CN1372593A - 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 with sterically protected epitope regions

Field of Invention

The present invention relates to chemically modified subtilisins useful in compositions such as personal care compositions, laundry compositions, hard surface cleaning compositions and delicates cleaning compositions.

Background of the Invention

Enzymes are the largest class of naturally occurring proteins. One such class is proteases that catalyze the hydrolysis of other proteins. Typically, this ability to hydrolyze proteins has been exploited by the incorporation of naturally occurring and genetically engineered proteases into cleaning compositions, especially those for laundry use.

In the field of cleaning, serine proteases are most commonly used. Most serine proteases are produced by bacteria, while a small number of them are produced by other microorganisms, such as fungi. See, Siezen et al. "Homology modeling and protein engineering strategies for subtilisins, a family of subtilisin-like serine proteases", Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of wild-type proteases in their natural environment often cannot be optimized for use in the non-native environment of cleaning compositions. In particular, protease properties, such as thermostability, pH stability, oxidative stability, and substrate specificity, are not necessarily optimal for use outside of the protease's natural environment.

Several methods have been used to alter the wild-type amino acid sequence of serine proteases in order to enhance the efficacy of the proteases in non-natural wash environments. These methods include genetic remodeling and/or chemical modification of proteases to enhance the thermal and oxidative stability of proteases under widely varying conditions.

However, since such proteases are foreign to mammals, they are potential antigens. As antigens, these proteases elicit an immunogenic and/or allergenic response in the mammal (collectively referred to herein as an immunogenic response).

In addition, while significant efforts have been made in the ongoing research to develop more efficient proteases for laundry use through genetic remodeling and chemical modification, such proteases have not been used commercially in personal care compositions and delicates middle. The main reason proteases are not added to products such as soaps, gels, body washes, shampoos and delicates detergents is due to the aforementioned problem of sensitization in humans leading to adverse immunogenic responses. It would therefore be of great interest to provide a personal care composition or delicates detergent which possesses the cleaning performance of proteases while minimizing the eliciting of an immunogenic response.

Currently, immunogenic responses to proteases can be minimized by immobilizing, pelleting, coating or dissolving chemically modified proteases to prevent them from becoming airborne. These methods, while taking into account consumer exposure to airborne proteases, still present the risk of persistent tissue contact with the final composition and worker exposure to protease-containing dust or aerosols during the manufacturing process.

It has also been suggested that reducing the immunogenicity of proteases can be achieved by linking polymers to the proteases. See, for example, U.S. Patent 4,179,337 issued December 18, 1979 to Davis et al; U.S. Patent 5,856,451 issued January 5, 1999 and assigned to Novo Nordisk by Olsen et al; published January 7, 1999 and WO 99/00489, assigned to Novo Nordisk by Olsen et al.; WO 98/30682, published 16 July 1998 and assigned to Novo Nordisk by Olsen et al.; and WO 98/30682, published 13 August 1998, by Von Der Osten et al. 98/35026. However, these proposals do not demonstrate the importance of linking polymers to specific amino acid regions of the protease in order to most effectively reduce the immunogenic response.

It has recently been discovered that subtilisins contain three epitope regions and that one or more polymers, polypeptides, or other groups should be attached to one or more of these regions to achieve a significant reduction in the immunogenicity of the protease . See, eg, US Patent Application 09/088912, Weisgerber et al., assigned to Procter & Gamble, filed June 2, 1998.

The inventors have discovered that steric protection near one or more epitope regions of the protease is an alternative mechanism to prevent or impede presentation of epitopes and reduce the immunogenicity of the protease. Accordingly, the inventors provide chemically modified subtilisins, wherein said modification is at a region in spatial proximity to one or more epitope regions. Accordingly, the present inventors have discovered subtilisins capable of eliciting attenuated immunogenic responses yet retaining their activity as potent and active proteases. Accordingly, the protease conjugates described herein are suitable for use in several types of compositions including, but not limited to, laundry, dishwashing, hard surface cleaning, skin care, hair care, cosmetic, oral care, and contact lens cleaning. combination.

Summary of Invention

The present invention relates to protease conjugates comprising a protease moiety and one or more additional moieties, wherein each additional moiety is covalently linked to an epitope protected site of said protease moiety, wherein:

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

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

其中所述X不存在或为一连接部分；R₁不存在或选自第一多肽和第一聚合物；R₂不存在或选自第二多肽和第二聚合物；其中所述的X、R₁和R₂中至少有一个不存在。(c) the epitope protection positions in the third epitope region are selected from the group consisting of 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157 corresponding to subtilisin BPN', 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275; and wherein each additional moiety independently has the following structure:

Wherein said X does not exist or is a connecting part; _R does not exist or is selected from the first polypeptide and the first polymer; _R does not exist or is selected from the second polypeptide and the second polymer; wherein said At least one of X, _R1 and _R2 is absent.

The protease conjugates of the invention have reduced 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 compositions, dishwashing, hard surface cleaning, skin care, hair care, cosmetic, oral care, and contact lens cleaning compositions.

Detailed description of the invention

The main components of the present invention are described below. Also included are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.

The present invention may include, or consist of, or consist essentially of, any of the essential or optional components and/or limitations described herein.

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

All component or composition levels are referenced to the active content of that component or composition and exclude impurities such as residual solvents or by-products that may be present in commercially sourced products.

All documents, including all patents, patent applications, and printed publications, mentioned in this specification are hereby incorporated by reference in their entirety.

Materials used in the present invention under trade names include, but are not limited to, enzymes. The inventors do not intend to be limited by a certain trade name material in the present invention. Equivalents of a trade name material (eg, material obtained from a different source with a different name or catalog (reference) number) may be substituted and used in the protease conjugates and compositions described herein.

The present invention uses abbreviations to describe amino acids. A series of abbreviations used in the present invention are provided in Table 1:

Table I

Amino Acids Three-letter abbreviations Single-letter abbreviations

Alanine Ala A

Arginine Arg R

Asparagine Asn Asn N

Aspartic Acid Asp D

Cysteine Cys C

Glutamine Gln Q

Glutamic Acid Glu E

Glycine Gly G

Histidine His H

Isoleucine Ile

Leucine Leu L

Lysine K

Methionine M

Phenylalanine Phe F

Proline P

Serine S

Threonine T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

Definition

The term "mutation" used in the present invention refers to a change in a gene sequence and/or an amino acid sequence resulting from the gene sequence. Mutations include deletions, substitutions and additions of amino acid residues in the wild-type protein sequence.

The term "parent" as used herein refers to a protein (wild type or variant) that can be used for further modification to form a protease conjugate of the invention.

The term "wild-type" as used herein refers to a protein, such as a protease or other enzyme, produced by a naturally occurring organism.

The term "variant" as used herein refers to a protein whose amino acid sequence differs from that of the corresponding wild-type protein.

All polymer molecular weights used in the present invention are expressed in terms of weight average molecular weight.

Although the conjugates described in the present invention are not limited to those containing subtilisin BPN' and its variants, all amino acid numbering refers to the amino acid sequence of subtilisin BPN' represented by SEQ ID NO: 1 . The amino acid sequence of subtilisin BPN' is further described in Wells et al., Nucleic Acids Res., Vol. 11, pp. 7911-7925 (1983).

      Protease conjugates of the present invention

A protease conjugate of the invention is a compound comprising a protease moiety and one or more additional moieties, wherein the protease moiety and the additional moiety are joined by a covalent linkage (ie, a covalent bond). protease part

The protease moiety of the present invention is a subtilisin-like protease, wild type or a variant thereof. The term "subtilisin-like protease" as used in the present invention refers to a protease having at least 50%, preferably 80%, amino acid sequence homology to the sequence of subtilisin BPN'. Wild-type subtilisin-like proteases are produced, for example, by microorganisms of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus, and Bacillus subtilis. A discussion of subtilisin-like serine proteases and their homology can be found in Siezen et al., "Homology modeling and protein engineering strategies for subtilisin-like serine proteases, the subtilisin-like serine protease family", Protein Engineering, Vol.4, No. 7, pp. 719-737 (1991).

