MXPA00009385A - Protease conjugates - Google Patents

Abstract

The present invention relates to subtilisin protease conjugates comprising a protease moiety and one or more addition moieties wherein the protease moiety has a modified amino acid sequence of a parent amino acid sequence, the parent amino acid sequence comprising a first epitope region, a second epitope region, and a third epitope region, wherein the modified amino acid sequence comprises a substitution by a substituting amino acid at one or more positions in one or more of the epitope regions and wherein each addition moiety is covalently attached to one of the substituting moieties. The present invention further relates to cleaning and personal care compositions comprising such protease conjugates.

Description

CONJUGATES OF PROTEASE FIELD OF THE INVENTION The present invention relates to subtilisin-protease conjugates and compositions comprising conjugates having decreased immunogenicity in relation to their corresponding progenitor proteases.

BACKGROUND OF THE INVENTION Enzymes are the largest class of proteins that occur naturally. One class of enzyme includes proteases that catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has typically been exploited by incorporating naturally occurring proteases and proteases engineered into cleaning compositions, particularly those pertinent to laundry applications. In cleaning techniques, the most widely used of these proteases are serine proteases. Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen et. al., "Homology Modeling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases", Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the effectiveness of wild-type proteases in their natural environment often does not translate to the environment of the unnatural cleaning composition. Specifically, the characteristics of the protease such as, for example, thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized by using the protease outside the natural environment. Various methods have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the wash environment that is not natural. These approaches include the genetic redesign and / or chemical modification of proteases to increase thermal stability and to improve oxidation stability under very diverse conditions. However, because such proteases are foreign to mammals, they are potential antigens. As antigens, these proteases produce immunogenic and allergic responses (collectively described herein as immunogenic responses) in mammals. In fact, sensitization to serine proteases has been observed in environments where humans are regularly exposed to proteases. Such environments include manufacturing facilities, where employees are exposed to proteases through vehicles such as uncontrolled dust or aerosolization. Aerosolization can occur by introducing the protease into the lung, which is the route of exposure of the protease that causes the most dangerous response. Protease sensitization can also occur in the market, where the repeated use of products containing proteases by consumers can cause an immounogenic response. In addition, although genetic redesign and chemical modification of proteases has been prominent in the continuing search for more highly effective proteases to be used in laundry applications, such proteases have been used to a minimum in personal care compositions and light duty detergents. A major reason for the absence of these proteases in products such as, for example, soaps, gels, body washes and shampoos is due to the aforementioned problem of human sensitization leading to undesirable immunogenic responses. Therefore, it would be highly advantageous to provide a personal care composition that would provide the cleaning properties of the proteases without the provocation of immunogenic responses. Currently, immunogenic responses to proteases can be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases to prevent them from being transported through the air. These methodsAlthough they deal with consumer exposure to airborne proteases, they still present the risks associated with extended tissue contact with the finished composition and worker exposure to powder or aerosol containing protease during manufacture. In the medical field, suggestions have been made to decrease the immunogenicity of enzymes through another method. This method involves linking polymers to enzymes. See, for example, US patent. Do not. 4,179,337, Davis, et al., Issued December 18, 1979 and the application PCT WO 96/17929, Pisen, et al., Published June 13, 1996. One method that leads to the decrease of the immunogenic activity of a protease is through the alleviation of the immunogenic properties of the epitopes. The epitopes are those regions of amino acids of an antigen that induce an immune response through the binding of antibodies or the presentation of processed antigens to T cells through a major histocompatibility complex protein (MHC). Changes in epitopes can affect its efficiency as an antigen. See Walsh, B. J. and M.E.H. Howden. "A Method for the Detection of IgE Binding Sequences of Allergens Based on a Modification of Epitope Mapping", Journal of Immunological Methods, Vol. 121, pp. 275-280 (1989). The inventors of the present invention have discovered that those serine proteases commonly known as subtilisins, including subtilisin BPN ', have prominent epitope regions at positions 70-84 of amino acids corresponding to subtilisin BPN'. The inventors of the present invention have chemically modified such subtilisins here in one or more of these epitope regions to remediate the immunogenic properties of the protease. By doing so, the active site of the protease is affected to a minimum. Therefore, the inventors of the present invention have discovered subtilisin type proteases that induce a decreased immunogenic response but maintain their activity as an efficient and active protease. Accordingly, the protease conjugates of the present invention are suitable for use in various types of compositions including, but not limited to, laundry, dishwashing, hard surfaces, skin care, hair care, beauty care, oral compositions. and compositions for contact lenses.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to subtilisin-protease conjugates comprising a protease portion and one or more addition portions wherein: (a) The protease portion has a modified amino acid sequence of a progenitor amino acid sequence, the progenitor amino acid sequence it comprises a first epitope region, a second epitope region, and a third epitope region, wherein the modified amino acid sequence comprises a substitution by an amino acid substituent at one or more positions in one or more epitope regions wherein: (i) ) When a substitution occurs in the first epitope region, substitution occurs in one or more positions corresponding to positions 70-84 of the subtilisin BPN '; (ii) when a substitution occurs in the second epitope region, substitution occurs in one or more positions corresponding to positions 103-126 of the subtilisin BPN '; and (iii) when a substitution occurs in the third epitope region, substitution occurs in one or more positions corresponding to positions 217-252 of the subtilisin BPN '; and (b) wherein each of the addition portions is covalently bound to one of the substituent amino acids present in the protease portion and has the structure: rv 1 \ X- where X is selected from the group consisting of null and a linking portion; Ri is selected from the group consisting of null, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of null, a second polypeptide, and a second polymer; where at least one of X, Ri, and R2 is not null. The present invention also relates to cleaning and personal care compositions comprising said protease conjugates.

DETAILED DESCRIPTION OF THE INVENTION The essential components of the present invention are described below. Also included are non-limiting descriptions of various optional and preferred components useful in the embodiments of the present invention. The present invention may comprise, consisting of or consisting 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 levels of component or composition are in reference to the active level of that component or composition, and are exclusive of impurity, for example, residual solvents or by-products, which may be present in commercially available sources. All documents referred to herein, including all patents, patent applications and printed publications, are hereby incorporated by reference in their entirety. In the present reference is made to commercial names for materials including, but not limited to, enzymes. The inventors herein do not intend to be limited by materials under a certain trade name. Equivalent materials (e.g., those obtained from a different source under a name or catalog number (reference) other than those referenced by trade name may be substituted and used in the protease conjugates and compositions herein. As used herein, abbreviations will be used to describe amino acids Table 1 provides a list of abbreviations used in the present invention.

