and R₂ are each, independently, polypeptide moieties or polymer moieties, R] and R₂ may be identical or different. Preferably, wherein R] is a polypeptide moiety, R₂ is selected from nil and a polypeptide moiety, and is most preferably nil Most preferably, wherein R_! is a polypeptide moiety, R₂ is selected from nil and an identical polypeptide moiety, and is most preferably ml. Preferably, wherein Ri is a polymer moiety, R₂ is selected from ml and a polymer moiety. Most preferably, wherein R] is a polymer moiety, R₂ is selected from nil and an identical polymer moiety. Wherein at least one of R] and R₂ are respectively, the first polymer and the second polymer, then X is preferably not nil. Polypeptide Moieties

The polypeptide moieties descπbed herein include those compπsmg two or more ammo acid residues Preferred polypeptide moieties are selected from proteins, including enzymes Preferred enzymes include proteases, cellulases, lipases, amylases, peroxidases, microperoxidases, hemicellulases, xylanases, phosphohpases, esterases, cutmases, pectmases, keratmases, reductases (including, for example, NADH reductase), oxidases, phenoloxidases, hpoxygenases, hgnmases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabmosidases, hyaluromdase, chondroitmase, laccases, transferases, isomerases (including, for example, glucose isomerase and xylose isomerase), lyases, ligases, synthetases, and fruit-based enzymes (including, for example, papam). More preferred enzymes for use as polypeptide moieties include proteases, cellulases, amylases, lipases, and fruit-based enzymes, with proteases being even more preferred.

Examples of lipases for use as a polypeptide moiety include those deπved from the following microorganisms. Humicola, Pseudonomas, Fusarium, Mucor, Chromobacterium, Aspergillus. Candida, Geotricum, Penicillium, Rhizopus, and Bacillus.

Examples of commercial lipases include Lipolase^®, Lipolase Ultra^®, Lipozyme^®, Palatase . Novozym435 , and Lecitase^® (all of which are commercially available from Novo Nordisk, Copenhagen, Denmark), Lumafast^® (commercially available from Genencor, Int , Rochester, NY), and Lipomax^® (Genencor, Int.).

Examples of proteases for use as the polypeptide moiety include seπne proteases, chymotrypsin, and elastase-type enzymes. The most preferred proteases for use as a polypeptide moiety include seπne proteases, as were defined herein above in the discussion of "protease moieties"

Most preferably, wherein the polypeptide moiety is a serme protease, the polypeptide moiety carπes, independently, the definition of a protease moiety as described herein above Preferably, as descπbed above, the polypeptide moiety has a modified ammo acid sequence of a parent ammo acid sequence wherein the modification is in one or more of the epitope protection positions as descπbed herein above (which parent ammo acid sequence may be referred to as a "second" parent amino acid sequence). In this instance, one of the linking moiety (wherein the linking moiety is not nil) or the protease moiety (wherein the linking moiety is nil) is covalently attached to the polypeptide moiety through one of the substituting ammo acids present m one of the epitope protection positions of the polypeptide moiety. Wherein the polypeptide moiety is a seπne protease, the same preferred groupings of epitope protection positions apply as are descπbed herein above for protease moieties and their corresponding parent ammo acid sequences

Most preferably, wherein the polypeptide moiety is a serme protease, the polypeptide moiety and the protease moiety are equivalent moieties. In this instance, the polypeptide moiety and the protease moiety are most preferably attached through a disulfide bπdge, wherein X is nil, and most preferably, R₂ is ml. Polymer Moieties

The addition moieties herein may compπse a polymer moiety. As used herein, the term polymer moiety means any molecule which comprises two or more identical (preferably five or more identical) monomer units. Examples of suitable polymer moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarboxylates, polyvmylpyrrohdones, celluloses, dexfrans, starches, glycogen, agaroses, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectm, algmic acid hydrosylates, and hydrosylates of chitosan. Preferred polyalkylene oxides include polyethylene glycols, methoxypolyethylene glycols, and polypropylene glycols. Preferred dexfrans include carboxymethyldextrans Preferred celluloses include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethyl cellulose, carboxyethyl cellulose, and hydroxypropylcellulose. Preferred starches include hydroxyethyl starches and hydroxypropyl starches. The more preferred polymers are polyalkylene oxides. The most preferred polymer moiety is polyethylene glycol.

Wherein Rj and R₂ are each, independently, polymer moieties, R_λ and R₂ preferably has a collective molecular weight (i.e , molecular weight of R] plus molecular weight of R₂) of from about 0.2 kD (kilodaltons) to about 40kD, more preferably from about 0.5 kD to about 40 kD, even more preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD

Wherein Ri and R₂ are each polymer moieties, R_l and R₂ each, independently, preferably have a molecular weight of about 0.1 kD to about 20kD, more preferably from about 0.25 kD to about 20 kD, even more preferably from about 0.5 kD to about 10 kD, and most preferably from about 0.5 kD to about 5 kD. Wherein Ri and R₂ are each polymer moieties, the ratio of the molecular weights of Rj to R2 preferably ranges from about 1: 10 to about 10: 1, more preferably from about 1 : 5 to about 5:1, and most preferably from about 1 :3 to about 3:1.

