WO2001007576A2 - Intramolecularly cross-linked subtilisin proteases having reduced immunogenicity - Google Patents

Abstract

The present disclosure describes subtilisin proteases comprising an intramolecular cross-link, wherein the intramolecular cross-link comprises a covalent linkage between an amino acid of a first residue of the protease and an amino acid of a second residue of the protease. The protease have decreased immunogenicity relative to a parent protease. Accordingly, such proteases are suitable for use in several types of compositions including, but not limite to, laundry, dishes, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.

Description

INTRAMOLECULARLY CROSS-LINKED SUBTILISIN PROTEASES HAVING REDUCED IMMUNOGENICITY

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

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

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

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

However, because such modified proteases are foreign to mammals, they are potential antigens As antigens, these proteases cause an immunogenic and / or allergemc response (herein collectively descnbed as immunogenic response) in mammals

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

Presently, immunogenic response to proteases may be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne proteases, still present the nsks associated with extended tissue contact with the finished composition.

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

The present inventors have surpnsmgly discovered that by mtramolecularly cross-linking the protease, the lmmunogemcity of the protease is reduced. It has further been discovered that these mtramolecularly cross-linked proteases maintain the activity of the respective parent protease. In addition, the present inventors have discovered defined residues withm the protease which are most preferable to participate in cross-linking due to, for example, stenc location to the epitope regions (i e , locations of the protease which are responsible for the immune response) or clip sites (i.e., locations of the protease where hydrolysis occurs in vivo) of the protease.

The present inventors have therefore discovered subtihsm proteases which evoke a decreased immunogenic response yet maintain their activity as an efficient and active protease. Accordingly, the present proteases are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.

SUMMARY OF THE INVENTION The present invention is directed to subtihsm proteases compnsmg an intramolecular cross-l k, wherein the intramolecular cross-lmk compπses a covalent linkage between an ammo acid of a first residue of the protease and an amino acid of a second residue of the protease.

The proteases of the present mvention have decreased lmmunogemcity relative to the parent protease. Accordingly, such proteases are suitable for use m several types of compositions including, but not limited to, laundry, dish, hard surface, skm care, hair care, beauty care, oral care, and contact lens compositions.

DETAILED DESCRIPTION OF THE INVENTION

The essential components of the present mvention are herein descnbed below. Also included are non-hmitmg descnptions of various optional and prefeπed components useful in embodiments of the present invention

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

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

All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impunties, for example, residual solvents or byproducts, which may be present in commercially available sources.

All documents referred to herein, including all patents, patent applications, and publications, are hereby incorporated by reference in their entirety.

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

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

Table I

Definitions

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

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

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

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

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

As referred to herein, while the mtramolecularly cross-linked proteases of the present invention are not limited to those comprising subtihsm BPN' and vanants thereof, all ammo acid numbenng is with reference to the amino acid sequence for subtihsm BPN' which is represented by SEQ ID NO: l. The ammo acid sequence for subtihsm BPN' is further descnbed by Wells et al, Nucleic Acids Research, Vol. II, pp. 7911 - 7925 (1983).

As used herein, wherein a hyphen is utilized to define a region of ammo acid residues, it is intended that this region is inclusive of the listed residues and each residue occurπng between the listed residues. For example, wherein the region 70 - 84 is set forth, this is intended to convey the region of ammo acid residues 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84. mtramolecularly Cross-Linked Proteases of the Present Invention The present invention is directed to subtihsm proteases compnsmg an intramolecular cross-lmk, wherein the intramolecular cross-lmk compπses a covalent linkage between an amino acid of a first residue of the protease and an ammo acid of a second residue of the protease. Without intending to be limited by theory, it is believed that the resulting cross-lmk decreases the rate of protease degradation in the endosomal compartments of the antigen presenting cells (i.e , increases stability) which, in turn, impedes presentation of the epitopes of the protease.

The proteases which may be mtramolecularly cross-linked according to the present invention are subtilism-hke proteases, either wild-type or vaπants thereof. As used herein, the term "subtihsin-like protease" means a protease which has at least 50%, and preferably 80%, ammo acid sequence identity with the sequences of subtihsm BPN'. Wild-type subtihsin-like proteases are produced by, for example, Bacillus alcalophύus , Bacillus amylohquefaciens . Bacillus amylosaccharicus, Bacillus hcheniformis , Bacillus lentus, and Bacillus subtύis microorganisms A discussion relating to subtihsin-hke senne proteases and their homologies may be found in Siezen et al. "Homology Modelling and Protein Engmeenng Strategy of Subtilases, the Family of Subtihsm-Like Serme Proteases", Protein Engineering, Vol. 4, No. 7, pp. 719 - 737 (1991).