TABLE 1 Definitions As used herein, the term "mutation" refers to alterations in gene sequences and amino acid sequences produced by those gene sequences. The mutations can be deletions, substitutions or additions of amino acid residues to the sequence of wild-type proteins. As used herein, the term "parent" refers to an enzyme, wild-type or variant. As used herein, the term "wild type" refers to a protein, for example a protease or other enzyme, produced by non-mutated organisms. As used herein, the term "variant" means an enzyme having an amino acid sequence that differs from that of the corresponding wild-type enzyme due to the genetic mutation of the nucleotide sequences encoding that enzyme or the mutation. of the wild-type enzyme itself. As used herein, all molecular weights of polymer are expressed as weight average molecular weights. As referred to herein, although the conjugates of the present invention are not limited to those comprising subtilisin BPN 'and variants thereof, all amino acid numbering is with reference to the amino acid sequence for subtilisin BPN' which is represented by SEQ ID NO: 1. The amino acid sequence for subtilisin BPN 'is also described by Walls et al., Nucleic Acids Research, Vol. II, p. 7911-7925 (1983).

Protease conjugates of the present invention The protease conjugates of the present invention are compounds comprising a protease portion and one or more addition portions, wherein the protease portion and the addition portions are connected by covalent bond.

Protease portions The protease portions of the present have a modified amino acid sequence of a progenitor amino acid sequence. The progenitor amino acid sequences herein are serine proteases, either wild type or variants thereof. As used herein, the term "serine protease" means a protease having at least 50%, and preferably 80%, amino acid sequence identity with the sequences of one or more serine proteases type subtilisin. Serine proteinases type wild-type subtilisin are produced by, for example, microorganisms Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus, and Bacillus subtilis. An exposition concerning serine proteases type subtilisin and its homologies can be found in Siezen et al., "Homology Modeling and Protein Engineering Strategy of SubtHases, the Family of Subtilisin-Like Serine Proteases", Protein Engineering, Vol. 4, No. 7 , pp. 719-737 (1991). Preferred progenitor amino acid sequences for use herein include, for example, those obtained from Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis, subtilisin BPN, subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K , and termitase, including A / S Alcalase® (commercially available from Novo Industries, Copenhagen, Denmark), Esperase® (Novo Industries), Savinase® (Novo Industries), Maxatase® (commercially available from Gist-Brocades, Delft, The Netherlands). Low) Maxacal® (Gist-Brocades), Maxapem 15® (Gist-Brocades), and variants of the above. Especially preferred proteases for use herein include those obtained from Bacillus amyloliquefaciens and variants thereof. The proteases that are even more preferred for use as protease moieties herein are subtilisin BPN 'and variants thereof. Especially preferred variants of subtilisin BPN ', hereinafter referred to as "Protease A" for use as progenitor amino acid sequences herein are described in the US patent. No. 5,030,378, Venegas, issued July 9, 1991, as characterized by the amino acid sequence of subtilisin BPN 'with the following mutations: a) Gly in position 166 is substituted with an amino acid residue selected from Asn, Ser, Lys, Arg, His, Gln, Ala and Glu; Gly in position 169 is replaced by Ser; and Met at position 222 is substituted with an amino acid residue selected from Gln, Phe, His, Asn, Glu, Ala and Thr; b) Gly at position 160 is replaced with Ala, and Met at position 222 is replaced with Ala. Additionally preferred variants of subtilisin BPN ', hereinafter referred to as "Protease B", for use as progenitor amino acid sequences herein are disclosed in EP-B-251, 446, assigned to Genencor International, Inc., published on January 7, 1988, granted on December 28, 1994, as characterized by the amino acid sequence of wild-type subtilisin BPN 'with mutations one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45 , Ala48, Ser49, MetdO, His67, Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Methyl 24, Gly127, Gly128, Pro129, Leu135, Lys170, Tyr171, Pro172, Asp197, Meth 99, Ser204 , Lys213, Tyr214, Gly215, and Ser221; or two or more of the positions listed above combined with one or more mutations in the selected positions of Asp32, Ser33, Tyr104, Alai 52, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222. Other preferred variants of subtilisin BPN 'for use as progenitor amino acid sequences herein, hereinafter referred to as "Protease C", and described in WO 95/10615, assigned to Genencor International Inc., published on 20 April 1995, as characterized by the amino acid sequence of the wild-type subtilisin BPN 'with a mutation at the Asn76 position, in combination with mutations at one or more selected positions of Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107 , Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265 and Ala274. Other variants of the BPN 'subtilisin preferred for use as progenitor amino acid sequences herein, hereinafter referred to as "Protease D", are described in the US patent. No. 4,760,025, Estell et al., July 26, 1988, as characterized by the amino acid sequence of wild-type subtilisin BPN 'with mutations to one or more amino acid positions selected from the group consisting of Asp32, Ser33, His64, Tyr104, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222. The proteases that are most preferred for use as progenitor amino acid sequences herein are selected from the group consisting of Alcalase®, subtilisin BPN ', Protease A, Protease B, Protease C, and Protease D, with Protease D being most preferred. In accordance with the present invention, the progenitor amino acid sequence is substituted at one or more of the parent amino acid residues with an amino acid substituent to produce a (precursor for a) protease portion suitable to bind with one more of the addition portions present The substitution must be made in one or more positions in one or more of the epitope regions that have been discovered by the inventors herein. The present inventors have discovered three regions of epitope, one occurring at positions 70-84 corresponding to subtilisin BPN '(the first epitope region), one occurring at positions 103-126 corresponding to subtilisin BPN' (the second epitope region), and one occurring at positions 217-252 of the subtilisin BPN '(the third epitope region). In another embodiment of the invention, the protease portion comprises a substitution at one or more positions in two or more of the epitope regions (i.e., one or more substitutions occur in each of two or all three epitope regions). In another embodiment of the invention, the protease comprises a substitution at one or more positions in each of the three epitope regions (i.e., one or more substitutions occur in each of the three epitope regions). Most preferably, the progenitor amino acid sequence is substituted on one or more of the progenitor amino acid residues wherein at least one of the substitutions occurs in the first epitope region. Where a substitution occurs in the first epitope region, substitution occurs in one or more of positions 70-84, preferably one or more of positions 73-81, and most preferably in position 78. Where a substitution occurs in the second epitope region, substitution occurs in a or more of the positions 106-126, preferably one or more of the positions 106-120, and most preferably in the 116 position. Where a substitution occurs in the third epitope region, substitution occurs in one or more of the positions 217 -254, preferably one or more of the positions 236-254, and most preferably in the 240 position. In order to better obtain the selective binding (i.e., selective binding in one or more of the epitope regions) of one or more addition portions of the present invention to the protease portion, the substitution must be with an amino acid substituent that does not occur in (is unique to) the progenitor amino acid sequence. In this regard, any amino acid substituent that is unique to the progenitor amino acid sequence can be used. For example, since a cysteine residue does not occur in the wild-type amino acid sequence for subtilisin BPN ', a substitution of subtilisin BPN' with one or more cysteine residues in one or more of the epitope regions is appropriate for the present invention. Where a cysteine residue occurs outside the epitope regions of the progenitor amino acid sequence, it is preferable to substitute another amino acid residue in each of said positions to allow selective coupling with one or more addition portions in the region (s) (is) epitope. Cysteine is the most preferred amino acid substituent for substitution in one or more of the epitope regions. Other preferred substituent amino acids include lysine. Where the amino acid substituent is lysine, it is preferred to mutate lysine residues that occur outside the epitope regions of the progenitor amino acid sequence to another amino acid residue so that the functionalization of one or more of the lysine residues in the regions of epitope is selective. For example, a lysine residue occurs at position 237 of the subtilisin BPN 'that is in the third epitope region. Selective mutation in situ of the other lysine residues occurring in the subtilisin BPN 'sequence can be carried out followed by selective functionalization of the lysine residue in the third epitope region with an addition portion. Alternatively, positions in the epitope regions can be mutated to lysine followed by selective functionalization at said positions by a polymer moiety.