Wherein Ri is a polymer moiety and R₂ is ml, R preferably has a molecular weight of from about 0.1 kD to about 40kD, more preferably about 0.5 kD to about 40 kD, even more preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD. Linking Moieties

As used herein, X may be nil or a linking moiety which is optionally covalently attached to one or more polypeptide moieties or one or more polymer moieties, or both, and is also covalently attached to an ammo acid residue at one of the epitope protection positions of the protease moiety The linking moiety may be, generally, any small molecule, i.e , a molecule having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300. The most preferred linking moieties include those capable of being covalently bound to a cysteme residue or a lysme residue, most preferably a cysteme residue.

Examples of linking moieties and related chemistry are disclosed m U.S. Patent No. 5,446,090, Hams, issued August 29, 1995; U.S. Patent No. 5,171,264, Mernll. issued December 15, 1992; U.S. Patent No 5,162,430, Rhee et al. issued November 10, 1992; U.S. Patent No 5,153,265, Shadle et al.. issued October 6, 1992, U.S. Patent No. 5,122,614, Zahpsky. issued June 16, 1992; Goodson et al., "Site-Directed Pegylation of Recombmant Interleukm-2 at its Glycosylation Site", Biotechnology, Vol. 8, No. 4, pp. 343 - 346 (1990); Kogan. "The Synthesis of Substituted Methoxy-Poly(ethylene glycol) Deπvatives Suitable for Selective Protein Modification", Synthetic Communications, Vol. 22, pp. 2417 - 2424 (1992); and Ishπ et al., "Effects of the State of the Succmimido-Rmg on the Fluorescence and Structural Properties of Pyrene Maleimide-Labeled αα-Tropomyosm", Biophysical Journal, Vol. 50, pp. 75 - 80 (1986) The most preferred linking moiety is substituted (for example, alkyl) or unsubstituted succmimide.

As further examples, the following non-limitmg reagents may be utilized to form the linking moiety. N-[alpha-maleιmιdoacetoxy]succmιmιde ester, N-5-azιdo-2- nitrobenzoyloxysuccmimide, bismaleimidohexane, N-[beta-maleιmιdopropyloxy]succιmmιde ester; bιs[2-(succιmmιdyloxycarbonyloxy)-ethyl]sulfone; bιs[sulfosuccmιmιdyl]suberate, 1,5- dιfluoro-2,4-dmfrobenzene, dimethlyadipimate • 2 HCI, dimethylpimehmidate • 2 HCI, dimethylsubeπmidate • 2 HCI; disuccmimidyl glutarate; disuccimmidyl suberate; m- maleιmιdobenzoyl-N-hydroxysuccmιmιde ester; N-hydroxysuccmιmιdyl-4-azιdosalιcylιc acid; N- succmιmιdyl-6-[4'-azιdo-2'-mtrophenylammo]hexanoate; N-hydroxysuccimmidyl 2,3 - dibromopropionate; succmimidyl 4-[N-maleιmιdomethyl]cyclohexane-l-carboxylate; succinimidyl 4-(f>-maleιmιdophenyl)-butyrate; succιmrmdyl-6-[(beta- maleιmιdopropιonamιdo)hexanoate]; bιs[2-(sulfosuccmιmιdyloxycarbonyloxy)-ethyl]sulfone; N- [gamma-maleimidobutyryloxy] sulfosuccmimide ester; N-hydroxysulfosuccmιmιdyl-4- azidobenzoate: N-[kappa-maleιmιdoundecanoyloxy]sulfosuccmιmιde ester; m- maleimidobenzoyl-N-hydroxysulfosuccimmide ester; sulfosuccmιmιdyl[4- azidosahcylamido]hexanoate, sulfosuccmimidyl 7-azιdo-4-methylcoumaπn-3-acetate, sulfosuccmimidyl 6-[4'-azιdo-2'-mtrophenylammo]hexanoate; sulfosuccmimidyl A-\p- azιdophenyl]butyrate; sulfosuccιmmιdyl[4-ιodoacetyl]ammobenzoate; sulfosuccmimidyl 4-[N- maleιmιdomethyl]cyclohexane-l -carboxylate; and sulfosuccmimidyl 4-(p-maleιmιdophenyl)- butyrate. Each of these reagents is commercially available from Pierce Chemical Co., Rockford, IL. Optional Moieties

The protease conjugate may additionally compπse one or more other chemical structures, including (for example) one or more small molecules, polypeptides, and / or polymers attached to other residues of the protease not herein exemplified or even at an epitope protection position not beaπng an addition moiety (herein referred to as "supplementary moieties"). Supplementary moieties may include polypeptide moieties, polymer moieties, and linking moieties as descπbed herein above. Additionally, for example, one or more polymers (most preferably polyethylene glycol) having a molecular weight of from about 100 Da to about 5000 Da, preferably from about 100 Da to about 2000 Da, more preferably from about 100 Da to about 1000 Da, still more preferably from about 100 Da to about 750 Da, and most preferably about 300 Da may be covalently attached to the protease moiety herein at residues other than those exemplified herein. Such polymer moieties may be attached directly to the protease moiety herein, at any location of the protease moiety, using techniques as described herein and as well-known m the art (including through a linking moiety as described herein). Non-limiting examples of polymer conjugation of this optional type is set forth in WO 99/00849, Olsen et al.. Novo Nordisk A S, published January 7, 1999.