Preferred protease moieties for use in the present invention include, for example, proteases obtained from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis, subtilisin BPN, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K and thermase, including A/S ^Alcalase® (available from Novo Industries, Copenhagen, Denmark), ^Esperase® (Novo Industries), Savinase ) ^® (Novo Industries), ^Maxatase® (commercially available from Genencor International Inc.), ^Maxacal® (Genencor International Inc.), Maxapem ^15® (Genencor International Inc.), and variants of the above enzymes. Particularly preferred protease moieties for use in the present invention include proteases obtained from Bacillus amyloliquefaciens and variants thereof. The most preferred protease moiety in the present invention is subtilisin BPN' and variants thereof.

A particularly preferred subtilisin BPN' variant for use in the present invention, hereinafter referred to as "Protease A", is disclosed in U.S. Patent 5,030,378, issued to Venegas on July 9, 1991, and is characterized in that The amino acid sequence of protease BPN' has the following mutations:

(a) Gly at position 166 is substituted by an amino acid residue selected from: Asn, Ser,

Lys, Arg, His, Gln, Ala, and Glu; Gly at position 169 is replaced by Ser

Substitution; and Met at position 222 is substituted by an amino acid residue selected from the group consisting of:

Gln, Phe, His, Asn, Glu, Ala, and Thr; or

b) Gly at position 160 is replaced by Ala, and Met at position 222 is replaced by Ala.

Another preferred subtilisin BPN' variant, hereinafter referred to as "Protease B", for use as a parent amino acid sequence in the present invention is disclosed in EP-B251, assigned to Genencor International Corporation published on January 7, 1988 ,446, the above variant is characterized by mutations in the amino acid sequence of wild-type subtilisin BPN' at one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67 , Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Met124, Gly127, Gly128, Pro129, Leu 135, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Ser251, Lys2114, and Tyr2 or at two or more of the above positions combined with one or more mutations selected from the following positions: Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222 .

Other preferred variants of subtilisin BPN' for use in the present invention, hereinafter referred to as "Protease C", are described in WO 95/10615, published April 20, 1995, assigned to Genencor International, Inc. Characterized in that the amino acid sequence of wild-type subtilisin BPN' has a mutation at the Asn76 position combined with one or more mutations selected from the following positions: Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glul95, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265 and Ala274.

Other preferred subtilisin BPN' variants for use in the present invention, hereinafter referred to as "Protease D", are described in U.S. Patent 4,760,025, issued to Estell et al. on July 26, 1988, and are characterized in The wild-type subtilisin BPN' amino acid sequence has one or more mutations selected from the following amino acid positions: Asp32, Ser33, His64, Tyr104, Asn155, Glul56, Gly166, Gly169, Phe189, Tyr217 and Met222.

More preferred protease moieties of the present invention are selected from the group consisting of subtilisin BPN', Protease A, Protease B, Protease C, and Protease D, with Protease D being most preferred.

Without being bound by theory, the protease moiety of the present invention has 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 Corresponds to positions 220-246 of subtilisin BPN'. See, eg, U.S. Patent Application 09/088,912, filed June 2, 1998, by Weisgerber et al., and assigned to Procter &Gamble; co-pending U.S. Patent Application 60/144991, filed July 22, 1999, by Rubingh et al. "Serine protease variants having amino acid substitutions and deletions in the epitope region"; and co-pending U.S. Patent Application 60/144980, "Serine having amino acid substitutions in the epitope region" filed July 22, 1999 by Sikorski et al. protease variants".

The inventors have surprisingly found epitope protection positions in spatial proximity to at least one of the above-mentioned epitope regions. It has also been found that by covalently linking one or more additional moieties to amino acids of the protease moiety at epitope protection positions these epitopes are protected from hydrolysis and thus from exposure of the epitopes.

The epitope protection positions suitable for covalent modification with additional moieties depend on the epitope one desires to protect. More preferably, at least one additional moiety is covalently linked to an epitope protecting position in the first epitope region.

The epitope protection positions in the first epitope region have been found to be 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. More preferably, the epitope protection positions in 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 found that the epitope protection positions in the second epitope region are 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99 corresponding to subtilisin BPN' , 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145. Preferably, the epitope protection positions in the second epitope region are 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138, and 141.

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

In a preferred embodiment of the invention, the protease moiety comprises a modified sequence of the parent amino acid sequence. The parent amino acid sequence can be any of the proteases described above, with the same preferred constraints as described above. In this embodiment, the parental amino acid sequence is substituted at one or more parental amino acid residues with substituting amino acids to generate a protease moiety suitable for attachment to one or more additional moieties of the invention. According to the invention, the substitutions should be made at one or more epitope protected positions. Epitope protection positions, and their preferred constraints, are described above.

To best achieve selective linkage of one or more additional moieties to the protease moiety at one or more epitope protected positions, the substitutions should be made with (parental amino acid sequence unique) to substitute amino acids. In this regard, any substituted amino acid unique to the parent amino acid sequence may be used. For example, since cysteine residues are not present in the wild-type amino acid sequence of subtilisin BPN', one or more cysteine residues are used at one or more epitope protection positions of subtilisin BPN' Substitution of amino acid residues is suitable for the present invention. Where the cysteine residue exists at a position other than the epitope protection position of the parental amino acid sequence, it is preferably substituted with another amino acid residue at each of the above positions to ensure that the epitope protection position is compatible with one or multiple additional moieties are selectively coupled. Cysteine is the most preferred substituting amino acid for substitution at one or more epitope protected positions.

Other preferred substituting amino acids include lysine. Where the substituting amino acid is lysine, it is preferred to mutate a lysine residue present at a position other than an epitope-protected position in the parent amino acid sequence to another amino acid residue such that the epitope-protected position The functionalization of one or more lysine residues on is selective. For example, a lysine residue is present at position 170 of subtilisin BPN', which is an epitope protected position as defined in the present invention. Site-selective mutagenesis of all other lysine residues occurring in the subtilisin BPN' sequence can be performed, followed by selective functionalization of the lysine residue at position 170 with an additional moiety. In addition, amino acid residues at any of the epitope protection positions can be mutated to lysine (for example), followed by selective functionalization at those positions with additional moieties. additional part

The protease conjugates of the present invention comprise one or more additional moieties, wherein each of said additional moieties is covalently linked to an amino acid residue at an epitope protected position as described above. The additional moiety can be of any chemical structure. Preferably, the additional moiety sterically blocks the epitope protection site to which it is linked, or any other epitope protection site as defined herein. Non-limiting examples of additional moieties include, but are 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. The term "polypeptide" as used in the present invention refers to a molecule comprising two or more amino acid residues. The term "polymer" as used in the present invention refers to any molecule comprising two or more identical (preferably 5 or more identical) monomeric units.

Preferably, the additional part has the following structure: Wherein said X does not exist or is a connecting part; R ₁ does not exist or is selected from the first polypeptide and the first polymer; R ₂ does not exist or is selected from the second polypeptide and the second polymer; wherein X, At least one of R ₁ and R ₂ is absent.

Preferably, 1 to about 15, more preferably about 2 to about 10, most preferably about 1 to about 5 additional moieties are included in the protease conjugate.

Wherein said R ₁ and R ₂ are each independently a polypeptide moiety or a polymer moiety, R ₁ and R ₂ may be the same or different. Preferably, where said R ₁ is a polypeptide moiety, R ₂ is absent or is a polypeptide moiety, and most preferably absent. Most preferably, where R ₁ is a polypeptide moiety, R ₂ is absent or is the same polypeptide moiety, and most preferably absent. Preferably, when said R ₁ is a polymer moiety, R ₂ does not exist or is a polymer moiety. Most preferably, where said R ₁ is a polymer moiety, R ₂ is absent or is the same polymer moiety. Wherein at least one of R ₁ and R ₂ is the first polymer and the second polymer respectively, X is preferably absent. Polypeptide part

The polypeptide portion of the present invention includes a polypeptide portion comprising 2 or more amino acid residues. Preferred polypeptide moieties are selected from proteins including enzymes. Preferred enzymes include proteases, cellulases, lipases, amylases, peroxidases, microperoxidases, hemicellulases, xylanases, phospholipases, esterases, cutinases, pectinases, Keratinases, reductases (including, for example, NADH reductase), oxidases, phenoloxidases, lipoxygenases, lignases, pullulanases, tannases, pentosan polysulfatases, malanases, beta - glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, transferases, isomerases (including, for example, glucose isomerase and xylose isomerase), lyases, Ligase, synthetase, and enzymes of fruit origin (including, eg, papain). More preferred enzymes for use as part of the polypeptide include proteases, cellulases, amylases, lipases and enzymes of fruit origin, with proteases being more preferred.