Addition portions The protease conjugates of the present invention further comprise one or more addition portions wherein each of the addition portions is covalently bound to one of the substituent amino acids present in one of the epitope regions and has the structure: wherein X is selected from null and a link portion; Ri is selected from the group consisting of null, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of null, a second polypeptide, and a second polymer, wherein at least one of X, Ri and R2 is not zero. Preferably, from 1 to about 15, preferably from about 2 to about 10, and most preferably from about 1 to about 5 addition portions comprise the protease conjugate. Where Ri and R2 are each, independently, polypeptide portions or polymer portions Ri and R2 may be equivalent or different. Preferably, where Ri is a polypeptide moiety, R2 is selected from null and a polypeptide moiety, and most preferably it is null. Most preferably, where Ri is a polypeptide moiety, R2 is selected from null and an equivalent polypeptide moiety, and most preferably is null. Preferably, where Ri is a polymer moiety, R2 is selected from null and a polymer moiety. Most preferably, where Ri is a polymer moiety, R2 is selected from null and an equivalent polymer moiety. Where at least one of Ri and R2 are respectively the first polymer and the second polymer, then X preferably is not zero.

Polypeptide Portions The polypeptide portions described herein include those comprising two or more amino acid residues. Preferred polypeptide portions 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, ligninases, pullulanases , tanases, pentosanas, malanases, ß-glucanases, arabinosidases, hyaluron? dasat chondroitinase, laccases, transferases, isomerases (including, for example, glucose isomerase and xylose isomerase), lyases, ligases, synthetases, and fruit-based enzymes ( including, for example, papain). The most preferred enzymes to be used as polypeptide portions include proteases, cellulases, amylases, lipases, and fruit-based enzymes, with proteases being even more preferred. Examples of lipases to be used as a polypeptide moiety include those derived 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 A / S, Copenhagen, Denmark), Lumafast® (commercially available from Genencor, Int., Rochester, NY), and Lipomax® (Genencor, Int.). Examples of proteases to be used as the polypeptide moiety include serine proteases, chymotrypsin, and elastase-like enzymes. The most preferred proteases to be used as a polypeptide moiety include serine proteases, as defined hereinbefore in the "protease portion" exposure. Most preferably, where the polypeptide moiety is a serine protease, the polypeptide moiety independently carries the definition of a protease moiety as described hereinbefore, ie, the polypeptide moiety has a modified amino acid sequence of an amino acid sequence progenitor in one or more of the epitope regions as described hereinbefore (whose progenitor acid sequence can be called a "second" progenitor amino acid sequence). In this case, one of the binding portion (where the binding portion is not null) or the protease portion (where the binding portion is null) is covalently bound to the polypeptide portion through one of the substituent amino acids present in a of the epitope regions of the polypeptide portion. Where the polypeptide moiety is a serine protease, the same preferred, more preferred, and highly preferred moieties apply as described hereinbefore for protease moieties and their corresponding progenitor amino acid sequences. Most preferably, where the polypeptide portion is a serine protease, the polypeptide portion and the protease portion are equivalent portions. In this case, the polypeptide portion and the protease portion are preferably linked through a disulfide bridge, where X is zero, and most preferably, R2 is zero.

Polymer portions The addition portions herein may comprise a polymer portion. Examples of suitable polymer moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidones, polyamino acids, celluloses, dextrans, starches, glucogen, agarose, guar gum, swarm, inulin, xanthan gum, carrageenan, pectin, biopolymers, Hydrosylates of alginic acid, and hydrosylates of chitosan. Preferred polyalkylene oxides include polyethylene glycols, methoxypolyethylene glycols, and propylene glycols. Preferred dextrans include carboxymethyldextrans. Preferred celluloses include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose, and hydroxypropylcellulose. Preferred starches include hydroxyethyl starches and hydroxypropyl starches. The most preferred polymers are polyalkylene oxides. The most preferred polymer portion is polyethylene glycol. Where Ri and R2 are each, independently, polymer portions, Ri and R2 preferably have a collective molecular weight (ie, the molecular weight of Ri plus the molecular weight of R2) or from about 0.5 kD (kilodaltons) to about 40 kD, preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD. Where Ri and R2 are each polymer portions, Ri and R2 each, independently, preferably have a molecular weight of 0.25 kD to about 20 kD, preferably of about 0.5 kD to about 10 kD, and most preferably of about 0.5 kD at approximately 5 kD.

Where R ^ and R2 are each polymer moieties, the ratio of the molecular weights of Ri and R2 preferably ranges from about 1: 10 to about 10: 1, preferably from about 1: 5 to about 5: 1, and most preferably from about 1: 3 to about 3: 1. Where Ri is a polymer moiety and R2 is zero, Ri preferably has a molecular weight of from about 0.5 kD to about 40 kD, preferably from about 0.5 kD to about 20 kD, and most preferably from about 1 kD to about 10 kD.

Linking Portions As used herein, X may be (null or) a binding moiety that is covalently bound to a polypeptide moiety or a polymer moiety and is also covalently linked to a single amino acid substituent present in a moiety. of the epitope regions of the protease portion. The linkage portion is any small molecule, ie, a molecule having a molecular weight of less than about 800, preferably less than about 400, most preferably less than about 300. The most preferred linking portions include those capable of binding in a manner covalent to a cysteine residue or a lysine residue, most preferably a cysteine residue. Examples of linked portions and related chemistry are described in the U.S. patent. No. 5,446,090, Harris, issued August 29, 1995; patent of E.U.A. No. 5.171, 264, Merril. issued on December 15, 1992; patent of E.U.A. No. 5,162,430, Rhee et al., Issued November 10, 1992; patent of E.U.A. No. 5,153,265, Shadle et al., Issued October 6, 1992, patent of E.U.A. No. 5,122,614, Zalipskv, issued June 16, 1992; Goodson et al., "Site-Directed Pegylation of Recombinat lnterleukin-2 at its Glycosylation Site", Biotechnology, Vol. 8, No. 4, p. 343-346 (1990); Kogan.'The Synthesis of Substituted Methoxy-Poly (ethylene glycol) Derivatives Suitabie for Selective Protein Modification ", Synthetic Communications, Vol. 22, pp. 2417-2424 (1992), and Ishii et al .." Effects of the State of Succinimido-Ring on the Fluorescence and structural Properties of Pyrene Maleimide-Labeled aa-Tropomyosin ", Biophysical Journal, Vol 50, pp. 75-80 (1986) The most preferred binding portion is substituted succinimide (eg, alkyl) ) or not replaced.