Preferred proteases which may be mtramolecularly cross-linked in accordance with the present invention include, for example, those obtained from Bacillus amylohquefaciens, Bacillus hcheniformis, and Bacillus subtihs, subtihsm BPN, subtihsm BPN', subtihsm Carlsberg. subtihsm DY, subtihsm 309, protemase K, and thermitase, including A/S Alcalase®

(commercially available from Novo Industries, Copenhagen, Denmark), Esperase® (Novo

Industπes), Savinase® (Novo Industries), Maxatase® (commercially available from Genencor

International Inc.), Maxacal® (Genencor International Inc ), Maxapem 15® (Genencor International Inc.), and variants of the foregoing. Especially preferred proteases which may be mtramolecularly cross-lmked in accordance with the present invention are those obtained from Bacillus amylohquefaciens and variants thereof The most preferred proteases herein are subtihsm BPN' and vaπants thereof.

Especially preferred variants of subtihsm BPN', hereinafter referred to as "Protease A", for use herein are disclosed in U.S. Patent No 5,030,378, Venegas. issued July 9, 1991, as characterized by the subtihsm BPN' ammo acid sequence with the following mutations-

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

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

Additionally preferred vanants of subtihsm BPN', hereinafter referred to as "Protease B", for use in intramolecular cross-lmkmg herein are disclosed in EP 251 ,446, assigned to Genencor International, Inc., published January 7, 1988, as characterized by the wild-type subtihsm BPN' ammo acid sequence with mutations at one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, Hιs67, Ser87, Lys94, Val95, Gly97, SerlOl, Gly 102, Gly 103, Ilel07, Gly 110, Met 124, Glyl27, Glyl28, Prol29, Leul35, Lysl70, Tyrl71, Prol72, Aspl97, Metl99, Ser204, Lys213, Tyr214, Gly215, and Ser221 ; or two or more of the positions listed above combined with one or more mutations at positions selected from Asp32, Ser33, Tyrl04, Alal52, Asnl55, Glul56, Glyl66, Glyl69, Phel89, Tyr217, and Met222.

Other preferred subtihsm BPN' variants for use m intramolecular cross-lmkmg herein are hereinafter referred to as "Protease C", and are descnbed in WO 95/10615, assigned to Genencor International Inc., published April 20, 1995, as characterized by the wild-type subtihsm BPN' ammo acid sequence with a mutation to position Asn76, in combination with mutations in one or more other positions selected from Asp99, SerlOl, Glnl03, Tyrl04, Serl05, Ilel07, Asnl09, Asnl23, Leul26, Glyl27, Glyl28, Leul35, Glul56, Glyl66, Glul95, Aspl97, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265, and Ala274.

Other preferred subtihsm BPN' variants for use in intramolecular cross-lmkmg herein, hereinafter referred to as "Protease D", are descnbed in U.S. Patent No. 4,760,025, Estell et al., issued July 26, 1988, as characterized by the wild-type subtihsm BPN' ammo acid sequence with mutations to one or more ammo acid positions selected from the group consisting of Asp32, Ser33, Hιs64, Tyrl04, Asnl55, Glul56, Gly 166, Glyl69, Phel89, Tyr217, and Met222.

The more preferred proteases which may be mtramolecularly cross-linked herein are selected from the group consisting of subtihsm BPN', Protease A, Protease B, Protease C, and Protease D, with Protease D being the most preferred.

The mtramolecularly cross-linked proteases of the present invention compπse a covalent linkage between two ammo acids of a foregoing protease. These ammo acids are referred to independently herein as the ammo acid of a first residue and the amino acid of a second residue. As used herein, the terms "first residue" and "second residue" should not be construed as referπng to position 1 and position 2, respectively, corresponding to subtihsm BPN'. Rather, these terms are used simply to claπfy references to one residue or the other. For example, the first residue may correspond to position 27 of subtihsm BPN' and the second residue may correspond to position 118 of subtihsm BPN'. Of course, it is certainly permissible withm the scope of the present invention that the first residue corresponds to position 1 of subtihsm BPN' or that the second residue corresponds to position 2 of subtihsm BPN'.