Method of Making The protease moieties having a substitution in one or more of the epitope protection positions (or any other location of the moiety) are prepared by mutating the nucleotide sequences that code for a parent ammo acid sequence. Such methods are well-known in the art; a non- limitmg example of one such method is set forth below:

A phagemid (pSS-5) containing the wild-type subtilisin BPN' gene (Mitchison. C. and J.A. Wells, "Protein Engmeeπng of Disulfide Bonds m Subtilisin BPN'", Biochemistry, Vol. 28, pp. 4807 - 4815 (1989) is transformed into Eschenchia coh dut- ung- strain CJ236 and a single stranded uracil -containing DNA template is produced using the VCSM13 helper phage (Kunkel et al , "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection", Methods in Enzymolog} , Vol 154, pp. 367 - 382 (1987), as modified by Yuckenberg et al., "Site-Directed in vitro Mutagenesis Using Uracil-Contammg DNA and Phagemid Vectors", Directed Mutagenesis - A Practical Approach. McPherson, M. J. ed., pp. 27 - 48 (1991) Primer site-directed mutagenesis modified from the method disclosed in Zoller. M. J.. and M. Smith. "Ohgonucleotide - Directed Mutagenesis Using Ml 3 - Deπved Vectors: An Efficient and General Procedure for the Production of Point Mutations in any Fragment of DNA", Nucleic Acids Research, Vol. 10, pp. 6487 - 6500 (1982) is used to produce all mutants (essentially as presented by Yuckenberg et al.. supra).

Ohgonucleotides are made using a 380B DNA synthesizer (Applied Biosystems Inc.) Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (Ameπcan Type Culture Collection E. coli 33625). All mutations are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain PG632 (Saunders et al., "Optimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34-Ammo Acid Fragment of Human Parathyroid Hormone", Gene, Vol. 102, pp. 277 - 282 (1991) and Yang et al.. "Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an in vitro - Deπved Deletion Mutation", Journal of Bacteriology, Vol. 160, pp. 15 - 21 (1984).

Fermentation is as follows Bacillus subtilis cells (PG632) containing the protease of interest are grown to mid-log phase in one liter of LB broth containing 10 g/L glucose, and inoculated into a Biostat C fermentor (Braun Biotech, Inc., Allentown, PA) in a total volume of 9 liters. The fermentation medium contains yeast extract, casein hydrosylate, soluble - partially hydrolyzed starch (Malfrm M-250), antifoam, buffers, and trace minerals (see "Biology of Bacilli: Applications to Industry", Doi, R. H. and M. McGloughlm, eds (1992)). The broth is kept at a constant pH of 7.5 duπng the fermentation run. Kanamycm (50 μg/mL) is added for antibiotic selection of the mutagemzed plasmid. The cells are grown for 18 hours at 37 °C to an Aβoo of about 60 and the product harvested.

The fermentation broth is taken through the following steps to obtain pure protease. The broth is cleared of Bacillus subtilis cells by tangential flow against a 0.16 μm membrane The cell-free broth is then concenfrated by ulfrafilfration with a 8,000 molecular weight cut-off membrane The pH is adjusted to 5.5 with concenfrated MES buffer (2-(N- morpholmo)ethanesulfonιc acid). The protease is further purified by cation exchange chromatography with S-sepharose and elution with NaCl gradients. See Scopes, R. K . "Protein Puπfication Principles and Practice", Spπnger-Verlag, New York (1984)

A /?NA assay (DelMar et al.. Analytical Biochemistry, Vol. 99, pp. 316 - 320 (1979)) is used to determine the active protease concentration for fractions collected duπng gradient elution. This assay measures the rate at which /j-nitroamlme is released as the protease hydrolyzes the soluble synthetic substrate, succinyl-alanme-alanine-prolme-phenylalanine-/?- nitroanilme (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a specfrophotometer and is proportional to the active protease moiety concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The active protease/total-protem ratio gives the protease puπty, and is used to identify fractions to be pooled for the stock solution.

To avoid autolysis of the protease duπng storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the puπfication procedure the puπty of the stock protease 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 trypsm inhibitor type IJ-T turkey egg white (Sigma Chemical Company, St. Louis, Missouπ).

In preparation for use, the protease stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer m the enzyme stock solution is exchanged for 0.01 M KH₂P0₄ solution, pH 5.5.

With the protease prepared it may be utilized for functionahzation with one or more addition moieties to produce the protease conjugate The precursor to the addition moiety (the precursor to the addition moiety reacts with the precursor to the protease moiety to form the protease conjugate which is compnsed of the addition moiety and the protease moiety) is preferably activated to enhance reactivity with the precursor to the protease moiety Such activation is well-known in the art. Non-limitmg examples of methods of protease conjugate preparation are provided below.

Example 1

A protease comprising a cysteme residue at one of the epitope protection positions is coupled with a polymer moiety according to the above scheme using the following method (wherein "P" represents the protease moiety minus the thiol group resulting from the cysteme substitution and n is the number of repeating monomer units of the polyethylene glycol (for example, n = 77).