MANUFACTURING METHOD The protease portions are prepared by mutation of the nucleotide sequences encoding a progenitor amino acid sequence. Such methods are well known in the art; a non-limiting example of one of these methods is set forth below: A phagemid (pSS-5) containing the wild-type BNP 'subtilisin gene (Mitchison, C. and JA Wells, "Protein Engineering of Disulfide Bonds in Subtilisin BPN '", Biochemistry, Vol. 28, pp. 4807-4815 (1989) is transformed into CJ236 strain of Escherichia coli dut-ung and a single DNA template containing single chain uracil is produced using the auxiliary phage of VCSM13 ( Kunkel et al., "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection", Methods in Enzymology, Vol 154, pp. 367-382 (1987), as modified by Yuckenbert et al., "Site-Directed in In vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors ", Directed Mutagenesis - A Practical Approach, McPherson, MJ ed., pp. 27-48 (1991). Site-directed mutagenesis modified from the method described in Zoller, MJ and M. Smith, "Oligonucleotide-Directed Mutagenesis Using M13-Deriv ed 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). The oligonucleotides are made using a DNA synthesizer from 380B (Applied Biosystems Inc.). The mutagenesis reaction products are transformed into the Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations are confirmed by DNA sequencing and the isolated DNA is transformed into the expression strain PG632 of Bacillus subtilis (Saunders et al .. "Optimization of the Signal-Sequience Cleavage Site for Secretion from Bacillus subtilis of a 34-Amino Acid Fragment of Human Parathyroid Homone "; 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 Créate an in 'íro -Derived Deletion Mutation ", Journal of Bacteriology, Vol. 160, pp. 15-21 (1984) .The fermentation is done as follows: Bacillus subtilis cells (PG632) containing the protease of interest are cultured at mid phase -log in one liter of LB broth containing 10 g / L glucose, and inoculated in a Biostat C fermentor (Braun Biotech, Inc., Allentown, PA) in a total volume of 9 liters.The fermentation medium contains extract of yeast, casein hydrosylate, partially hydrolyzed starch, solubility le (Maltrin M-250), anti-foam, pH regulators and trace minerals (see "Biology of Bacilli: Aplications to Industry", Doi ,. R. H. and M. McGloughlin, eds. (1992)). The broth is maintained at a constant pH of 7.5 during the fermentation operation. Kanamycin (50 μg / mL) is added for antibiotic selection of the mutagenized plasmid. The cells are cultured for 18 hours at 37 ° C at an A6oo of about 60 and the product is harvested. The fermentation broth is collected through the following steps to obtain pure protease. Bacillus subtilis cells are removed from the broth by tangential flow against a membrane of 0.16 μm. The cellless broth is then concentrated by ultrafiltration with a molecular weight cut-off membrane of 8000. The pH is adjusted to 5.5 with concentrated MES pH regulator (2- (N-mofolino) ethanesulfonic acid). The protease is then purified by cation exchange chromatography with S-sepharose and eluted with NaCl gradients. (see Scopes, R.K., "Protein Purification Principles and Practice," Springer-Verlag, New York (1984).

A pNA test (DelMar et al .. Analytical Biochemistry, Vol. 99, pp. 316-320 (1979)) is used to determine the concentration of active protease for fractions collected during gradient elution. This test measures the rate at which p-nitroaniline is released as the protease hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (sAAPF-pNA). The yellow production rate of the hydrolysis reaction is measured at 410 nm in a spectrophotometer and is proportional to the concentration of active protease portion. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The ratio of active protease / total protein gives the purity of the protease, and is used to identify fractions that have to be deposited for the supply solution. To avoid autolysis of the protease during storage, equal weight of propylene glycol is added to the deposited fractions obtained from the chromatographic column. Upon completion of the purification procedure, the purity of the supply protease solution is verified with SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined through a site titration method active using type II-T trypsin inhibitor: turkey egg white (Sima Chemical Company, St. Louis, Missouri). During preparation for use, the protease supply solution is eluted through a Sephadex-G25 size exclusion column (Pharmacia, Piscataway, New Jersey) to remove the propylene glycol and exchange the pH regulator. The pH regulator MES in the enzyme supply solution is exchanged by KH2PO40.01 M solution at a pH of 5.5. With the prepared protease, it can be used for functionalization with one or more addition portions to produce the protease conjugate. The precursor for the addition portion (the precursor for the addition portion reacts with the precursor for the protease portion to form the protease conjugate comprising the addition portion and the protease portion) is preferably activated to increase the reactivity with the precursor for the protease portion. Such activation is well known in the art. The following are non-limiting examples of methods of preparation of protease conjugates.

EXAMPLE 1 P) SH A protease containing a cysteine residue in one of the epitope regions is coupled with a polymer portion according to the above scheme using the following method (wherein "P" represents the protease portion minus the thiol group resulting from the substitution of cysteine and n is the number of repeating monomer units of the polyethylene glycol (eg, n = 77) A variant of the subtilisin BPN 'is prepared with a substitution of leucine for tyrosine at position 217 and a substitution of cysteine for serine in position 78. A concentration of approximately 2 mg / mL is obtained in the phosphate pH regulator (pH 5.5) of the variant, then the pH is elevated to 7.5 with dilute sodium hydroxide .The variant is mixed with the maleimide of monomethylpolyethylene glycol to a 25: 1 activated polymer to the excess of the variant.After 1 hour of mixing at room temperature, the pH of the mixture is adjusted to 5.5 with diluted phosphoric acid and filtered. through a molecular weight cutting ultrafilter to remove the excess polymer. The concentrate contains the purified protease conjugate.

EXAMPLE 2 A protease portion comprising a cysteine residue in one of the epitope regions is coupled with a polymer portion according to the above scheme using the following method (wherein "P" represents the protease portion minus the thiol group resulting from the cysteine substitution and n is the number of repeating monomer units of each polyethylene glycol (eg, n = 77) A variant of the subtilisin BPN 'is prepared with a substitution of leucine for tyrosine at position 217 and a substitution of cysteine by serine in position 78. A concentration of approximately 2 mg / mL is obtained in the phosphate pH regulator (pH 5.5) of the variant, then the pH is raised to 7.5 with diluted sodium hydroxide. the maleimide of dimethylpolyethylene glycol to a 25: 1 activated polymer to the excess of the variant.After 1 hour of mixing at room temperature, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and it is filtered through a molecular weight cutting ultrafilter to remove the excess polymer. The concentrate contains the purified protease conjugate.