Examples of lipases useful as polypeptide moieties include lipases from the following microorganisms: Humicola, Pseudomonas, Fusarium, Mucor, Chromobacter, Aspergillus, Candida, Geothrix Genus, Penicillium, Rhizopus and Bacillus.

Examples of commercially available lipases include ^Lipolase® , Lipolase ^Ultra® , ^Lipozyme® , ^Palatase® , Novozym ^435® , and ^Lecitase® (all commercially available from Novo Nordisk A/S, Copenhagen, Denmark), ^Lumafast® (available from Genencor, Int., Rochester, NY), and ^Lipomax® (Genencor, Int.).

Examples of proteases useful as moieties of polypeptides include serine proteases, chymotrypsin, and elastase-type enzymes. Most preferred proteases for use as polypeptide moieties include serine proteases, as defined above in the discussion of "protease moieties" herein.

Most preferably, when said polypeptide part is a serine protease, said polypeptide part independently comprises the definition of protease part mentioned above in the present invention. Preferably, as mentioned above, the amino acid sequence of the polypeptide part is a modified sequence of the parent amino acid sequence, wherein the modification is at one or more epitope protection positions as described above (the parent amino acid sequence can be referred to as "second" parental amino acid sequence). In this case, one of said linking moiety (wherein said protease moiety is absent) or protease moiety (wherein said linking moiety is absent) is protected by an epitope present at 1 of the polypeptide moieties. One substituted amino acid is covalently linked to the polypeptide moiety. Where said polypeptide moiety is a serine protease, equally preferred, more preferred, and most preferred groups of epitope protection positions are as described above for the protease moiety and their corresponding parent amino acid sequences.

Most preferably, where said polypeptide moiety is a serine protease, said polypeptide moiety and said protease moiety are equivalent moieties. In this case, said polypeptide moiety and said protease moiety are preferably linked by a disulfide bridge, wherein X is absent, and most preferably, _R2 is absent. polymer part

The additional moieties of the present invention may comprise polymeric moieties. The term "polymer moiety" as used in the present invention refers to any molecule comprising two or more identical (preferably 5 or more identical) monomeric units. Examples of suitable polymeric moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarbonates, polyvinylpyrrolidone, polyamino acids, cellulose, dextran, starch, glycogen, agarose, guacamole, Gum, pullulan, inulin, xanthan gum, carrageenan, pectin, biopolymers, hydrolysates of alginic acid and hydrolysates of chitosan. Preferred polyalkylene oxides include polyethylene glycol, methoxypolyethylene glycol and polypropylene glycol. Preferred dextrans include carboxymethyl dextran. Preferred celluloses include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose. Preferred starches include hydroxyethyl starch and hydroxypropyl starch. More preferred polymers are polyalkylene oxides. The most preferred polymer moiety is polyethylene glycol.

Where R ₁ and R ₂ are each independently a polymer moiety, preferably, the combined molecular weight of R ₁ and R ₂ (i.e., the molecular weight of R ₁ plus the molecular weight of R ₂ ) is from about 0.2 kD (kilodaltons) to About 40 kD, more preferably about 0.5 kD to about 40 kD, more preferably about 0.5 kD to about 20 kD, and most preferably about 1 kD to about 10 kD.

Where R _and R are _each a polymer moiety, preferably, R and _R each independently have a molecular weight of 0.1 kD to _about 20 kD, more preferably a molecular weight of 0.25 kD to about 20 kD, more preferably about 0.5 kD ~ about 10 kD, and most preferably about 0.5 kD - 5 kD.

Where R _and R are _each a polymer moiety, preferably, the molecular weight ratio of R and _R is from about 1:10 to about 10:1, more preferably from _about 1:5 to about 5:1, and most preferably Preferably it is about 1:3 to about 3:1.

wherein _R is a polymer moiety and _R is absent, preferably _R has a molecular weight of from about 0.1 kD to about 40 kD, more preferably from about 0.5 kD to about 40 kD, more preferably from about 0.5 kD to about 20 kD, And most preferably from about 1 kD to about 10 kD. connecting part

X used in the present invention may be absent or may be a linking moiety which is covalently linked to one or more polypeptide moieties or one or more polymer moieties or both, and at the same time is protected with an epitope of the protease moiety. The amino acid residues at the positions are covalently linked. The linking moiety may generally be any small molecule, ie, a molecule having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, more preferably less than about 300. Most preferred linking moieties include those covalently bondable to cysteine residues or lysine residues, most preferably cysteine residues.

Examples of linking moieties and related chemistry are disclosed in US Patent 5,446,090 issued August 29, 1995 to Harris; US Patent 55,171,264 issued December 15, 1992 to Merrill; issued November 10, 1992 to Rhee et al. US Patent 5,162,430 issued to Shadle et al. on October 6, 1992; US Patent 5,122,614 issued to Zalipsky on June 16, 1992; Goodson et al., "Recombinant Interleukin-2 in its Sugar Site-directed PEGylation at sylation sites", Biotechnology, Vol.8, No.4, pp.343-346 (1990); Kogan, "Substituted methoxy- Synthesis of Poly(ethylene glycol) Derivatives", Synthetic Communications, Vol. 22, pp. 2417-2424 (1992); and Ishii et al., "Succinimide Ring State Effects on Pyrenemaleimide-labeled αα - Influence of fluorescence and structural properties of tropomyosin", Journal of Biophysics, Vol.50, pp.75-80 (1986). Most preferred linking moieties are substituted (eg, alkyl) or unsubstituted succinimides.

As additional examples, the following non-limiting reagents can be used to form the linking moiety: N-[α-maleimidoacetoxy]succinimidyl ester; N-5-azido-2 -Nitrobenzoyl succinimide; bismaleimidohexane; N-[β-maleimidopropionyl]succinimide ester; bis[2-(succinimide Aminooxycarbonyloxy)-ethyl]sulfone; bis[sulfosuccinimidyl]suberate; 1,5-difluoro-2,4-dinitrobenzene; dimethyladipyl Dimethylpimelimidate·2HCl; Dimethylpimelimidate·2HCl; Dimethylsuberimidate·2HCl; Glutaric Acid Disuccinimidyl Ester; Suberic Acid Disuccinimidyl Amino ester; m-maleimidobenzoyl-N-hydroxysuccinimidyl ester; N-hydroxysuccinimidyl-4-azidosalicylic acid; N-succinimidyl- 6-[4'-Azido-2'nitroanilino] ester; N-hydroxysuccinimide 2,3-dibromopropionate; 4-[maleimidomethyl] ring Hexane-1-carboxylic acid succinimidyl ester; 4-(p-maleimidotolyl)butyric acid succinimidyl ester; succinimidyl-6-[(β-maleyl iminopropionylamino)hexanoate]; bis[2-(sulfosuccinimidyloxycarbonyloxy)-ethyl]sulfone; N-[γ-maleimidobutyrate) ]sulfosuccinimidyl ester; N-hydroxysulfosuccinimidyl-4-azidobenzoate; N-[κ-maleimidoundecanoyl]sulfosuccinate Imide Esters; m-Maleimidobenzoyl-N-Hydroxysulfosuccinimidyl Ester; Sulfosuccinimidyl [4-Azidosalicylamino]hexanoate; Sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate; Sulfosuccinimidyl-6-[4'-azido-2'-nitrobenzene sulfosuccinimidyl-4-[p-azidophenyl]butyrate; sulfosuccinimidyl[4-sulfoacetyl]aminobenzoate; sulfo Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate; and sulfosuccinimidyl-4-[p-maleimido phenyl]-butyrate. Each of these reagents was purchased from Pierce Chemical Company, Rockford, IL.

optional part

The protease conjugates of the present invention may additionally comprise one or more other chemical structures, including, for example, with other residues of proteases not exemplified in the present invention or even in the presence of epitope protection without additional moieties. Positionally linked one or more small molecules, polypeptides and/or polymers (referred to herein as "complementary moieties"). Supplemental moieties may include polypeptide moieties, polymer moieties, and linking moieties as described above. In addition, for example, a molecular weight of about 100 Da to about 5000 Da, preferably about 100 Da to about 2000 Da, more preferably about 100 Da to about 1000 Da, more preferably about 100 Da to about 750 Da, and most preferably about 300 Da One or more polymers, most preferably polyethylene glycol, may be covalently attached to residues of the protease moiety of the invention other than those enumerated herein. Such polymeric moieties may be linked directly to the protease moieties of the invention at any position on the protease moiety using techniques described herein and well known in the art, including via linking moieties as described herein. . A non-limiting example of this optional type of polymer conjugate is described in WO 99/00849, published January 7, 1999 (Olsen et al., Novo Nordisk A/S).