Additionally, use of the term "corresponding to position" with reference to a particular position coπespondmg to subtihsm BPN' should not be construed as reqmπng the ammo acid which naturally occurs at that position. Rather, such language is intended to refer to the position number, rather than a particular ammo acid residue which occurs at that position. Indeed, in accordance with the present invention, cysteme (which does not naturally occur in subtihsm BPN') is a highly preferred ammo acid residue of the first residue or the second residue.

In accordance with the present invention, the intramolecular cross-lmk of the subtihsm protease comprises a covalent linkage between an ammo acid of a first residue of the protease and an ammo acid of a second residue of the protease. Such covalent linkage compπses a linking moiety which bridges the first residue and the second residue. The linking moiety may be any structural mechanism through which the first residue and the second residue may be covalently bndged (attached). As used herein, however, the linking moiety is not simply a covalent bond such as that which forms, for example, a mere disulfide bπdge. Without intending to be limited by theory, the present inventors have discovered that such disulfide bπdges are ineffective m providing an mtramolecularly cross-linked protease having decreased lmmunogemcity relative to the parent

For example, the linking moiety may be any small molecule, i e , a molecule having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300.

The most preferred linking moieties include those capable of being covalently bound to one or more cysteme residues, lysine residues, and / or the ammo terminus of the protease. For example, m a preferred embodiment of the present invention, the ammo acid of the first residue compnses an amme group (e.g , as non-limiting examples, the ammo acid of the first residue is lysine (either through natural occurrence or mutation) or the ammo acid of the first residue is the ammo terminus of the protease) Additionally, m this preferred embodiment, the ammo acid of the second residue comprises a thiol group, e g , as a non-limiting example, the ammo acid of the second residue is cysteme (either through natural occurrence or mutation). For example, the following non-hmitmg reagents may be utilized to form the covalent linkage of the mtramolecularly cross-linked proteases of the present invention: N-[alpha- maleιmιdoacetoxy]succmιmιde ester; N-5-azιdo-2-nιtrobenzoyloxysuccmιmιde; bismaleimidohexane; N-[beta-maleιmidopropyloxy]succmιmιde ester; bιs[2-

(succιmmιdyloxycarbonyloxy)-ethyl]sulfone; bιs[sulfosuccmιmιdyl]suberate; 1 ,5-dιfluoro-2,4- dmtrobenzene; dimethlyadipimate • 2 HCl; dimethylpimelimidate • 2 HCl; dimethyl subeπmidate • 2 HCl; disuccmimidyl glutarate; disuccmimidyl suberate; w-maleιmιdobenzoyl-N- hydroxysuccinimide ester; N-hydroxysuccmιmιdyl-4-azιdosalιcyhc acid; N-succmιmιdyl-6-[4'- azιdo-2'-nιtrophenylammo]hexanoate; N-hydroxysuccmimidyl 2,3-dιbromopropιonate; succmimidyl 4- N-maleιmιdomethyl]cyclohexane-l-carboxylate; succmimidyl 4-(p- maleιmιdophenyl)-butyrate; succmιmιdyl-6-[(beta-maleιmιdopropιonamιdo)hexanoate] ; bιs[2- (sulfosuccιmmιdyloxycarbonyloxy)-ethyl]sulfone; N-[gamma- maleιmιdobutyryloxy]sulfosuccmιmιde ester; N-hydroxysulfosuccιmmιdyl-4-azιdobenzoate; N- [kappa-maleimidoundecanoyloxy] sulfosuccmimide ester; w-maleιmιdobenzoyl-N- hydroxysulfosuccmimide ester; sulfosuccmιmιdyl[4-azιdosahcylamιdo]hexanoate, sulfosuccmimidyl 7-azιdo-4-methylcoumann-3-acetate; sulfosuccimmidyl 6-[4'-azιdo-2'- nitrophenylammojhexanoate; sulfosuccimmidyl 4-[p-azιdophenyl]butyrate; sulfosuccimmidyl [4- ιodoacetyl]amιnobenzoate; sulfosuccimmidyl 4-[N-maleιmιdomethyl]cyclohexane-l-carboxylate; and sulfosuccimmidyl 4-(p-maleιmιdophenyl)-butyrate. Each of these reagents is commercially available from Pierce Chemical Co., Rockford, IL. Non-hmitmg examples of preparation of the present mtramolecularly cross-linked proteases using similar reagents is set forth herein below.