EXAMPLE 3 A polymer protected with succinimide is selectively coupled to lysine in one or more epitope regions (where "MPEG" and "PEGM" are equivalent and represent monomethylpolyethylene glycols and wherein "P" represents the protease portion minus the amino group of lysine shown): pH 8.5 EXAMPLE 4 A carbodiimide protected polymer is selectively coupled to the lysine in one or more epitope regions (where "MPEG" and "PEGM" are equivalent and represent monomethyl polyethylene glycols, "P" represents the protease portion minus the amino group of the lysine shown, and X and X 'are side chains comprising the carbodiimide portion, eg, alkyls): EXAMPLE 5 A protease portion comprising a cysteine residue in one of the epitope regions is coupled to an alkylmaleimide using the following method (where "P" represents the protease portion minus the thiol group resulting from the cysteine and "R" substitutions). it is an alkyl group). In this example, Ri and R2 are each null and the linking portion is derived from the alkylamaleimide. A variant of subtilisin BPN 'is prepared with a substitution of leucine for tyrosine at position 217 and a substitution of cysteine for serine at position 78. A solution of 20 mL of the variant is prepared at a concentration of approximately 1 mg / l. mL in pH regulator of KH2PO4 0.01 M (pH 7). To this solution, 1.5 equivalents of alkylmaleimide (for example, methylmaleimide) are added. The solution is mixed gently at room temperature for about 1 hour. The resulting protease conjugate is obtained from the solution by standard methods.

JEJEMPLO 6 2 Equivalents 1 Equivalent A protease portion comprising a cysteine residue in one of the epitope regions forms a dimer using the following method (wherein "P" represents the protease portion minus the thiol group resulting from the cysteine substitution). In this example, the protease portion and the polypeptide portion are equivalent (and X is zero). A variant of subtilisin BPN1 is prepared with a substitution of leucine for tyrosine at position 217 and a substitution of cysteine for serine at position 78. A solution of 20 mL of the variant is prepared at a concentration of approximately 1 mg / mL in pH regulator of KH2P04 0.01 M (pH8.6). Oxygen is gently bubbled through the solution at room temperature for about 1 hour to form the desired protease conjugate dimer. The resulting protease conjugate is obtained from the solution by standard methods.

Analytical methods The protease conjugates of the present can be tested for enzymatic activity and immunogenic response using the following methods, which are known to the person skilled in the art. Other methods well known in the art may alternatively be used.

Activity of a protease conjugate The protease activity of a protease conjugate of the present invention can be tested by methods that are well known in the art. Two of these methods are discussed below: Skin flake activity method Using Scotch® # 3750G tape, flakes of human skin are separated from the legs of a subject repeatedly until the tape is substantially opaque with flakes. Then the ribbon is cut into 2.54 cm x 2.54 cm squares and set aside. In a 10 mm by 35 mm petri dish, 2 mL of 0J5 mg / mL of a control enzyme (for example, subtilisin BPN ') or the protease conjugate to be tested is added the pH regulator of KH2P04 0.01 M at a pH of 5.5. To this solution is added 1 mL of 2.5% sodium laurate at a pH of 8.6. The solution is mixed gently on a platform shaker. The previously prepared tape box is soaked in the solution (the leaflet upwards) for ten minutes continuing the smooth mixing. The tape box is then gently rinsed in tap water for fifteen seconds. In a clean petri dish, it is applied with Stevenel blue dye pipette (3 ml, commercially available from Sigma Chemical Co., St. Louis, MO). The rinsed tape box is placed in the dye for 3 minutes (with the leaflet facing up) with gentle mixing. The tape box is removed from the dye and rinsed consecutively in two 300 mL beakers with distilled water, for 15 seconds by rinsing. The tape box is allowed to air dry. The color intensity between the tape frame obtained from the control enzyme and the tape frame obtained from the protease conjugate is visually compared or using a chromameter. In relation to the control enzyme tape frame, a protease conjugate tape frame showing less color intensity indicates a protease conjugate that has higher activity.

Collagen-stained activity method 50 mL of 0.1 M tris pH-regulator (tris-hydroxymethylaminomethane) containing 0.01 M CaCl2 is combined to give a pH of 86, and 0.5 grams of azocol (collagen impregnated with azo dye, commercially available from Sigma Chemical Co., St. Louis, MO). This mixture is incubated at 25 ° C while mixing gently with a platform shaker. 2 mL of the mixture is filtered through a 0.2 micron syringe filter and the absorbance of the mixture is read at 520 nm to zero a spectrophotometer. One ppm of a control enzyme (eg, subtilisin BPN ') of the protease conjugate to be tested is added to the remaining 48 mL of tris / azocol mixture. 2 mL of the control / protease conjugate containing solution is filtered through a syringe filter of 2 microns every two minutes for a total of ten minutes. For each filtered sample, the absorbance is read immediately at 520 nm. The results are plotted against time. The slopes of the control and the test conjugate indicate relative activities of the samples. A higher slope indicates a higher activity. The activity of the test protease conjugate (slope) can be expressed as percent of the control activity (slope). The immunogenic potential of the protease conjugates of the present invention can be determined using methods known in the art or by the T Cell Proliferation Test presented below. This test is a variation of the test described in Bungy Poor Fard et al .. "T Cell Epitopes of the Major Fraction of Rye Grass Lolium perennial (Lol p I) Defined Using Overlapping Peptides in vitro and in vivo", Clinical Experimental Immunology, Vol. 94, pp. 111-116 (1993). The blood of subjects allergic to subtilisin BPN '(positive to prick test) and control subjects (negative to prick test) are used in this test. The blood (~ 60 ml) of each subject is collected and the mononuclear cells are harvested using ficoll-hypaque (available from Pharmacia, Piscataway, New Jersey). Cells are washed twice in RPMI 1640 (available from Gibco, Grand Island, New York) and then resuspended in complete RPMI medium supplemented with 10% 10% human AB serum, L-glutamine 2 mM and 25 μg / mL of gentamicin (which can be obtained from Gibco). The cells are cultured at a concentration of 2 x 10 5 cells / well in 0.2 mL of complete medium in 96-well microtitre plates with U-bottom. The potential antigen to be tested (either subtilisin BPN 'inactivated as positive control or a protease conjugate of the present invention) is added to a final concentration of up to 40 μg / ml. The cultures are incubated at 37 ° C in 5% C02. After 5 days, 1 μCi / cavity of methyl-3 H -thymidine is added and 18 cells are harvested later. The incorporation of 3H-thymidine by the cells is evaluated as a measurement of T cell proliferation by liquid scintillation counting.

Compositions of the present invention The protease conjugates of the present invention can be used in any application that is suitable for the respective progenitor protease. An example of this type includes cleaning compositions. Due to the desirable reduced immunogenicity properties of the protease conjugates of the present invention, the protease conjugates can be further used in applications that benefit from a minimum of the use of proteases. Examples of such applications include those in which the protease conjugate is necessarily in close contact with the skin of mammals (especially human skin), such as with the use of personal care compositions.