Preparation

Said protease moiety having substitutions at one or more epitope protection positions (or any other position of the protease moiety) is prepared by mutating the nucleotide sequence encoding the parental amino acid sequence. Such methods are well known in the art; a non-limiting example of one such method is set forth below:

Genes containing wild-type subtilisin BPN' (Mitchison, C. and J.A. Wells, "Protein Engineering of Disulfide Bonds in Subtilisin BPN'", Biochemistry, Vol.28, pp.4807-4815 (1989)) phagemid (pSS-5) transformed into E. coli dut-ung-strain CJ236, and to Yuckenberg et al., "In vitro site-directed mutagenesis using uracil-containing DNA and phagemid vectors", Site-directed mutagenesis-a A practical approach, McPherson, M.J. eds., pp.27-48 (1991) modified the method described in the article, using VCSM13 helper phage (Kunkel et al., "Rapid and efficient site-directed mutagenesis without phenotypic selection" , Enzymatic Methods, Vol 154, pp.367-382 (1987)) to prepare a uracil-containing single-stranded DNA template. From Zoller, M.J. and M.Smith, "Oligonucleotide site-directed mutagenesis with M13-derived vectors: an efficient and general method for generating point mutations in any DNA segment", Nucleic Acids Research, Vol.10, Primer-directed mutagenesis modified from the method disclosed in pp. 6487-6500 (1982) was used to generate all mutants (essentially as Yuckenberg et al., supra).

Oligonucleotides were prepared using a 380B DNA synthesizer (Applied Biosystems Inc.). The mutagenesis reaction product was transformed into E. coli MM294 strain (American Type Culture Collection E. coli 33625). All mutations were confirmed by DNA sequencing and the isolated DNA was transformed into the Bacillus subtilis expression strain PG632 (Saunders et al., "Optimization of the signal sequence cleavage site for the secretion of a 34-amino acid fragment of human parathyroid hormone from Bacillus subtilis", pp. Gene, Vol.102, pp.277-282 (1991) and Yang et al., "The cloning of the Bacillus subtilisin gene and the application of the cloned gene in the establishment of in vitro-derived deletion mutations", Journal of Bacteriology, Vol. 160, pp. 15-21 (1984).

Fermentation was carried out as follows. Bacillus subtilis cells (PG632) containing the protease of interest were cultivated to the mid-log phase with one liter of LB broth containing 10 g/L glucose, and inoculated into a Biostat C fermenter (Braun Biotech, Inc., Allentown) with a total volume of 9 liters. , PA). The fermentation medium contains yeast extract, casein hydrolyzate, water-soluble partially hydrolyzed starch (Maltrin M-250), antifoaming agents, buffers, and trace minerals (see "Bacillus Biology: Industrial Applications", pp. Doi, RH and M. McGloughlin, eds. (1992)). The pH of the broth was kept constant at 7.5 during the fermentation. Add kanamycin (500 μg/mL) for antibiotic selection of mutagenized plasmids. The cells were incubated at 37°C for 18 hours until the _A600 was about 60, and the product was harvested.

Pure protease was obtained by processing the fermentation broth as follows. Bacillus subtilis cells in the broth were filtered by tangential flow with a 0.16 μm filter. The cell-free broth was then concentrated by ultrafiltration through an 8,000 molecular weight cut-off filter. The pH was adjusted to 5.5 with concentrated MES buffer (2-(N-morpholino)ethanesulfonic acid). The protease was further purified by cation exchange chromatography on S-Sepharose and eluted with a NaCl gradient. See Scopes, R.K. "Principles and Operations of Protein Purification", Springer-Verlag, New York (1984)

Active protease concentrations in fractions collected during gradient elution were determined by the pNA assay (DelMar et al., Analytical Chemistry, Vol. 99, pp. 316-320 (1979)). This assay measures the release of p-nitroaniline when the protease hydrolyzes the soluble synthetic substrate succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (sAAPF-pNA). rate. The rate at which the yellow color is produced by the hydrolysis reaction is measured spectrophotometrically at 410 nm and is proportional to the concentration of the active protease moiety. In addition, total protein concentration was determined using absorbance measurements at 280 nm. The ratio of active protease/total protein gives protease purity and can be used to identify fractions in the collected stock solution.

To avoid self-dissolution of the protease during storage, an equal weight of propylene glycol was added to the pooled fractions obtained from the chromatography column. After the purification process was completed, the purity of the protease stock solution was checked by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and type II trypsin inhibitor-T: turkey egg white (Sigma Chemical Company, St. Louis, Missouri) to determine absolute enzyme concentrations by active site titration.

In preparation for use, the protease stock solution was eluted through a Sephadex-G25 (Pharmacia, Piscataway, NeW Jersey) size exclusion column to remove propylene glycol, and the buffer was exchanged. The MES buffer in the enzyme stock solution was replaced with 0.01M KH ₂ PO ₄ solution, pH 5.5.

Protease conjugates can be produced by functionalizing the prepared protease with one or more additional moieties. Preferably, the add-on moiety precursor is activated to enhance its reactivity with the protease moiety precursor (reaction of the add-on moiety precursor with the protease moiety precursor to form a protease conjugate consisting of the add-on moiety and the protease moiety). Methods of such activation are well known in the art. Non-limiting examples of methods for the preparation of protease conjugates are given below.

Example 1

According to the protocol above, a protease containing a cysteine residue at an epitope protected position is coupled to a polymer moiety (where "P" is minus the sulfhydryl group generated by cysteine substitution) using the following method: The protease moiety, n is the number of polyethylene glycol monomer repeating units (eg, n=77)).

Subtilisin BPN' variants were prepared by substituting leucine for tyrosine at position 217 and cysteine for serine at position 161. The concentration of this variant reaches about 2 mg/mL in phosphate buffer (pH 5.5). Then, the pH was raised to 7.5 with dilute sodium hydroxide solution. The above variants were mixed with monomethyl polyethylene glycol maleimide at a ratio of activated polymer to variant of 25:1. After mixing for 1 hour at ambient temperature, the pH of the mixture was adjusted to 5.5 with dilute phosphoric acid and excess polymer was removed by filtration with a molecular weight cutoff ultrafilter. The concentrate contains the purified protease conjugate.

Example 2

According to the above protocol, a protease moiety containing a cysteine residue at an epitope protected position is coupled to a polymer moiety (where "P" minus the resulting cysteine substitution) using the following method: The protease moiety of the sulfhydryl group, n is a sufficient number of repeating units of polyethylene glycol monomers (eg n=77)).

Subtilisin BPN' variants were prepared by substituting leucine for tyrosine at position 217 and cysteine for phenylalanine at position 261. The variant reaches a concentration of approximately 2 mg/mL in phosphate buffer (pH 5.5). Then, the pH was raised to 7.5 with dilute sodium hydroxide solution. The above variants were mixed with dimethyl polyethylene glycol maleimide at a ratio of activated polymer to variant of 25:1. After mixing for 1 hour at ambient temperature, the pH of the mixture was adjusted to 5.5 with dilute phosphoric acid and excess polymer was removed by filtration with a molecular weight cutoff ultrafilter. The concentrate contains the purified protease conjugate.

Example 3

Selective coupling of succinimide-protected polymers to lysines at one or more epitope-protected positions (where "MPEG" and "PEGM" are equivalent, representing monomethylpolyethylene alcohol, where "P" represents the protease moiety minus the lysine amine group shown):

Example 4

Selective coupling of carbodiimide-protected polymers to lysines at one or more epitope-protected positions (where "MPEG" and "PEGM" are equivalent, representing monomethylpolyethylene alcohol, "P" represents the protease moiety minus the illustrated lysine amine group, and X and X' are side chains containing carbodiimide moieties, such as alkyl):

Example 5

Protease moieties containing cysteine residues at one of the epitope protected positions were coupled to alkylmaleimides as follows (where "P" represents minus the sulfhydryl group generated by cysteine substitution protease moiety, "R" is an alkyl group). In this embodiment, each of _R1 and _R2 is absent, and the linking moiety is derived from an alkylmaleimide.