A vaπant of subtilisin BPN' with a substitution of leucme for tyrosine at position 217 and a substitution of cysteme for seπne at position 3 is prepared. A concentration of approximately 2 mg / mL in phosphate buffer (pH 5.5) of the variant is achieved. The pH is then raised to 7.5 with dilute sodium hydroxide. The vaπant is mixed with the monomethyl polyethylene glycol maleimide at a 25: 1 activated polymer to vaπant excess. After one hour of mixing at ambient temperature, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and filtered through a molecular weight cut-off ultrafilter to remove excess polymer. The concentrate contains the puπfied protease conjugate.

Example 2

A protease moiety compπsmg a cysteme residue at one of the epitope protection positions is coupled with a polymer moiety according to the above scheme using the following method (wherein "P" represents the protease moiety minus the thiol group resulting from the cysteme substitution and n is the number of repeating monomer units of each polyethylene glycol (e.g., n = 77)).

A vaπant of subtilisin BPN' with a substitution of leucme for tyrosine at position 217 and a substitution of cysteme for phenylalanme at position 17 is prepared. A concenfration of approximately 2 mg / mL m phosphate buffer (pH 5.5) of the variant is achieved. The pH is then raised to 7.5 with dilute sodium hydroxide. The vaπant is mixed with the dimethyl polyethylene glycol maleimide at a 25: 1 activated polymer to vaπant excess. After one hour of mixing at ambient temperature, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and filtered through a molecular weight cut-off ultrafilter to remove excess polymer. The concentrate contains the purified protease conjugate. Example 3 Succmimide -protected polymer is coupled selectively to lysine in one or more of the epitope protection positions (wherein "MPEG" and "PEGM" are equivalent and represent monomethyl polyethylene glycols and wherein "P" represents the protease moiety minus the lysine amme group shown):

pH 8 5

Example 4 Carbodπmide-protected polymer is coupled selectively to lysine in one or more of the epitope protection positions (wherein "MPEG" and "PEGM" are equivalent and represent monomethyl polyethylene glycols, "P" represents the protease moiety minus the lysine amme group shown, and X and X' are side chains compπsmg the carbodnmide moiety, for example, alkyls):

Example 5

A protease moiety compπsmg a cysteme residue in one of the epitope protection positions is coupled with an alkyl maleimide using the following method (wherein "P" represents the protease moiety minus the thiol group resulting from the cysteme substitution and "R" is an alkyl group). In this example, Ri and R are each ml and the linking moiety is deπved from the alkyl maleimide.

A vaπant of subtilisin BPN' with a substitution of leucme for tyrosine at position 217 and a substitution of cysteme for seπne at position 86 is prepared. A 20 mL solution of the variant is prepared at a concentration of approximately 1 mg / mL in 0.01 M KH₂P0₄ buffer (pH 7). To this solution, an 1.5 equivalents of alkyl maleimide (for example, methyl maleimide) is added to the solution The solution is gently mixed at ambient temperature for approximately one hour. The resulting protease conjugate is obtained from the solution by standard methods. Example 6

2 Equivalents 1 Equivalent

A protease moiety comprising a cysteme residue at one of the epitope protection positions forms a dimer using the following method (wherein "P" represents the protease moiety minus the thiol group resulting from the cysteme substitution). In this example, the protease moiety and the polypeptide moiety are equivalent (and X is nil).

A vaπant of subtilisin BPN' with a substitution of leucme for tyrosine at position 217 and a substitution of cysteme for serme at position 214 is prepared. A 20 mL solution of the vaπant is prepared at a concenfration of approximately 1 mg / mL in 0.01 M KH₂P0₄ buffer (pH 8.6). Oxygen is gently bubbled through the solution at ambient temperature for approximately one hour to form the desired protease conjugate dimer. The resulting protease conjugate is obtained from the solution by standard methods.

Analytical Methods

The present protease conjugates may be tested for enzymatic activity and lmmunogenic response using the following methods, both of which are known to one skilled in the art. Other methods well-known in the art may alternatively be used.

Protease Comugate Activity

The protease activity of a protease conjugate of the present invention may be assayed by methods which are well-known m the art. Two such methods are set forth herein below: Skm Flake Activity Method

Using Scotch^® #3750G tape, human skm flakes are stripped from the legs of a subject repeatedly until the tape is substantially opaque with flakes. The tape is then cut into 1 inch by 1 inch squares and set aside. In a 10 mm by 35 mm pern dish, 2 mL of 0.75 mg / mL of a control enzyme (for example, subtilisin BPN') or the protease conjugate to be tested is added in 0.01 M

KH P0₄ pH 5.5 buffer. To this solution 1 mL of 2.5% sodium laurate pH 8.6 solution is added

The solution is gently mixed on a platform shaker. The previously prepared tape square is soaked m the solution (flake side up) for ten minutes continuing gentle mixing. The tape square is then πnsed gently in tap water for fifteen seconds. Stevenel Blue Stain (3 mL, commercially available from Sigma Chemical Co., St. Louis, MO) is pipetted into a clean pern dish. The πnsed tape square is placed into the stam for three minutes (flake side up) with gentle mixing. The tape square is removed from the stam and rinsed consecutively m two beakers of 300 mL distilled water, for fifteen seconds per nnse. The tape square is allowed to air-dry. The color intensity between the tape square obtained from the control enzyme and the tape square obtained from the protease conjugate is compared visually or by using a chromameter. Relative to the control enzyme tape square, a protease conjugate tape square showing less color intensity is indicative of a protease conjugate having higher activity. Dyed Collagen Activity Method