Other examples of linking moieties and related chemistry are disclosed in U.S. Patent No 5,446,090. Hams, issued August 29, 1995; U.S. Patent No. 5,171,264, Merπll, issued December 15, 1992; U.S. Patent No. 5,162,430, Rhee et al.. issued November 10, 1992; U.S. Patent No. 5,153,265, Shadle et al. issued October 6, 1992; U.S. Patent No. 5,122,614, Zahpskv, issued June 16, 1992; Goodson et al., "Site-Directed Pegylation of Recombinant Interleukm-2 at its Glycosylation Site", Biotechnology, Vol. 8, No. 4, pp. 343 - 346 (1990); Kogan, "The Synthesis of Substituted Methoxy-Poly(ethylene glycol) Derivatives Suitable for Selective Protein Modification", Synthetic Communications, Vol. 22, pp. 2417 - 2424 (1992); and Ishn et al., "Effects of the State of the Succinmiido-Rmg on the Fluorescence and Structural Properties of Pyrene Maleimide-Labeled aa-Tropomyosm", Biophysical Journal, Vol. 50, pp. 75 - 80 (1986). The most preferred linking moiety is substituted (for example, alkyl) or unsubstituted succimmide In a preferred embodiment of the present invention, the ammo acid of the first residue compnses an amine group (e.g., as non-hmiting examples, the ammo acid of the first residue is lysine (either through natural occurrence or mutation) or the ammo acid of the first residue compnses the amino terminus of the protease). It is therefore preferred to mutate lysine residues which occur at positions other than the first and second residues to one or more other ammo acid residues such that cross-lmkmg of a lysine residue at, e.g. , the first residue, is selective. For example, a lysine residue occurs at position 43 of subtihsm BPN' which is in the first proximal epitope region as defined herein. Site-selective mutation of all other lysme residues occurnng m the protease may be performed followed by selective cross-lmkmg of the lysine residue at position 43 to the second residue. Alternatively, a naturally occurring amino acid of the first residue may be mutated to lysine (for example), followed by site-selective mutation of all other lysine residues occurnng in the protease, and selective cross-lmkmg of the lysine residue to the second residue.

In this same preferred embodiment, it is preferred that the ammo acid of the second residue compπses a thiol group, e g., as a non-hmitmg example, the ammo acid of the second residue is cysteme (either through natural occurrence or mutation). In this example, wherein a cysteme residue occurs at a position other than the second residue, it is preferable to substitute another ammo acid residue for cysteme in each of those positions to enable selective cross- linking between the first residue and the second residue.

In an alternate preferred embodiment of the present invention, the ammo acid of the first and second residues each comprise a thiol group. As a non-hmiting example, the ammo acid of the first residue is cysteme (either through natural occurrence or mutation) and the ammo acid of the second residue is cysteme (either through natural occurrence or mutation). In this example, wherein a cysteme residue occurs at a position other than the first and second residues, it is preferable to substitute another ammo acid residue for cysteme in each of those positions to enable selective cross-lmkmg between the first residue and the second residue.

In a further preferred embodiment of the present invention, the ammo acid of the first and second residues each comprise an amine group. As a non-hmitmg example, the ammo acid of the first residue may be lysine or the ammo terminus (either through natural occurrence or mutation) and the ammo acid of the second residue is lysine (either through natural occurrence or mutation). In this example, wherein lysine occurs at a position other than the first and second residues, it is preferable to substitute another ammo acid residue for lysine in each of those positions to enable selective cross-linking between the first residue and the second residue. Accordingly, in a preferred embodiment of the present invention, the ammo acid of the first residue is lysine or comprises the ammo terminus and the ammo acid of the second residue is cysteme. In an alternate preferred embodiment of the present mvention, the amino acid of the first residue is cysteme and the ammo acid of the second residue is cysteme. In a further preferred embodiment of the present invention, the ammo acid of the first residue is lysme or the ammo terminus and the ammo acid of the second residue is lysme.