Cleaning compositions Protease conjugates can be used in cleaning compositions including, but not limited to laundry compositions, hard surface cleaning compositions, light duty cleaning compositions including tableware cleaning compositions and automatic laundry detergent compositions. crockery. The cleaning compositions of the present invention comprise an effective amount of one or more protease conjugates of the present invention and a vehicle of cleaning composition. As used herein, "effective amount of protease conjugate," or the like, refers to the necessary amount of protease conjugate to achieve the necessary proteolytic activity in the specific cleaning composition. Said effective amounts are easily investigated by one of ordinary skill in the art and are 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 composition is desired ( for example, granulated, in bar), and the like. Preferably, the cleaning compositions are comprised of from about 0.0001% to about 10%, most 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. this invention. Several examples of different cleaning compositions in which protease conjugates can be used are discussed in more detail below. In addition to these protease conjugates, these cleansing compositions further consist of a vehicle of the cleansing composition containing one or more materials of the cleansing composition compatible with the protease conjugate. The term 'cleaning composition material', as used herein, means any material selected for the particular type of cleaning composition desired and the shape of the product (eg, liquid, granulate, stick, aerosol, pellet). , in paste, in gel), whose materials are also compatible with the protease conjugate used in the composition. The specific selection of materials of the cleaning composition is carried out with ease considering the material to be cleaned and the desired form of the composition for cleaning conditions during use (for example, through the use of washing detergent). ). The term 'compatible', as used herein, means that the materials of the cleaning composition do not reduce the proteolytic activity of the protease conjugate to such an extent that the protease is not as effective as desired during situations of normal use. The specific materials of the cleaning composition are exemplified in detail below.

The protease conjugates of this invention can be used in a variety of detergent compositions in which it is desired to obtain a high degree of foaming and a good cleaning activity. Therefore, protease conjugates can be used with several common ingredients in order to provide hard surface cleaners of complete formulation, dishwashing compositions, fabric washing compositions, and the like. Said compositions may be in liquid, granulated, bar and the like form. Such compositions can be formulated as 'concentrated' detergents containing from about 30% to about 60% by weight of surfactants. The cleaning compositions herein may optionally contain, and preferably, various surfactants (for example, anionic, nonionic or zwitterionic surfactants). Such surfactants are typically present at levels of about 5% to about 35% of the compositions. Non-limiting examples of the surfactants useful herein include the Cn-C? 8 aiquilbenzene sulphonates and the common primary and random alkyl sulfates, the secondary alkyl sulfates (2.3) of do-C-is of the formulas CH3 (CH2) x (CHOSO3) 1vl +) CH3 and CH3 (CH2) and (CHOSO3"M +) CH2CH3, where x and (y +1) are integers of at least about 7, preferably at least about 9, and M is a cation soluble in water, especially sodium; C10-C18 alkylalkoxy sulfates (especially EO 1-5 ethoxysulfates), C10-C18 alkylalkoxycarboxylates (especially EO 1-5 ethoxycarbpxylates), alkyl polyglycosides of C? oC- | 8 and their corresponding sulfated polyglucosides, esters of C12-C18 a-sulphonated fatty acids, C12-C18 alkyl and alkylphenolalcoxylates (especially ethoxylates and ethoxy / mixed propoxy), betaines and sulfobetaines ('sultaines') of C? 2-C? s, oxides of amine C10-C18, and the like. Preferred herein are alkylalkoxy sulfates (AES) and alkylalkoxycarboxylates (AEC). The use of such surfactants in combination with the amine oxide and / or betaine or sultaine surfactants is also preferred, depending on what the formulator desires. Other common useful surfactants are mentioned in normal texts. Particularly useful surfactants include the C 10 -C 18 N-methyl glucamides described in U.S. Pat. 5,194,639, Connor et al., Issued March 16, 1993. A wide variety of other ingredients useful in detergent cleansing compositions can be included in the compositions herein, including, for example, other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, and solvents for liquid formulations. If it is desired to obtain an additional increase in foaming, they can be incorporated into foam-enhancing compositions, such as C 10 -C 16 -alcolamides, typically at levels of about 1% to about 10%. C-io-C monoethanolamides and diethanolamides illustrate a typical class of such foam enhancers. It is also convenient to use said foam boosters with auxiliary foaming surfactants, such as the amine oxides, betaines and sultaines mentioned above. If desired, soluble magnesium salts, such as MgCl 2, MgSO 4 and the like, can be added, typically at levels of from about 0.1% to about 2% to provide additional foaming. The liquid detergent compositions herein may contain water and other solvents as carriers. The primary or secondary low molecular weight alcohols are exemplary, exemplified by methanol, ethanol, propanol and / so-propanol. Monohydric alcohols are preferred for solubilizing surfactants, but polyols, such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (eg, 1,3-propanediol, ethylene glycol), can also be used. , glycerin and 1,2-propanediol). The compositions may contain from about 5% to about 90%, typically from about 10% to about 50%, of said vehicles. The detergent compositions herein will be formulated, preferably, such that during use in aqueous cleaning operations, the wash water contains a pH between about 6.8 and about 11. The finished products are generally formulated on this scale. Techniques for controlling pH at recommended levels of use include the use of, for example, pH, alkali and acid regulators. Such techniques are well known to those skilled in the art.

In formulating the hard surface cleaning compositions and fabric cleaning compositions of this invention, the formulator may wish to employ various builders at levels of from about 5% to about 50% by weight. Typical builders include zeolites of 1 to 10 microns, polycarboxylates, such as citrates and oxydisuccinates, layered silicates, phosphates and the like. Other common detergency builders are mentioned in the normal forms. Also, the formulator may wish to employ several additional enzymes, such as cellulases, lipases, amylases and proteases in said compositions, typically at levels from about 0.001% to about 1% by weight. Various detersive and fabric care enzymes are well known in the laundry detergent art. Various bleaching compounds, such as percarbonates, perborates and the like, can be used in such compositions, typically at levels of about 1% to about 15% by weight. If desired, said compositions may contain bleaching activators, such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonate and the like, which are also known in the art. Usage levels vary, generally, from about 1% to about 10% by weight. Dirt releasing agents, especially of the anionic oligoester type; chelating agents, especially aminophosphonates and ethylenediamine disuccinates; clay soil removal agents, especially ethoxylated tetraethylenepentamine; dispersing agents, especially polyacrylates and polyaspartates; brighteners, especially anionic brighteners, suds suppressors, especially silicones and secondary alcohols; Fabric softeners, especially smectite clays, and the like, can be used in said compositions at levels ranging from about 1% to about 35% by weight. Normal forms and published patents contain multiple detailed descriptions of such common materials. Also, enzyme stabilizers can be used in the cleaning compositions. Said 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 variants of the present invention are useful in hard surface cleaning compositions. The expression 'hard surface cleaning compositions', as used herein, refers to liquid and granular detergent compositions for cleaning hard surfaces, floors, walls, bathroom tiles and the like. The hard surface cleaning compositions of this invention comprise an effective amount of one or more protease conjugates of this invention, preferably from about 0.001% to about 10%, most preferably from about 0.01% to about 5%, still of greater than Preferably from about 0.05% to about 1% by weight of protease conjugate of the composition. In addition to being one or more protease conjugates, said hard surface cleaning compositions are generally constituted by water soluble surfactants and builders. However, in certain specialized products, such as aerosol glass cleaners, the surfactants are sometimes not used, since they can produce a residue in the form of a layer and / or streaking on the surface of the glass. The surfactant component, when present, may comprise at least 0.1% of the compositions herein, but, generally, the compositions will contain from about 0.25% to about 10%, preferably from about 1% to about 5%. % surfactant. Commonly, the compositions will contain from about 0.5% to about 50% of a builder, preferably from about 1% to about 10%. Preferably, the pH should fall within the range of about 7 to about 12. Common pH adjusting agents, such as sodium hydroxide, sodium carbonate or hydrochloric acid, may be employed if an adjustment is required. Solvents may be included in the compositions. 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 ethylene glycol. , 2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. When used, said solvents are present, generally, at levels of from about 0.5% to about 15%, preferably from about 3% to about 11%. In addition, highly volatile solvents, such as / so-propanol or ethanol, can be used in these compositions to facilitate that the composition evaporates faster from the surfaces when the surface is not cleaned after a 'full concentration' application of the composition to the surface. When used, volatile solvents are usually present at levels of about 2% to about 12% in the compositions.