Combine 50 mL of 0.1 M tπs buffer (fris-hydroxymethyl-ammomethane) containing 0.01 M CaCl₂ to give pH 8.6, and 0.5 g azocoll (azo dye impregnated collagen, commercially available from Sigma Chemical Co., St. Louis, MO). Incubate this mixture at 25 °C while gently mixing with a platform shaker. Filter 2 mL of the mixture through a 0.2 micron syπnge filter and read absorbance of the mixture at 520 nm to zero a spectrophotometer. Add 1 ppm of a control enzyme (for example, subtilisin BPN') or the protease conjugate to be tested to the remaining 48 mL of tπs / azocoll mixture. Filter 2 mL of the control / protease conjugate containing solution through a 0.2 micron syπnge filter every two minutes for a total of ten minutes. For each filtered sample, read the absorbance immediately at 520 nm. Plot the results against time. The slopes of the control and the test conjugate are indicative of relative activities of the samples. A higher slope is indicative of a higher activity The test protease conjugate activity (slope) may be expressed as a percent of the control activity (slope).

Mouse Intranasal Test for Immunogenicity

The lmmunogenic potential of the protease conjugates of the present invention may be determined using a methods known m the art or by the Mouse Intranasal Test for Immunogenicity presented herein below. This test is similar to the assays descπbed in Robinson et al., "Specific Antibody Responses to Subtilisin Carlsberg (Alcalase) m Mice: Development of an Intranasal Exposure Model", Fundamental and Applied Toxicology, Vol. 34, pp. 15 - 24 (1996) and Robinson et al., "Use of the Mouse Intranasal Test (MINT) to Determine the Allergemc Potency of Detergent Enzymes: Compaπson to the Guinea Pig Intratracheal (GPIT) Test", Toxicological Science, Vol. 43, pp. 39 - 46 (1998), both of which assays may be utilized in place of the test set forth herein below.

Female BDF 1 mice (Charles River Laboratories, Portage, MI) weighing from about 18 to about 20 grams are utilized in the test. The mice are quarantined one week pπor to dosing The mice are housed in cages with wood chip bedding in rooms controlled for humidity (30 - 70%), temperature (67 - 77 °F) and 12 hour light and dark cycles. The mice are fed Puπna^® mouse chow (Punna Mills, Richmond, IN) and water ad libitum.

The potential antigen to be tested (either subtilisin BPN' as positive control or a protease con_jugate of the present invention) is dosed to a group of five mice. Pπor to dosmg, each mouse is anesthetized by an mfrapeπtoneal (i.p.) injection of a mixture of Ketaset (88.8 mg/kg) and Rompun (6.67 mg/kg). The anesthetized animal is held in the palm of the hand, back down, and dosed mfranasally with 5 μL protease in buffer solution (0.01 M KH₂P0₄, pH 5.5). While each group receives the same dosage, vanous dosages may be tested Dosmg solutions are gently placed on the outside of each nostril and inhaled by the mouse. Dosmg is repeated on days 3, 10, 17, and 24

Serum samples are collected on day 29. Enzyme-specific IgGl antibody in mouse serum is measured by an antigen capture ELISA method. Immunogenicities of the protease conjugate may be compared against those of subtilisin BPN' using standard ED₅₀ values.

Compositions of the Present Invention

The protease conjugates herein can be used m any application m which is suitable for the respective parent protease. One such example includes cleaning compositions. Because of the desirable reduced immunogenicity properties of the present protease conjugates, the protease conjugates may further be used m applications which have histoπcally minimally benefited from the use of proteases. Examples of such applications include those m which the protease conjugate necessaπly comes m close contact with mammalian skm (especially human sk ), such as with the use of personal care compositions.

Cleaning Compositions

The protease conjugates may be utilized in cleaning compositions including, but not limited to, laundry compositions, hard surface cleansmg compositions, light duty cleaning compositions including dish cleansing compositions, and automatic dishwasher detergent compositions.

The cleaning compositions herein compπse an effective amount of one or more protease con_jugates of the present invention and a cleaning composition earner.

As used herein, "effective amount of protease conjugate", or the like, refers to the quantity of protease conjugate necessary to achieve the proteolytic activity necessary m the specific cleaning composition Such effective amounts are readily ascertained by one of ordinary skill the art and is based on many factors, such as the particular protease conjugate used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e g , granular, bar) composition is required, and the like Preferably, the cleaning compositions compnse from about 0.0001% to about 10%, more preferably from about 0 001% to about 1%, and most preferably from about 0.01% to about 0.1% of one or more protease conjugates of the present invention. Several examples of vanous cleaning compositions wherein the protease conjugates may be employed are discussed m further detail below.

In addition to the present protease conjugates, the present cleaning compositions further compπse a cleaning composition earner compnsmg one or more cleaning composition mateπals compatible with the protease conjugate The term "cleaning composition matenal", as used herein, means any material selected for the particular type of cleaning composition desired and the form of the product (e g , liquid, granule, bar, spray, stick, paste, gel), which mateπals are also compatible with the protease conjugate used m the composition The specific selection of cleaning composition materials is readily made by consideπng the surface matenal to be cleaned, the desired form of the composition for the cleaning condition duπng use (e g , through the wash detergent use). The term "compatible", as used herein, means the cleaning composition matenals do not reduce the proteolytic activity of the protease conjugate to such an extent that the protease is not effective as desired during normal use situations Specific cleaning composition matenals are exemplified in detail hereinafter.