In a preferred embodiment of the present invention, at least one of the first and second residues occur withm a defined region. It has been discovered that the subtihsm protease compπses three epitope regions Accordingly, without intending to be limited by theory, it is believed that wherein a residue of one or more of these epitope regions participates in cross- lmkmg, lmmunogemcity is reduced because presentation of the epitope is impeded For example, wherein a residue of one or more of the epitope regions is cross-linked to another residue of the protease, multiple cleavages of the protease will be required pπor to presentation of the epitope. It has been discovered that the first epitope region corresponds to positions 70 - 84 of subtihsm BPN'; the second epitope region corresponds to positions 103 - 126 of subtihsm BPN'; and the third epitope region corresponds to positions 220 - 246 of subtihsm BPN'. See, e.g., U.S. Patent Application Seπal No. 09/088,912, Weisgerber et al., assigned to The Procter & Gamble Co., filed June 2, 1998, copendmg U.S. Provisional Patent Application Seπal No. 60/144,991, Rubingh et al., "Seπne Protease Vanants Having Ammo Acid Substitutions and Deletions m Epitope Regions" filed July 22, 1999; and copendmg U S. Provisional Patent Application Senal No. 60/144,980, Sikorski et al., "Serme Protease Vaπants Having Ammo Acid Substitutions in Epitope Regions" filed July 22, 1999

It has additionally been discovered that certain residues of the protease are m close proximity to the epitope regions of the protease. The present inventors have discovered that residues of the first, second, and third proximal epitope regions are in close proximity to the first, second, and third epitope regions, respectively. Accordingly, without intending to be limited by theory, it is believed that wherein a residue of one or more of a first, second, or third proximal epitope region participates in cross-linking, lmmunogenicity is reduced because presentation of the epitope is impeded. It has been discovered that the first proximal epitope region corresponds to positions 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 of subtihsm BPN'; the second proximal epitope region corresponds to positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145 of subtihsm BPN'; and the third proximal epitope region corresponds to positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275 of subtihsm BPN' See, e.g., U.S. Provisional Patent Application Seπal No. 60/144,979, Rubingh et al., "Protease Conjugates Having Steπcally Protected Epitope Regions", filed July 22, 1999.

Additionally, also as a preferred embodiment of the present mvention, it has been discovered that the subtihsm protease compπses a first "clip site" region and a second "clip site" region (i e , locations of the protease where hydrolysis occurs in vivo). Without intending to be limited by theory, it is believed that wherein one or more residues m these clip site regions participates in cross-lmkmg, hydrolysis of the protease is impeded and thus presentation of an epitope is similarly impeded. It has been discovered that the first clip site region corresponds to positions 156 - 165, 170, 186, 191 - 196, and 259 - 262 of subtihsm BPN' and the second clip site region corresponds to positions 12 - 24, 27, 84 - 88, 271, and 274 of subtihsm BPN' See, e.g , U.S Provisional Patent Application Serial No. 60/144,981, Weisgerber et al., "Protease Conjugates Having Stencally Protected Clip Sites", filed July 22, 1999.

Accordingly, m a preferred embodiment of the present mvention, the present protease compπses an intramolecular cross-link, wherein the intramolecular cross-lmk compnses a covalent linkage between an ammo acid of a first residue of the protease and an amino acid residue of a second residue of the protease, wherein at least one of the residues occurs withm a region selected from'

(a) an amino terminus region corresponding to position 1 of subtihsm BPN';

(b) a first epitope region corresponding to positions 70 - 84 of subtihsm BPN',

(c) a second epitope region corresponding to positions 103 - 126 of subtihsm BPN'.

(d) a third epitope region corresponding to positions 220 - 246 of subtihsm BPN',

(e) a first clip site region corresponding to positions 156 - 165, 170, 186, 191 - 196, and 259 - 262 of subtihsm BPN';

(f) a second clip site region corresponding to positions 12 - 24, 27, 84 - 88, 271, and 274 of subtihsm BPN';

(g) a first proximal epitope region corresponding to positions 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 of subtihsm BPN',

(h) a second proximal epitope region corresponding to positions 25, 26, 27, 46, 47. 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133. 134, 136, 137, 138, 140, 141, 144, and 145 of subtihsm BPN', and (i) a third proximal epitope region corresponding to positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275 of subtihsm BPN'.

Wherein only one of the first residue and second residue occurs withm a foregoing region, the other residue is selected from any other position of the protease.