Subtilisin BPN' variants were prepared by substituting leucine for tyrosine at position 217 and cysteine for serine at position 163. 20 mL of a solution of the above variant at _a concentration of approximately 1 mg/mL was prepared in 0.01 M _KH2PO4 buffer (pH 7). To this solution was added 1.5 equivalents of alkylmaleimide. The solution was mixed gently for 1 hour at ambient temperature. The resulting protease conjugate is obtained from the above solution by standard methods.

Example 6

Protease moieties containing cysteine residues at 1 epitope protection position were dimerized by the method described below (where "P" represents the protease moiety minus the sulfhydryl group generated by cysteine substitution). In this example, the protease moiety and the polypeptide moiety are equivalent (and X is absent).

Subtilisin BPN' variants were prepared by substituting leucine for tyrosine at position 217 and cysteine for serine at position 163. 20 mL of a solution of the above variant at a concentration of approximately 1 mg/mL was prepared in 0.01 M KH ₂ PO ₄ buffer (pH 8.6). Oxygen was gently bubbled through the solution for about 1 hour at ambient temperature to generate the desired protease conjugate dimer. The resulting protease conjugate is obtained from the above solution by standard methods.

Analytical method

The enzymatic activity and immunogenic response of the protease conjugates of the invention can be tested by the following methods, both of which are known to those skilled in the art. Other methods well known in the art may be used instead.

Protease Conjugate Activity

The protease activity of the protease conjugates of the present invention can be determined by methods well known in the art. Two such methods are described below: Skin Debris Active Method

Using a Scotch(R) #3750G tape, scraps of human skin were repeatedly stripped from the subject's legs until the tape was substantially covered with debris. Then, cut the strips into 1-inch squares and set aside. In a 10 mm x 35 mm Petri dish, 2 mL of 0.75 mg/mL of the control enzyme (eg, subtilisin BPN') or the protease conjugate to be tested is added to _0.01 M _KH2PO4 pH 5.5 buffer. To this solution was added 1 mL of 2.5% sodium laurate pH 8.6 solution. The solution was mixed gently on a platform shaker. The strip squares prepared above were soaked in the solution (crumb side up) with constant gentle mixing for 10 minutes. Then, gently rinse the squares with tap water for 15 seconds. Pipette Stevenel blue stain (3 mL, purchased from Sigma Chemical Co., St. Louis, MO) into a clean Petri dish. Place the rinsed strip squares in the stain solution (crumb side up) for 3 minutes with gentle mixing. Remove the strip squares from the dye solution and rinse them consecutively in two beakers filled with 300 mL of distilled water for 15 s each. Air dry the strip of squares. The difference in color intensity between the band squares obtained from the control enzyme and the band squares obtained from the protease conjugate was compared visually or with a colorimeter. The color intensity of the protease conjugate squares is less intense compared to the control enzyme squares, indicating higher activity of the protease conjugate. Pigmented Collagen Activity Method

50 m of 0.1 M tris buffer (tris-hydroxymethyl-aminomethane) (pH 8.6) containing 0.01 M CaCl ₂ and 0.5 g of azocoll (collagen impregnated with azo pigments, purchased from Sigma Chemical Co., St. Louis, MO) mixed. The mixture was incubated at 25°C with gentle mixing on a plate shaker. Filter 2 mL of the mixture through a 0.2 micron syringe filter, read the absorbance of the mixture at 520 nm and adjust the regulator of the spectrophotometer to zero. To the remaining 48 ml of tris/azocoll mixture was added 1 ppm of the control enzyme (eg, subtilisin BPN') or the protease conjugate to be tested. For a total of 10 minutes, filter 2 mL of the solution containing the control/protease conjugate every 2 minutes with a 0.2 micron syringe filter. Absorbance at 520 nm was read immediately for each filtered sample. Plot the results versus time. The slopes for the control and test conjugates indicate the relative activity of the samples. A larger slope indicates higher activity. The activity (slope) of the protease conjugate tested can be expressed as a percentage of the activity (slope) of the control.

Mice Intranasal Assay for Determination of Immunogenicity

The immunogenic potential of the protease conjugates of the invention can be determined by methods known in the art or by the mouse intranasal assay described below for determining immunogenicity. This assay is similar to Robinson et al., "Specific antibody response to subtilisin Carlsberg (Alcalase) in mice: development of an intranasal exposure model", Fundamental and Applied Toxicology, Vol. 34 , pp. 15-24 (1996) and Robinson et al., "Determination of allergenic potency of detergent enzymes using the mouse intranasal test (MINT): comparison with the guinea pig intratracheal (GPIT) test", Toxicological Sciences, Vol. .43, the assays described on pages 39-46 (1998), these two assays may be used in place of the tests described below.

BDF1 female mice (Charles River Laboratories, Portage, MI) weighing approximately 18 to approximately 20 grams were used in this experiment. The mice were isolated for one week prior to dosing. The mice were housed in woodchip bedding cages in a humidity-controlled (30-70%) and temperature (67-77°F) room that underwent a 12-hour light-dark cycle . The mice received Purina ^(R) mouse chow (Purina Mills, Richmond, IN) and water ad libitum.

A group of 5 mice were administered with the potential antigen to be tested (Bacillus subtilis BPN' as a positive control or an intermolecularly cross-linked protease of the present invention). Prior to dosing, each mouse was anesthetized by intraperitoneal (ip) injection of a mixture of Ketaset (88.8 mg/kg) and Rompun (6.67 mg/kg). The anesthetized mouse was held in the palm with its back facing down, and intranasally administered with 5 mL of protease-dissolved buffer solution (0.01M KH ₂ PO ₄ , pH 5.5). When each group is given the same dose, experiments can be conducted with different doses. The dosing solution was dabbed gently outside the nostrils of each mouse and inhaled by the mouse. Dosing was repeated on days 3, 10, 17 and 24.

Serum samples were collected on day 29. Enzyme-specific IgG1 antibodies in mouse sera were determined by antigen capture ELISA. The immunogenicity of protease conjugates and subtilisin BPN' can be compared using standard _ED50 values.

Compositions of the present invention

The protease conjugates of the invention can be used in any situation where the use of the respective parent protease is suitable. One such example includes cleaning compositions. Due to the desirably attenuated immunogenicity of the protease conjugates of the present invention, the protease conjugates may also be used in situations that have previously benefited minimally from the use of proteases. Examples of such uses include where the protease conjugate must come into intimate contact with mammalian skin, especially human skin, for example where a personal care composition is used.

Cleaning Compositions

The protease conjugates are useful in cleaning compositions including, but not limited to, laundry compositions, hard surface cleaning compositions, delicates cleaning compositions including dishwashing compositions, and automatic dishwasher detergent compositions.

The cleaning composition of the present invention comprises an effective amount of one or more protease conjugates of the present invention and a carrier of the cleaning composition.

As used herein, an "effective amount of a protease conjugate" or similar expressions refers to an amount of a protease conjugate sufficient to achieve the desired proteolytic activity in a particular cleaning composition. The effective amount can be readily determined by one of ordinary skill in the art and depends on various factors, such as the particular type of protease conjugate used, the purpose of cleaning, the particular composition of the cleaning composition, and whether liquid or Dry (eg, granules, sticks) compositions, and the like. Preferably, the cleaning composition comprises from about 0.0001% to about 10%, more preferably from about 0.001% to 1%, and most preferably from about 0.01% to about 0.1% of one or more protease conjugates of the present invention. compound. Several examples of different cleaning compositions in which the protease conjugates can be used are discussed in more detail below.

In addition to the protease conjugates described herein, the cleaning compositions of the present invention also include a cleaning composition carrier comprising one or more cleaning composition materials compatible with the protease conjugate. As used herein, the term "cleaning composition material" refers to any material selected for a particular type of desired composition and product form (e.g., liquid, granule, block, spray, stick, paste, gel). materials that are also compatible with the protease conjugate used in the composition. The specific choice of cleaning composition materials can readily be made by considering the surface material to be cleaned, the desired form of the composition for the cleaning conditions during use (eg, by use of detergents). As used herein, the term "compatible" means that the cleaning composition material does not reduce the proteolytic activity of the protease conjugate to such an extent that the protease is not as effective as desired under normal use conditions. Specific cleaning composition materials are exemplified in detail below.