The protease conjugates of the present invention may be used in a variety of detergent compositions wherein high sudsmg and good cleansing is desired. Thus the protease conjugates can be used with vanous conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabπc laundeπng compositions, and the like Such compositions can be in the form of liquids, granules, bars, and the like Such compositions can be formulated as "concenfrated" detergents which contain as much as from about 30% to about 60% by weight of surfactants.

The cleaning compositions herein may optionally, and preferably, contain vanous surfactants (e g , amonic, noniomc, or zwittenonic surfactants) Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.

Nonhmitmg examples of surfactants useful herein include the conventional Ci i -Ci g alkyl benzene sulfonates and primary and random alkyl sulfates, the Ci o-Cjg secondary (2,3) alkyl sulfates of the formulas CH₃(CH₂)_x(CHOS03)-M⁺)CH3 and CH₃(CH₂)_y(CHOS03-M⁺) CH2CH3 wherein x and (y+1) are integers of at least about 7, preferably at least about 9, and M is a water-solubihzmg cation, especially sodium, the C_{j Q}-Ci g alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), Ci ø-Cig alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the Ci ø-Cjg alkyl polyglycosides, and their corresponding sulfated polyglycosides, C^-Cj α-sulfonated fatty acid esters, C^- _j alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), Ci^-C_jg betames and sulfobetames ("sultames"), CiQ-Cj am e oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. The use of such surfactants in combination with the amme oxide and / or betame or sultame surfactants is also preferred, depending on the desires of the formulator. Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C_jQ-C_jg N-methyl glucamides disclosed m U.S. Pat. No. 5, 194,639, Connor et al.. issued March 16, 1993

A wide vanety of other ingredients useful in detergent cleaning compositions can be included m the compositions herein including, for example, other active ingredients, earners, hydrofropes, processing aids, dyes or pigments, and solvents for liquid formulations. If an additional increment of sudsmg is desired, suds boosters such as the Ci _Q-C^g alkolamides can be incorporated into the compositions, typically at about 1% to about 10% levels. The CJQ-C^ monoethanol and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsmg adjunct surfactants such as the amme oxides, betames and sultames noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2,

MgSO and the like, can be added at levels of, typically, from about 0.1% to about 2%. to provide additional sudsmg.

The liquid detergent compositions herein may contain water and other solvents as earners. Low molecular weight pnmary or secondary alcohols exemplified by methanol, ethanol, propanol, and *so-propanol are suitable. Monohydnc alcohols are preferred for solubilizmg surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g , 1,3-propanedιol, ethylene glycol, glyceπne, and 1,2- propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.

The detergent compositions herein will preferably be formulated such that duπng use in aqueous cleaning operations, the wash water will have a pH between about 6 8 and about 11

Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of, for example, buffers, alkalis, and acids Such techniques are well known to those skilled m the art. When formulating the hard surface cleaning compositions and fabnc cleaning compositions of the present invention, the formulator may wish to employ vanous builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccmates, layered silicates, phosphates, and the like Other conventional builders are listed m standard formulaπes

Likewise, the formulator may wish to employ vanous additional enzymes, such as cellulases, lipases, amylases, and proteases in such compositions, typically at levels of from about 0.001% to about 1% by weight Vanous detersive and fabric care enzymes are well-known in the laundry detergent art.

Vanous bleaching compounds, such as the percarbonates, perborates and the like, can be used m such compositions, typically at levels from about 1% to about 15% by weight If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamme, nonanoyloxybenzene sulfonate, and the like, which are also known m the art. Usage levels typically range from about 1% to about 10% by weight.

Soil release agents, especially of the anionic o goester type, chelatmg agents, especially the aminophosphonates and ethylenediammedisuccmates, clay soil removal agents, especially ethoxylated tefraethylene pentamme, dispersing agents, especially polyacrylates and polyasparatates, bnghteners, especially anionic bπghteners, suds suppressors, especially sihcones and secondary alcohols, fabnc 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 formulanes and published patents contain multiple, detailed descπptions of such conventional matenals

Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about 1%)

The present variants are useful in 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 present invention compπse an effective amount of one or more protease conjugates of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 5%, and more preferably still from about 0.05% to about 1% by weight of protease conjugate of the composition In addition to compπsmg one or more of the protease conjugates, such hard surface cleaning compositions typically compπse a surfactant and a water-soluble sequestenng builder In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy and / or streaky residue on the glass surface.

The surfactant component, when present, may compπse as little as 0.1% of the compositions herem, but typically the compositions will contain from about 0.25% to about 10%. more preferably from about 1% to about 5% of surfactant.

Typically the compositions will contain from about 0.5% to about 50% of a detergency builder, preferably from about 1% to about 10%.

Preferably the pH should be m the range of about 7 to 12. Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate, or hydrochloπc acid can be used if adjustment is necessary.

Solvents may be included m the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-tπmethyl-l,3- pentanediol and 2-ethyl-l,3-hexanedιol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, more preferably from about 3% to about 11%.