Preferably, at least one of the residues occurs withm a region selected from the ammo terminus region, the first epitope region, the second epitope region, the third epitope region, the first clip site region, and the second clip site region. More preferably, at least one of the residues occurs withm a region selected from the ammo terminus region, the first epitope region, the second epitope region, and the third epitope region. Even more preferably, at least one of the residues occurs withm a region selected from the first epitope region, the second epitope region, and the third epitope region. Most preferably, at least one of the residues occurs within the first epitope region.

In a further preferred embodiment of the present invention, each of the first and second residues of the protease occur withm a region selected from the ammo terminus region, the first epitope region, the second epitope region, the third epitope region, the first clip site region, the second clip site region, the first proximal epitope region, the second proximal epitope region, and the third proximal epitope region. Still preferably, the first residue corresponds to position 1 of subtihsm BPN' and the second residue occurs withm a region selected from the group consisting of the first epitope region, the second epitope region, the third epitope region, the first clip site region, the second clip site region, the first proximal epitope region, the second proximal epitope region, and the third proximal epitope region

Wherein at least one of the residues occurs withm the first epitope region, the region preferably corresponds to positions 75 - 83 of subtihsm BPN'. More preferably, the region corresponds to position 78 of subtihsm BPN'.

Wherein at least one of the residues occurs withm the second epitope region, the region preferably corresponds to positions 109, 114, and 118 of subtihsm BPN'. most preferably 118.

Wherein at least one of the residues occurs within the third epitope region, the region preferably corresponds to position 240 of subtihsm BPN'

Wherein at least one of the residues occurs withm the first clip site region, the region preferably corresponds to positions 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, and 262 of subtihsm BPN'. More preferably, the region corresponds to positions 158, 159, 160, 161, 162, 163, 164, 165, 170, 191, 192, 193, 194, 261, and 262 of subtihsm BPN'. Even more preferably, the region corresponds to positions 158, 159, 160, 161, 162, 163, 164, 192, 193, 194, 261, and 262 of subtihsm BPN'. Most preferably, the region corresponds to positions 160, 161, 162, 163, and 261 of subtihsm BPN'.

Wherein at least one of the residues occurs withm the second clip site region, the region preferably corresponds to positions 13, 14, 15, 16, 18, 19, 20, and 21 of subtihsm BPN'. More preferably, the region corresponds to positions 14, 15, 16, 18, 19, 20, and 21 of subtihsm BPN'. Most preferably, the region corresponds to positions 18, 19, 20, and 21 of subtihsm BPN'.

Wherein at least one of the residues occurs withm the first proximal epitope region, the region preferably corresponds to positions 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86, 87, 89, 206, 209, 214, and 215 of subtihsm BPN'. Most preferably, the region corresponds to positions 2, 3, 4, 5, 17, 40, 41, 43, 67, 86, 87, and 214 of subtihsm BPN'.

Wherein at least one of the residues occurs withm the second proximal epitope region, the region preferably corresponds to positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145 of subtihsm BPN'. Most preferably, the region corresponds to positions 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138, and 141 of subtihsm BPN'.

Wherein at least one of the residues occurs within the third proximal epitope region, the region preferably corresponds to positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157,

172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272, and 275 of subtihsm BPN'. Most preferably, the region corresponds to positions 22, 23, 24, 143, 146, 155.

173, 189, 197, 203, 204, 253, 254, 265, and 275 of subtihsm BPN'

Table 2 below sets forth non-hmitmg examples of preferred mtramolecularly cross-linked subtihsm proteases wherein, m each example, the position numbers listed indicate the first residue and second residue, respectively, i.e , through which residues the intramolecular crosslink occurs Consistent with the disclosure herein, all residue numbenng corresponds to subtihsm BPN' numbenng.

Table 2

Optional Moieties

The mtramolecularly cross-linked proteases of the present invention may additionally compnse one or more other chemical structures, including (for example) one or more small molecules, polypeptides, and / or polymers covalently attached to any of the residues of the protease (herein referred to as "supplementary moieties"), particularly those residues which do not participate in intramolecular cross-lmkmg. As used herein, the term "small molecule" means a molecule having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300. As used herein, the term "polypeptide" means a molecule compnsmg two or more amino acid residues). As used herein, the term "polymer" means any molecule which compπses two or more identical (preferably five or more identical) monomer units.