The protease conjugate of the present invention can be used in various detergent compositions where rich foam and good cleaning effect are desired. Thus, the protease conjugates can be used with a variety of conventional ingredients to provide fully formulated hard surface cleaners, dishwashing compositions, fabric cleaning compositions, and the like. Such compositions can be used in the form of liquids, granules, sticks and the like. Such compositions can be formulated as "concentrated" detergents containing up to about 30% to about 60% by weight of surfactant.

The cleaning compositions described herein may optionally, and preferably, comprise various surfactants (eg, anionic, nonionic or zwitterionic surfactants). Typically, such surfactants comprise from about 5% to about 35% of the composition.

Non-limiting examples of surfactants useful in the present invention include conventional C ₁₁ -C ₁₈ alkylbenzene sulfonates as well as primary and random alkyl sulfates; the general formula CH ₃ (CH ₂ ) _X (CHOSO ₃ ) C ₁₀ -C ₁₈ secondary (2,3) alkyl ^sulfates of - M + ) CH ₃ and _{CH 3} ⁽ CH ₂ ) _y (CHOSO ₃ ^- M ⁺ ) CH ₂ CH ₃ , where x and (y+l) is an integer of at least about 7, preferably at least about 9, and M is a water-soluble cation, especially sodium; C ₁₀ -C ₁₈ alkyl alkoxy sulfate (especially EO 1-5 ethoxy sulfates); C ₁₀ -C ₁₈ alkyl alkoxy carboxylates (especially EO 1-5 ethoxy carboxylates); C ₁₀ -C ₁₈ alkyl polyglucosides and their corresponding sulfated polyglucosides Glycosides; C ₁₂ -C ₁₈ α-sulfonated fatty acid esters; C ₁₂ -C ₁₈ alkyl and alkylphenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy); C ₁₂ -C ₁₈ betaine and sultaine; C ₁₀ -C ₁₈ amine oxide, etc. Alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. In addition, it is also preferred to use such surfactants in combination with amine oxide and/or betaine or sultaine surfactants according to the desire of the formulator. Other conventional useful surfactants are listed in standard textbooks. Particularly useful surfactants include C ₁₀ -C ₁₈ N-methylglucamine, described in US Patent 5,194,639, issued March 16,1993 to Connor et al.

The compositions of the present invention may also include a variety of other ingredients that function in detergent cleaning compositions, including, for example, other active ingredients, carriers, hydrotropes, processing aids, dyes or colorants and Solvents for liquid formulations. If additional suds enhancement is desired, a suds booster such as a _C10 - _C16 alkanolamide can be added to the composition, typically in an amount of from about 1% to about 10%. The C ₁₀ -C ₁₄ monoethanolamides and diethanolamides represent a typical class of such suds boosters. It is also advantageous to use such suds boosters in combination with high sudsing co-surfactants such as the amine oxides, betaines and sultaines mentioned above. If desired, soluble magnesium salts such as _MgCl2 , _MgSO4 , typically from about 0.1% to about 2%, can be added to provide additional foam.

The liquid detergent composition of the present invention may contain water and other solvents as carriers. Examples of low molecular weight primary or secondary alcohols are methanol, ethanol, propanol and isopropanol, all of which are suitable. Monohydric alcohols are preferred for dissolving surfactants, but polyhydric alcohols containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxyl groups (e.g., 1,3-propanediol, ethylene glycol, glycerol, and 1,2 - propylene glycol) can also be used. The composition may comprise from about 5% to about 90%, typically from about 10% to about 50%, of such carriers.

Detergent compositions are preferably formulated so that the wash water has a pH of from about 6.8 to about 11 during use in aqueous cleaning operations. Thus, the finished product will typically be formulated within this range. Techniques for controlling pH at recommended usage levels include, for example, the use of buffers, bases and acids. Such techniques are well known to those skilled in the art.

When formulating the hard surface cleaning compositions and fabric cleaning compositions described herein, the formulator may wish to use from about 5% to about 50% by weight of builder. Typical builders include 1-10 micron zeolites, polycarboxylates such as citrates and oxodisuccinates, layered silicates, phosphates, and the like. See standard formula booklets for other conventional builders.

Likewise, the formulator can employ various additional enzymes in such compositions, such as cellulases, lipases, amylases and proteases, typically at levels of from about 0.001% to about 1% by weight. Various stain removal and fabric care enzymes are well known in the laundry detergent art.

Various bleaching compounds such as percarbonates, perborates, and the like can also be used in such compositions, typically at levels of from about 1% to about 15% by weight. Such compositions, if desired, can also contain bleach activators, such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonate, and the like, which are also known in the art. Typical levels are from about 1% to about 10% by weight.

Detergents, especially of the anionic oligoester type, chelating agents, especially phosphoramidates and ethylenediamine disuccinate, clay soil removers, especially ethoxylated tetraethylenepentamine, dispersants , especially polyacrylates and polyaspartates, brighteners, especially anionic brighteners, foam suppressors, especially siloxanes and secondary alcohols, fabric softeners, especially smectite clay, etc. For such compositions, the amount used is about 1% to 35% (weight). Extensive and detailed descriptions of such conventional materials are contained in standard recipe books as well as published patents.

Enzyme stabilizers can also be used in 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 protease conjugates of the invention may also be used 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 tiles, and the like. The hard surface cleaning composition of the present invention comprises an effective amount of one or more protease conjugates of the present invention, preferably the content of the protease conjugate in the composition is about 0.001% to about 10%, more preferably about 0.01% to about 5%, still more preferably about 0.05% to about 1% by weight. Such hard surface cleaning compositions typically include, in addition to one or more protease conjugates, a surfactant and a water-soluble chelating builder. However, these surfactants are sometimes not used in certain specialty products, such as spray window cleaners, due to the filmy and/or spotty residue they may cause on glass surfaces.

When present, a surfactant component may be present in the compositions of the present invention at levels as low as 0.1%, however, typically, the composition will contain surfactants at levels from about 0.25% to about 10%, more preferably about 1% to about 5%.

Typically, the compositions comprise from about 0.5% to about 50% detergency aid, preferably from about 1% to about 10%.

The preferred pH range should be about 7-12. If pH adjustment is required, common pH adjusters such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used.

Solvents may also be included in the composition. Useful solvents include, but are not limited to, glycol ethers such as diethylene glycol monohexyl ether, diethylene glycol monobutyl ether, ethylene glycol monobutyl ether, ethylene glycol monohexyl ether, propylene glycol monobutyl ether, Dipropylene glycol monobutyl ether, and diols such as 2,2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. Typical levels of such solvents, when used, are from about 0.5% to about 15%, more preferably from about 3% to about 11%.

In addition, when the surface to be cleaned is not cleaned after "full strength" application of the composition of the present invention, a highly volatile solvent such as isopropanol or ethanol may be used in the composition to speed the dissolution of the composition on the surface. evaporation. When used, volatile solvents are typically present in the composition at levels of from about 2% to about 12%.

Example 7-12

Liquid hard surface cleaning composition Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Protease conjugates in Example 3 0.05% 0.50% 0.02% 0.03% 0.30% 0.05% EDTA - - 2.90% 2.90% - - Sodium citrate - - - - 2.90% 2.90% SDBS 1.95% - 1.95% - 1.95% - Sodium dodecyl sulfate - 2.20% - 2.20% - 2.20% Sodium C ₁₂ (ethoxy)sulfate - 2.20% - 2.20% - 2.20% C ₁₂ dimethylamine oxide - 0.50% - 0.50% - 0.50% Sodium cumene sulfonate 1.30% - 1.30% - 1.30% - Hexyl Carbitol 6.30% 6.30% 6.30% 6.30% 6.30% 6.30% water 90.4% 88.3% 87.53% 85.87% 87.25% 85.85% All formulations were adjusted to pH7.

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

Example 13-16

liquid dish detergent Example 13 Example 14 Example 15 Example 16 Protease conjugates in Example 1 0.05% 0.50% 0.02% 0.40% C ₁₂ -C ₁₄ N-methylglucamine 0.90% 0.90% 0.90% 0.90% _C12 Ethoxy(1) Sulfate 12.0% 12.0% 12.0% 12.0% 2-Methylundecanoic acid 4.50% 4.50% 4.50% 4.50% C ₁₂ ethoxy(2) carboxylate 4.50% 4.50% 4.50% 4.50% _C12 Fatty Alcohol Ethoxylates (4) 3.00% 3.00% 3.00% 3.00% C ₁₂ amine oxide 3.00% 3.00% 3.00% 3.00% Sodium cumene sulfonate 2.00% 2.00% 2.00% 2.00% ethanol 4.00% 4.00% 4.00% 4.00% Mg ²⁺ (as MgCl ₂ ) 0.20% 0.20% 0.20% 0.20% Ca ²⁺ (as CaCl ₂ ) 0.40% 0.40% 0.40% 0.40% water 65.45% 65% 65.48% 65.1% All formulations were adjusted to pH7.