Additionally, highly volatile solvents such as zso-propanol or ethanol can be used m the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not nnsed after "full strength" application of the composition to the surface When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions.

Examples 7 - 12 Liquid Hard Surface Cleaning Compositions

All formulas are adjusted to pH 7.

In another embodiment of the present invention, dishwashing compositions compnse one or more vanants of the present invention. As used herein, "dishwashing composition" refers to all forms of compositions for cleaning dishes including, but not limited to, granular and liquid forms.

Examples 13 - 16 Liquid Dish Detergent

All formulas are adjusted to pH 7. Examples 17 - 19 Liquid Fabnc Cleaning Compositions

Personal Care Compositions

The present protease conjugates are particularly suited for use in personal care compositions such as, for example, leave-on and πnse-off hair conditioners, shampoos, leave-on and πnse-off acne compositions, facial milks and conditioners, shower gels, soaps, foaming and non-foammg facial cleansers, cosmetics, hand, facial, and body lotions, moistuπzers, patches, and masks, leave-on facial moisturizers, cosmetic and cleansing wipes, oral care compositions, catamemals, and contact lens care compositions. The present personal care compositions comprise one or more protease conjugates of the present invention and a personal care earner.

To illustrate, the present protease conjugates are suitable for inclusion in the compositions descnbed in the following references: U.S. Pat. No. 5,641,479, Lmares et al . issued June 24, 1997 (skm cleansers), U.S. Pat. No. 5,599,549, Wivell et al., issued February 4, 1997 (skin cleansers); U.S. Pat. No. 5,585,104, Ha et al.. issued December 17, 1996 (skm cleansers); U.S. Pat. No. 5,540,852, Kefauver et al„ issued July 30, 1996 (skm cleansers), U.S Pat. No. 5,510,050, Dunbar et al.. issued April 23, 1996 (skm cleansers); U.S. Pat. No. 5,612,324, Guang Lin et al., issued March 18, 1997 (anti-acne preparations); U.S. Pat. No. 5,587,176, Warren et al., issued December 24, 1996 (anti-acne preparations); U.S Pat. No. 5,549,888, Venkateswaran, issued August 27, 1996 (anti-acne preparations); U.S. Pat. No. 5,470,884, Corless et al., issued November 28, 1995 (anti-acne preparations), U.S. Pat. No. 5,650,384, Gordon et al., issued July 22, 1997 (shower gels); U.S. Pat. No. 5,607,678, Moore et al.. issued March 4, 1997 (shower gels); U.S. Pat. No. 5,624,666, Coffmdaffer et al., issued Apnl 29, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,618,524, Bolich et al , issued Apπl 8, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,612,301, Inman, issued March 18, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,573,709, Wells, issued November 12, 1996 (hair conditioners and / or shampoos); U.S. Pat. No. 5,482,703, Pings, issued January 9, 1996 (hair conditioners and / or shampoos); U.S. Pat. No. Re. 34,584, Grote et al.. Reissued Apπl 12, 1994 (hair conditioners and / or shampoos); U.S. Pat. No. 5,641,493, Date et al., issued June 24, 1997 (cosmetics); U.S. Pat. No. 5,605,894, Blank et al.. issued February 25, 1997 (cosmetics), U.S. Pat. No. 5,585,090, Yoshioka et al.. issued December 17, 1996 (cosmetics); U.S. Pat. No. 4,939,179, Cheney et al.. issued July 3, 1990 (hand, face, and / or body lotions); U.S. Pat. No. 5,607,980, McAtee et al. issued March 4, 1997 (hand, face, and / or body lotions); U.S. Pat. No. 4,045,364, Richter et al., issued August 30, 1977 (cosmetic and cleansing wipes); European Patent Application, EP 0 619 074, Touchet et al., published October 12, 1994 (cosmetic and cleansing wipes); U.S. Pat. No. 4,975,217, Brown-Skrobot et al., issued December 4, 1990 (cosmetic and cleansing wipes); U.S. Pat. No. 5,096,700, Seibel, issued March 17, 1992 (oral cleaning compositions); U.S. Pat. No. 5,028,414, Sampathkumar, issued July 2, 1991 (oral cleaning compositions); U.S. Pat. No. 5,028,415, Benedict et al.. issued July 2, 1991 (oral cleaning compositions); U.S. Pat. No. 5,028,415, Benedict et al.. issued July 2, 1991 (oral cleaning compositions); U.S. Pat. No. 4,863,627, Davies et al.. September 5, 1989 (contact lens cleaning solutions); U.S. Pat. No. Re. 32,672, Huth et al, reissued May 24, 1988 (contact lens cleaning solutions); and U.S. Pat. No. 4,609,493, Schafer. issued September 2, 1986 (contact lens cleaning solutions).

To further illustrate oral cleaning compositions of the present invention, a pharmaceutically-acceptable amount of one or more protease conjugates of the present invention is included m compositions useful for removing protemaceous stams from teeth or dentures. As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental freatment solutions, and the like. Preferably, the oral cleaning compositions compnse from about 0.0001% to about 20% of one or more protease conjugates of the present invention, more preferably from about 0.001% to about 10%, more preferably still from about 0.01% to about 5%, by weight of the composition, and a pharmaceutically-acceptable earner. As used herein, "pharmaceutically-acceptable" means that drugs, medicaments, or inert ingredients which the term descnbes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, lrπtation, allergic response, and the like, commensurate with a reasonable benefit / nsk ratio. Typically, the pharmaceutically-acceptable oral cleaning earner components of the oral cleaning components of the oral cleaning compositions will generally compπse from about 50% to about 99.99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99%, by weight of the composition.