EXAMPLES 7-12 Liquid hard surface cleaning compositions All formulas are adjusted to a pH of 7. In another embodiment of this invention, dishwashing compositions consist of one or more variants of this invention. The term 'dishwashing composition', as used herein, refers to, but is not limited to, all forms of dishwashing compositions, including granular and liquid forms.

EXAMPLES 13-16 Liquid dishwashing detergent All the formulas are adjusted to a pH of 7.

EXAMPLES 17-19 Liquid compositions for cleaning fabrics Compositions for personal care These protease conjugates are especially suitable for use in personal care compositions, such as rinse and rinse-off hair conditioners, shampoos, rinsing and non-rinsing anti-acne compositions, facial milks and conditioners, shower-bath gels , soaps, foaming and non-foaming facial cleansers, cosmetics, lotions and body, facial and hand moisturizers, rinsing facial moisturizers, cosmetic wipes and cleansers, oral care compositions and contact lens care compositions. These personal care compositions comprise one or more protease conjugates of this invention and a personal care vehicle. By way of illustration, these protease conjugates are suitable for inclusion in the compositions described in the following references: U.S. Patent No. 5,641, 479, Linares et al., Issued June 24, 1997 (cleansers for the skin); U.S. Patent No. 5,599,549 Wivell et al., Issued February 4, 1997 (cleansers for the skin); U.S. Patent No. 5,585,104, Ha et al., Issued December 17, 1996 (cleansers for the skin); U.S. Patent No. 5,540,852, Kefauver et al, issued July 30, 1996 (cleansers for the skin); U.S. Patent No. 5,510,050, Dunbar et al., Issued April 23, 1996 (cleansers for the skin); U.S. Patent No. 5,612,324, Guang Lin et al., Issued March 18, 1997 (anti-acne preparations); U.S. Patent No. 5,587,176, Warren et al., Issued December 24, 1996 (anti-acne preparations); U.S. Patent No. 5,549,888, Venkateswaran, issued on August 27, 1996 (anti-acne preparations); U.S. Patent No. 5,470,884, Corless et al., Issued November 28, 1995 (anti-acne preparations); U.S. Patent No. 5,650,384, Gordon et al., Issued July 22, 1997 (gels for shower bath); U.S. Patent No. 5,607,678, Moore et al., Issued March 4, 1997 (gels for shower bath); U.S. Patent No. 5,624,666, Coffindaffer et al., Issued April 29, 1997 (conditioners and / or shampoos for hair); U.S. Patent No. 5,618,524, Bolich et al., Issued April 8, 1997 (conditioners and shampoos for hair); U.S. Patent No. 5,612,301, Inman, issued March 18, 1997 (conditioners and shampoos for hair); U.S. Patent No. 5, 573,709, Wells, issued November 12, 1996 (conditioners and / or hair shampoos); U.S. Patent No. 5,482,703, Pings, issued January 9, 1996 (conditioners and / or shampoos for hair); U.S. Patent No. Re. 34, 584, Grote et al. Grote et al., Reissued on April 12, 1994 (conditioners and / or shampoos for hair); U.S. Patent No. 5,641, 493, Date et al., Issued June 24, 1997 (cosmetics); U.S. Patent No. 5,605,894, Blank et al., Issued February 25, 1997 (cosmetics); U.S. Patent No. 5,585,090, Yoshioka et al., Issued December 17, 1996 (cosmetics); U.S. Patent No. 4,939,179, Chenev et al., Issued July 3, 1990 (lotions for hands, face and / or body); U.S. Patent No. 5,607,980, McAtee et al., Issued March 4, 1997 (lotions for hands, face and / or body); U.S. Patent No. 4,045,364, Richter et al., Issued August 30, 1997 (cosmetic cloths and cleansers); European patent application EP 0619074, Touchet et al., published on October 12, 1994 (cosmetic cloths and cleansers); U.S. Patent No. 4,975,217, Brown-Skrobot et al., Issued December 4, 1990 (cosmetic cloths and cleansers); U.S. Patent No. 5,096,700, Seibel, issued March 17, 1992 (compositions for oral cleaning); U.S. Patent No. 5,028,414, Sampathkumar, issued July 2, 1991 (compositions for oral cleaning); U.S. Patent No. 5,028,415, Benedict et al., Issued July 2, 1991 (compositions for oral cleaning); U.S. Patent No. 5,028,415, Benedict et al., Issued July 2, 1991 (compositions for oral cleaning); U.S. Patent No. 4,863,627, Davies et al .. September 5, 1989 (cleaning solutions for contact lenses); U.S. Patent No. Re. 32,672, Huth et al., Reissued on May 24, 1988 (cleaning solutions for contact lenses); and U.S. Patent No. 4,609,493, Schafer, issued September 2, 1986 (cleaning solutions for contact lenses). To further illustrate the buccal cleaning compositions of this invention, an amount of one or more pharmaceutically acceptable protease conjugates of this invention is included in the compositions useful for removing proteinaceous stains from teeth or dentures. The term 'oral cleansing compositions', as used herein, refers to dentifrices, toothpastes, dental gels, dental powders, mouth rinses, mouth sprays, mouth gels, chewing gums, lozenges, pouches, tablets, biogels, prophylactic pastes, dental treatment solutions and the like. Preferably, the oral cleaning compositions consist of from about 0.0001% to about 20% of one or more protease conjugates of this invention, most preferably from about 0.001% to about 10%, most preferably from about 0.01% to about 5%. % by weight of composition, and a pharmaceutically acceptable vehicle. The term "pharmaceutically acceptable", as used herein, means that the drugs, medicaments or inert ingredients described by the term are suitable for use in contact with human and lower animal tissues without risking undue toxicity, incompatibility. , instability, irritation, allergic response and the like, in proportion to a reasonable benefit / risk ratio. Typically, the pharmaceutically acceptable components of the buccal cleaning vehicle of the buccal cleaning components of the buccal cleaning compositions will generally be comprised of from about 50% to about 99.99%, preferably from about 65% to about 99.99%, most preferably from about 65% to about 99%, by weight of the composition. The pharmaceutically acceptable components of the vehicle and the optional components that can be included in the buccal cleaning compositions of this invention are well known to those skilled in the art. A wide variety of types of compositions, vehicle components and optional components useful in buccal cleaning compositions is described in the references cited above. In another embodiment of this invention, denture cleansing compositions for cleaning dentures outside the buccal cavity comprise one or more protease conjugates of this invention. Said denture cleansing compositions consist of an effective amount of one or more protease conjugates, preferably from about 0.0001% to about 50% of one or more variants, most preferably from about 0.001% to about 35%, even more preferably from approximately 0.01% to approximately 20%, by weight of the composition, and a denture cleaning vehicle. Various formats of denture cleansing compositions, such as effervescent tablets and the like, are well known in the art (see, for example, U.S. Patent No. 5,055,305, Young) and, in general, are suitable for incorporating one or more conjugates of protease to remove proteinaceous spots from dentures. In another embodiment of this invention, the contact lens cleaning compositions consist of one or more protease conjugates of this invention. Said contact lens cleaning compositions contain an effective amount of one or more protease conjugates, preferably from about 0.01% to about 50% of one or more variants, most preferably from about 0.01% to about 20%, most preferably still from about 1% to about 5%, by weight of the composition, and a contact lens cleaner vehicle. Various formats of contact lens cleaning compositions, such as tablets, liquids and the like, are well known in the art and are generally suitable for incorporating one or more protease conjugates of this invention to remove proteinaceous stains from contact lenses.