Example 17-19

liquid fabric cleaning composition Example 17 Example 18 Example 19 Protease conjugates in Example 4 0.05% 0.03% 0.30% Sodium C ₁₂ -C ₁₄ Alkyl Sulfate 20.0% 20.0% 20.0% 2-Butyl octanoic acid 5.0% 5.0% 5.0% Sodium citrate 1.0% 1.0% 1.0% C ₁₀ Fatty Alcohol Ethoxylates (3) 13.0% 13.0% 13.0% monoethanolamine 2.50% 2.50% 2.50% Water/propylene glycol/ethanol (100:1:1) 58.45% 58.47% 58.20%

Personal Care Compositions

The protease conjugates of the present invention are particularly suitable for use in personal care compositions such as leave-on and rinse-off hair conditioners, shampoos, leave-on and rinse-off acne compositions, facial cleansers and conditioners, shower gels, Soaps, foaming and non-foaming cleansers, cosmetics, hand, face and body emollients, moisturizers, patches and masks, leave-in facial moisturizers, cosmetic and cleansing wipes, oral care compositions, catamenials and contact lens care compositions. The personal care compositions of the present invention comprise one or more protease conjugates of the present invention and a personal care carrier.

For ease of illustration, the protease conjugates of the present invention are suitable for inclusion in compositions described in the following references: U.S. Patent 5,641,479 (skin cleansers), issued June 24, 1997 to Linares et al; US Pat. U.S. Patent 5,540,852 (skin cleansers) issued to Kefauver et al.; U.S. Patent 5,510,050 (skin cleansers) issued Apr. 23, 1996 to Dunbar et al.; issued March 18, 1997 to Guang Lin et al. US Patent 5,612,324 (anti-acne formulations); US Patent 5,587,176 (anti-acne formulations) to Warren et al., issued Dec. 24, 1996; US Patent 5,549,888 (anti-acne formulations), issued Aug. 27, 1996 to Venkateswaran ; U.S. Patent 5,470,884 (anti-acne preparations) issued Nov. 28, 1995 to Corless et al.; U.S. Patent 5,650,384 (shower gel) issued Jul. 22, 1997 to Gordon et al.; March 4, 1997 U.S. Patent 5,607,678 (shower gel) issued on April 29, 1997 to Moore et al.; U.S. Patent 5,624,666 (hair conditioner and/or shampoo) issued April 29, 1997 to Coffindaffer et al. U.S. Patent 5,618,524 (Hair Conditioner and/or Shampoo) issued to Bolich et al.; U.S. Patent 5,612,301 (Hair Conditioner and/or Shampoo) issued to Inman on March 18, 1997; U.S. Patent 5,573,709 issued November 12, 1996 to Wells (hair conditioner and/or shampoo); U.S. Patent 5,482,703 issued January 9, 1996 to Pings (hair conditioner and/or shampoo ); Reissued April 12, 1994 to Grote et al. U.S. Patent Re. Cosmetics); U.S. Patent 5,605,894 (cosmetics) issued Feb. 25, 1997 to Blank et al.; U.S. Patent 5,585,090 (cosmetics) issued Dec. 17, 1996 to Yoshioka et al.; issued Jul. 3, 1990 U.S. Patent 4,949,179 (emollients for hands, face, and body) to Cheney et al; U.S. Patent 5,607,980 (emollients for hands, face, and body) to McAtee et al. U.S. Patent 4,045,364 (Cosmetic or Cleansing Liniments) to Richter et al., issued Aug. 30, 1977; European Patent Application, Touchet et al., published Oct. 12, 1994, EP 0 619 074 (Cosmetic or Cleansing Liniments); U.S. Patent 4,975,217 (Cosmetic or Cleansing Liniments) issued Dec. 4, 1990 to Brown-Skrobot et al.; issued Mar. 17, 1992 to Seibel US Patent 5,096,700 (oral cleaning composition); US Patent 5,028,414 issued July 2, 1991 to Sampathkumar (oral cleaning composition); US Patent 5,028,415 issued July 2, 1991 to Benedict et al. cleaning compositions); issued September 5, 1989 to Davies et al., U.S. Patent 4,863,627 (contact lens cleaning compositions); issued May 24, 1988 to Huth et al., U.S. Patent Re.32,672 (contact lens Cleaning Compositions); and US Patent 4,609,493 to Schafer, issued September 2, 1986 (Contact Lens Cleaning Compositions).

To further illustrate the oral cleaning composition of the present invention, a pharmaceutically acceptable amount of one or more protease conjugates of the present invention can be added to the composition to remove proteinaceous stains on teeth or dentures. "Oral cleaning composition" as used in the present invention refers to dentifrice, toothpaste, tooth powder, mouthwash, mouth spray, chewing gum, lozenge, sachet, tablet, biogel, preventive paste, tooth treatment solution etc. Preferably, the oral cleaning composition comprises about 0.0001% to about 20% (weight) of one or more protease conjugates of the present invention, more preferably about 0.001% to about 10%, even more preferably About 0.01% to about 5%, and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" as used herein means that the term describes a drug, agent or inert ingredient suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, Irritation, allergic reaction, etc., with a reasonable effect/risk ratio.

Typically, the pharmaceutically acceptable oral cleansing carrier component of the oral cleansing composition generally accounts for about 50% to about 99.99% by weight of the composition, preferably about 65% to about 99.99%, more preferably about 65% to about 99% %.

Pharmaceutically acceptable carrier ingredients and optional ingredients that can be added to the oral cleansing component of the oral cleansing compositions of the present invention are well known to those skilled in the art. A wide variety of composition types, carrier ingredients and optional ingredients for use in oral cleansing compositions are disclosed in the references cited above herein.

In another embodiment of the present invention, a denture cleaning composition for cleaning dentures outside the oral cavity comprises one or more protease conjugates according to the present invention. This type of denture cleaning composition contains an effective amount of one or more protease conjugates and a denture cleaning carrier, and the weight percentage of the protease conjugate in the composition is preferably about 0.0001% to about 50%, more preferably Preferably from about 0.001% to about 35%, still more preferably from about 0.01% to about 20%. Various denture cleaning composition forms, such as effervescent tablets and the like, are well known in the art (see, e.g., U.S. Pat. No. 5,055,305, Young), and are generally suitable for incorporating one or more protease conjugates for cleaning dentures. proteinaceous stains.

In another embodiment of the present invention, contact lens cleaning compositions include one or more protease conjugates described herein. This type of contact lens cleaning composition contains an effective amount of one or more protease conjugates and a contact lens cleaning carrier, and the weight percentage of the protease conjugate in the composition is preferably about 0.01% to 50%, more preferably Preferably from about 0.01% to about 20%, still more preferably from about 1% to about 5%. Various contact lens cleaning composition forms, such as tablets, liquids, etc., are well known in the art and are generally suitable for incorporating one or more protease conjugates for cleaning proteinaceous stains from contact lenses.