The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art. A wide vanety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in the references cited heremabove.

In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity compnse one or more protease conjugates of the present invention. Such denture cleaning compositions compnse an effective amount of one or more of the protease conjugates, preferably from about 0.0001% to about 50%, 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 earner. Vanous denture cleansing composition formats such as effervescent tablets and the like are well known m the art (see, e.g.. U.S. Pat. No. 5,055,305, Young), and are generally appropnate for incorporation of one or more of the protease conjugates for removing protemaceous stams from dentures.

In another embodiment of the present invention, contact lens cleaning compositions comprise one or more protease conjugates of the present invention. Such contact lens cleaning compositions compnse an effective amount of one or more of the protease conjugates, preferably from about 0.01% to about 50% of one or more of the protease conjugates, more preferably from about 0.01% to about 20%, more preferably still from about 1% to about 5%, by weight of the composition, and a contact lens cleaning earner. Vanous contact lens cleaning composition formats such as tablets, liquids, and the like are well known in the art and are generally appropπate for incorporation of one or more protease conjugates of the present invention for removing protemaceous stams from contact lens. Examples 20 - 23 Contact Lens Cleaning Solution

Examples 32 - 33 Leave-on Skin Moisturizing Composition

Example 34 Cleansing Wipe Composition

The above composition is impregnated onto a woven absorbent sheet comprised of cellulose and / or polyester at about 250%, by weight of the absorbent sheet.

Claims

What is claimed is:

1. A protease conjugate characteπzed in that said conjugate compπses a protease moiety and one or more addition moieties wherein the protease moiety compnses a first epitope region, a second epitope region, and a third epitope region, wherein each addition moiety is covalently attached to an epitope protection position of the protease moiety, wherein:

(a) the epitope protection positions for the first epitope region are selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215, and 216 corresponding to subtilisin BPN';

(b) the epitope protection positions for the second epitope region are selected from the group consisting of 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145 corresponding to subtilisin BPN'; and

(c) the epitope protection positions for the third epitope region are selected from the group consisting of 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275 corresponding to subtilisin BPN'.

2. A protease conjugate according to Claim 1 wherein each addition moiety, independently, has the structure:

wherein X is selected from the group consisting of ml and a Unking moiety; Ri is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R₂ is selected from the group consisting of nil, a second polypeptide, and a second polymer; wherein at least one of X, R and R₂ is not nil.

3. A protease conjugate according to Claim 2 wherein the protease moiety has a modified ammo acid sequence of a parent ammo acid sequence, wherein the modified ammo acid sequence compπses a substitution by a substituting ammo acid at one or more of the epitope protection positions and wherein each addition moiety is covalently attached to one of the substituting ammo acids.

4. A protease conjugate according to Claim 3 wherein the substituting ammo acid is cysteme.

5. A protease conjugate according to Claim 4 wherein the parent ammo acid sequence is selected from the group consisting of subtilisin BPN, subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, thermitase, Protease A, Protease B, Protease C, and Protease D, and vaπants thereof.

6. A protease conjugate according to Claim 5 wherein-

(a) the epitope protection positions for the first epitope region are selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86, 87, 89, 206, 209, 214, and 215 corresponding to subtilisin BPN';

(b) the epitope protection positions for the second epitope region are selected from the group consisting of 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138, and 141 corresponding to subtilisin BPN'; and

(c) the epitope protection positions for the third epitope region are selected from the group consisting of 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254, 265, and 275 corresponding to subtilisin BPN'.

7. A protease conjugate according to Claim 6 wherein R and R₂ are each nil.

8. A protease conjugate according to Claim 6 wherein Ri is the first polypeptide that is selected from the group consisting of subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, thermitase, Protease A, Protease B, Protease C, and Protease D, and vaπants thereof.

9. A protease conjugate according to Claim 8 wherein the first polypeptide is covalently attached to the linking moiety or the protease moiety at a position of the first polypeptide selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 17, 22, 23, 24, 25, 26, 27, 36, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 62, 63, 67, 86, 87, 89, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 143, 144, 145, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 206, 209, 210, 212, 213, 214, 215, 216 253, 254, 256, 265, 267, 269, 271, 272, and 275 corresponding to subtilisin BPN.

10. A protease conjugate according to Claim 9 wherein X is ml and wherein the protease moiety and the first polypeptide are covalently attached through a disulfide bπdge.

11. A protease conjugate according to Claim 6 wherein R₂ is nil and the first polymer is a polyethylene glycol.

12. A protease conjugate according to Claim 11 wherein at least one addition moiety is covalently attached to an epitope protection position for an epitope region selected from the group consisting of the first epitope region, the second region, and the third region

13. A cleaning composition compnsmg a protease conjugate according to Claim 1 and a cleaning composition earner.

14. A personal care composition compnsmg a protease conjugate according to Claim 1 and a personal care earner.