EXAMPLES 20-23 Contact lens cleaning solution EXAMPLES 24-27 Products for washing the body EXAMPLES 28-31 Products for washing the face EXAMPLES 32-33 Non-wettable skin moisturizing composition EXAMPLE 34 Composition in cleaning cloths The above composition is impregnated in an absorbent woven cloth composed of cellulose and / or polyester at about 250% by weight of the absorbent cloth.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: WEISGERBER, D. et al. (ii) TITLE OF THE INVENTION: MODIFIED PROTEASES WHICH HAVE DECREASED IMMUNOGENICITY (iii) NUMBER OF SEQUENCES: 1 (iv) ADDRESS TO SEND CORRESPONDENCE: (A) RECIPIENT: THE PROCTER & GAMBLE COMPANY (B) STREET: 11810 EAST MIAMI RIVER ROAD (C) CITY: ROSS (D) STATE: OH (E) COUNTRY: E.U.A. (F) POSTAL CODE: 45061 (v) COMPUTER LEADABLE FORM: (A): TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM COMPATIBLE PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) INFORMATION ABOUT THE POWDER / AGENT: (A) NAME: HERSKO, BART S. (B) REGISTRATION NUMBER: 32,572 (C) APPOINED NUMBER: (¡X) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 513-627-0633 (B) TELEFAX: 513-627-0260 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 275 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Wing Gln Ser Val Pro Tyr Gly Val Ser Gln He Lys Wing Pro Wing Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Wing Val He Asp 20 25 30 Ser Gly He Asp Ser Ser His Pro Asp Leu Lys Val Wing Gly Gly Wing 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Wing Gly Thr Val Wing Ala Wing Asn Asn Being He Gly 65 70 75 SO Val Leu Gly Val Ala Pro Ser Ala Be Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Wing Asp Gly Ser Gly Gln Tyr Ser Trp He He Asn Gly lie Glu 100 105 110 Trp Wing Wing Wing Asn Asn Met Asp Val He Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Wing Wing Leu Lys Wing Wing Val Asp Lys Wing Val Wing 130 135 140 Se Gly Val Val Val Val Wing Wing Wing Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Being Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val He Wing 165 170 175 Val Gly Ala Val Asp Ser Ser A = n Gln Arg Wing Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Wing Pro Gly Val Ser He Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Wing Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu He Leu Ser Lys Hi = Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu He Asn Val Gln Wing 260 265 270 Wing Wing Gln 275

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - A protease conjugate characterized by a protease portion and one or more addition portions wherein: (a) the protease portion has a modified amino acid sequence of a progenitor amino acid sequence, the progenitor amino acid sequence comprises a first epitope region , a second epitope region, and a third epitope region, wherein the modified amino acid sequence comprises a substitution by an amino acid substituent at one or more positions in one or more of the epitope regions wherein: (i) when a Substitution occurs in the first epitope region, substitution occurs in one or more positions corresponding to positions 70-84 of the subtilisin BPN '; (ii) when a substitution occurs in the second epitope region, substitution occurs in one or more positions corresponding to positions 103-126 of the subtilisin BPN '; and (iii) when a substitution occurs in the third epitope region, substitution occurs in one or more positions corresponding to positions 217-252 of the subtilisin BPN '; and (b) wherein each of the addition portions is covalently attached to one of the substituent amino acids present in the protease portion and has the structure:

R t \ * -en where X is selected from the group consisting of null and a link portion; R ^ is selected from the group consisting of null, a first polypeptide, and a first polymer; and R2 is selected from the group consisting of null, a second polypeptide, and a second polymer; where at least one of X, R-i, and R2 is not null. 2. The protease conjugate according to claim 1, further characterized in that the amino acid substituent is cysteine.

3. The protease conjugate according to any of the preceding claims, further characterized in that the progenitor amino acid sequence is selected from the group consisting of subtilisin BPN ', subtilisin Carisberg, subtilisin DY, subtilisin 309, proteinase K, termitase, Protease A, Protease B, Protease C, and Protease D, and variants thereof.

4. The protease conjugate according to any of the preceding claims, further characterized in that Ri is zero.

5. The protease conjugate according to any of claims 1, 2, or 3, further characterized in that Ri is the first polypeptide.

6. - The protease conjugate according to any of claims 1, 2, 3 or 5, further characterized in that the first polypeptide has a modified amino acid sequence of a second progenitor amino acid sequence, the second progenitor amino acid sequence comprises a first epitope region, a second epitope region, and a third epitope region, wherein the modified amino acid sequence of the second progenitor amino acid sequence * # • comprises a substitution by an amino acid substituent at one or more positions in one or more of the epitope regions of the second sequence 10 of progenitor amino acids wherein: (i) when a substitution occurs in the first epitope region, substitution occurs in one or more of positions corresponding to positions 70-84 of the subtilisin BPN '; (ii) when a substitution occurs in the second epitope region, substitution occurs in one or more positions corresponding to positions 103-126 of the 15 subtilisin BPN '; and (iii) when a substitution occurs in the third epitope region, substitution occurs in one or more positions corresponding to positions 217-252 of the subtilisin BPN '; and wherein one of the linking portion or the protease portion is covalently bound to the first polypeptide through one of the substituent amino acids present in the 20 first polypeptide.

7. The protease conjugate according to any of claims 1, 2, 3, 5, 0 6, further characterized in that X is null and further characterized in that the protease portion and the first polypeptide are covalently linked through a disulfide bridge.

8. The protease conjugate according to any of claims 1, 2, or 3, further characterized in that Ri is the first polymer and R2 is selected from the group consisting of null and the second polymer.

9. The protease conjugate according to any of the preceding claims, further characterized in that R2 is zero.

10. A composition for personal care comprising a protease conjugate according to any of the preceding claims and a vehicle for personal care.