Example 20-23

contact lens cleaning composition Example 20 Example 21 Example 22 Example 23 Protease conjugates in Example 5 0.01% 0.5% 0.1% 2.0% glucose 50.0% 50.0% 50.0% 50.0% Non-ionic surfactant (polyoxyethylene-polyoxypropylene copolymer) 2.0% 2.0% 2.0% 2.0% Anionic Surfactant (Sodium Polyoxyethylene-Alkyl Phenyl Ether Sulfate) 1.0% 1.0% 1.0% 1.0% Sodium chloride 1.0% 1.0% 1.0% 1.0% Borax 0.30% 0.30% 0.30% 0.30% water 45.69% 45.20% 45.60% 43.70%

Example 24-27

bath products Example 24 Example 25 Example 26 Example 27 water 62.62% 65.72% 57.72% 60.72% Disodium EDTA 0.2% 0.2% 0.2% 0.2% Glycerol 3.0% 3.0% 3.0% 3.0% Polyquatenium 10 0.4% 0.4% 0.4% 0.4% Sodium Lauryl Ether Sulfate 12.0% 12.0% 12.0% 12.0% Cocamide MEA 2.8% 2.8% 2.8% 2.8% Sodium lauroamphoacetate 6.0% 6.0% 6.0% 6.0% myristic acid 1.6% 1.6% 1.6% 1.6% Magnesium Sulfate Heptahydrate 0.3% 0.3% 0.3% 0.3% Trihydroxystearin 0.5% 0.5% 0.5% 0.5% PEG-6 Caprylic/Capric Triglycerides 3.0% - - - Rattinic Acid Fatty Acid Sucrose Polyester 3.0% - - - Behenic Acid Fatty Acid Sucrose Polyester 3.0% - 4.0% - Petrolatum - 4.0% 8.0% - mineral oil - - - 6.0% DMDM hydantoin 0.08% 0.08% 0.08% 0.08% Protease conjugates in Example 6 0.1% 2.0% 2.0% 5.0% citric acid 1.40% 1.40% 1.40% 1.40%

Example 28-31

facial cleanser Example 28 Example 29 Example 30 Example 31 water 66.52% 65.17% 68.47% 68.72% Disodium EDTA 0.1% 0.1% 0.2% 0.2% citric acid - - 1.4% 1.4% Sodium Laureth-3 Sulfate 3.0% 3.5% - - Sodium Lauryl Ether-4 Carboxylate 3.0% 3.5% - - Laureth-12 1.0% 1.2% - - Polyquaternium 10 - - 0.4% 0.4% Polyquaternium 25 0.3% 0.3% - - glycerin 3.0% 3.0% 3.0% 3.0% Sodium lauroamphoacetate - - 6.0% 6.0% Lauric acid 6.0% 6.0% 3.0% 3.0% myristic acid - - 3.0% 3.0% Magnesium Sulfate Heptahydrate 2.3% 2.0% 2.0% 2.0% Triethanolamine 4.0% 4.0% 4.0% 4.0% Trihydroxystearin 0.5% 0.5% 0.5% 0.5% Behenic Acid Fatty Acid Sucrose Polyester 2.0% 2.0% - - Rattinic Acid Fatty Acid Sucrose Polyester 3.0% 2.0% - - PEG-6 Caprylic/Capric Triglycerides - - - 2.0% Petrolatum - - 4.0% - mineral oil - - - 2.0% Cocamidopropyl Betaine 2.0% 3.0% 1.8% 1.8% lauryl dimethylamine oxide 1.0% 1.2% 1.2% 1.2% D-Panthenol 1.0% 0.25% 0.25% - DMDM hydantoin 0.08% 0.08% 0.08% 0.08% Protease conjugates in Example 2 1.0% 2.0% 0.5% 0.5% Spices 0.2% 0.2% 0.2% 0.2%

Example 32-33

Leave-on skin warming composition Example 32 Example 33 glycerin 5.0% - stearic acid 3.0% - C _11-13 isoparaffins 2.0% - Ethylene glycol stearate 1.5% - Propylene Glycol - 3.0% mineral oil 1.0% 10.0% sesame oil - 7.0% Petrolatum - 1.8% Triethanolamine 0.7% - cetyl acetate 0.65% - Glyceryl Stearate 0.48% 2.0% TEA Stearate - 2.5% cetyl alcohol 0.47% - lanolin alcohol - 1.8% DEA-Cetyl Phosphate 0.25% - Methylparaben 0.2% 0.2% Propylparaben 0.12% 0.1% Carbomer 934 0.11% - Disodium EDTA 0.1% - Protease conjugates in Example 14 0.1% 0.5% water 84.32% 71.1%

Example 34

cleaner composition Propylene Glycol 1.0% ammonium lauryl sulfate 0.6% Succinic acid 4.0% sodium succinate 3.2% ^Triclosan® 0.15% Protease conjugates in Example 1 0.05% water 91.0% The above composition is impregnated onto a woven absorbent layer composed of cellulose and/or polyester at about 250% composition by weight of the absorbent layer.

SEQUENCE LISTING <110> Procter & Gamble <120> Protease conjugates with sterically protected epitope regions <130> Epitope protection <140> PCT/US00/<141> 2000-07-11<160>1<170> Patentin Ver.2.0 <210> 1 <211> 275 <212> PRT <213> Dexy Stocal Bacillus <400> 1ALA Gln Ser Val Val Val Val Ser Gln Ile Lys Ala Ala Leu1 5 10 15HIS GLN GLY TYR THR THR Gly Ser Asn Val Lys Val Ala Val Ile Asp

20 25 30Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala

35 40 45Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His

50 55 60Gly Thr His Val Ala Gly Thr Val Ala Leu asn Serle Gly65 70 75 80VAL Leu Gly Val Ala Seru Tyr Ala Val Lys Val Leu

85 90 95Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu

100 105 110Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly

115 120 125Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala

130 135 140SER GLE VAL VAL VAL VAL ALA ALA Ala Gly Asn GLU GLY Thr Ser Gly145 150 155 160SER Servr Val Gly Tyr Pro Serle Ala

165 170 175Val Gly Ala Val Asp Ser Ser Asn Gln Ar9 Ala Ser Phe Ser Ser Val

180 185 190Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr

195 200 205Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser

210 215 220Pro his value ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His His Pro Asn225 230 235 240TRP THR Gln Val Serg Serg Ser Leu asn Thr THR LYSS

245 250 255Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala

260 265 270Ala Ala Gln

275

Claims (14)

1. A protease conjugate, characterized in that the conjugate comprises a protease moiety and one or more additional moieties, wherein the protease moiety comprises a first epitope region, a second epitope region and a third epitope region An epitope region, wherein each additional moiety is covalently linked to an epitope protecting position of the protease moiety, wherein: (a) the epitope protection positions in the first epitope region are selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43 corresponding to subtilisin BPN', 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215, and 216; (b) the epitope protection position in the second epitope region is selected from the group consisting of 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91 corresponding to subtilisin BPN', 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145; and (c) the epitope protection positions in the third epitope region are selected from the group consisting of 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157 corresponding to subtilisin BPN', 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275. 2. The protease conjugate according to claim 1, wherein each additional moiety independently has the following structure:

Wherein X does not exist or is a connecting moiety; _R does not exist or is selected from the first polypeptide and the first polymer; _R does not exist or is selected from the second polypeptide and the second polymer; wherein X, R _and At least one of _R2 is absent. 3. The protease conjugate according to claim 2, wherein said protease moiety has a modified amino acid sequence of the parent amino acid sequence, wherein said modified amino acid sequence comprises a substitution at one or more epitope protection positions Amino acid substitutions wherein each additional moiety is covalently linked to a substituted amino acid. 4. The protease conjugate according to claim 3, wherein said substituted amino acid is cysteine. 5. The protease conjugate according to claim 4, wherein said parent amino acid sequence is selected from the group consisting of subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, thermase, protease A, Protease B, Protease C, and Protease D, and variants thereof. 6. The protease conjugate according to claim 5, wherein: (a) the epitope protection positions in the first epitope region are selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 67, 86 corresponding to subtilisin BPN', 87, 89, 206, 209, 214, and 215; (b) the epitope protection position in the second epitope region is selected from the group consisting of 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138 corresponding to subtilisin BPN', and 141; and (c) the epitope protection position in the third epitope region is selected from the group consisting of 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254 corresponding to subtilisin BPN', 265, and 275. 7. The protease conjugate according to claim 6, wherein R ₁ and R ₂ are absent. 8. The protease conjugate according to claim 6, wherein _R is selected from subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, thermase, protease A, protease B, the first polypeptides of Protease C, and Protease D, and their variants. 9. The protease conjugate according to claim 8, wherein said first polypeptide is covalently linked to said linking moiety or said protease moiety at a position selected from the group consisting of: corresponding to subtilisin BPN '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. 10. The protease conjugate according to claim 9, wherein X is absent and wherein said protease moiety and first polypeptide are covalently linked by a disulfide bridge. 11. The protease conjugate according to claim 6, wherein _R2 is absent and the first polymer is polyethylene glycol. 12. The protease conjugate according to claim 11, wherein at least one additional moiety is covalently linked to an epitope protection position in an epitope region selected from a first epitope region, a second epitope region and a third epitope region . 13. A cleaning composition comprising the protease conjugate of claim 1 and a cleaning composition carrier. 14. A personal care composition comprising the protease conjugate of claim 1 and a personal care carrier.