Supplementary moieties may include polypeptide moieties, polymer moieties, and linking moieties as descnbed in, for example, WO 96/17929, Olsen et al., Novo Nordisk A S, published June 13, 1996; WO 96/40791, Olsen et al.. Novo Nordisk A/S, published December 19, 1996; WO 96/40792, Olsen et al.. Novo Nordisk A/S, published December 19, 1996; WO 97/30148, Bisgard-Frantzen et al.. Novo Nordisk A S, published August 21, 1997; WO 98/30682, Olsen et aJL, Novo Nordisk A S, published July 16, 1998; WO 98/35026, Von Per Osten et al.. Novo Nordisk A S, published August 13, 1998; WO 99/00849, Olsen et al.. Novo Nordisk A S, published January 7, 1999; U.S. Patent No. 5,856,451, Olsen et al.. Novo Nordisk A S, issued January 5, 1999; WO 97/37007, Bott et al., Genencor International Inc., published October 9, 1997: U.S. Patent Application Senal No. 09/088,912, Weisgerber et al.. The Procter & Gamble Co., filed June 2, 1998; U.S. Patent Application Seπal No. 08/903,298, Weisgerber et al.. The Procter & Gamble Co., filed July 30, 1997; U.S. Provisional Patent Application Seπal No. 60/144,979, Rubingh et al.. "Protease Conjugates Having Steπcally Protected Epitope Regions", filed July 22, 1999; and U.S. Provisional Patent Application Senal No. 60/144,981, Weisgerber et al., "Protease Conjugates Having Steπcally Protected Clip Sites", filed July 22, 1999.

Method of Making

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

A phagemid (pSS-5) containing the wild-type subtihsm BPN' gene is transformed into Escherichia coli dut- ung- strain CJ236 and a single stranded uracil-containmg DNA template is produced using the VCSM13 helper phage (Kunkel et al., "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection", Methods in Enzymology, Vol 154, pp. 367 - 382 (1987), as modified by Yuckenberg et al.. "Site-Directed in vitro Mutagenesis Using Uracil- Contammg DNA and Phagemid Vectors", Directed Mutagenesis - A Practical Approach, McPherson, M. J. ed., pp. 27 - 48 (1991). Pnmer site-directed mutagenesis modified from the method disclosed in Zoller, M. J., and M. Smith. "Ohgonucleotide - Directed Mutagenesis Using Ml 3 - Denved Vectors: An Efficient and General Procedure for the Production of Point Mutations m 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). Oligonucleotides are made using a 380B DNA synthesizer (Applied Biosystems Inc ) Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (Amencan Type Culture Collection E coli 33625) All mutations are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain PG632 (Saunders et al , "Optimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34- Ammo Acid Fragment of Human Parathyroid Hormone", Gene, Vol 102, pp. 277 - 282 (1991) and Yang et al , "Cloning of the Neutral Protease Gene oϊ Bacillus subtilis and the Use of the Cloned Gene to Create an in vitro - Deπved Deletion Mutation", Journal of Bacteriology, Vol 160, pp 15 - 21 (1984)

Fermentation is as follows Bacillus subtilis cells (PG632) containing the protease of interest are grown to mid-log phase m one liter of LB broth containing 10 g/L glucose, and inoculated into a Biostat C fermentor (Braun Biotech, Inc , Allentown, PA) in a total volume of 9 liters The fermentation medium contains yeast extract, casem hydrosylate, soluble - partially hydrolyzed starch (Maltnn M-250), antifoam, buffers, and trace minerals (see "Biology of Bacilli. Applications to Industry", Doi, R H and M. McGloughlm, eds (1992)) The broth is kept at a constant pH of 7 5 dunng the fermentation run Kanamycm (50 μg/mL) is added for antibiotic selection of the mutagemzed plasmid The cells are grown for 18 hours at 37 °C to an A_δoo of about 60 and the product harvested

The fermentation broth is taken through the following steps to obtain pure protease The broth is cleared oϊ Bacillus subtilis cells by tangential flow against a 0 16 μm membrane The cell-free broth is then concentrated by ultrafiltration with a 8,000 molecular weight cut-off membrane The pH is adjusted to 5 5 with concentrated MES buffer (2-(N- morpholιno)ethanesulfonιc acid) The protease is further purified by cation exchange chromatography with S-sepharose and elution with NaCl gradients See Scopes. R K , "Protein Puπfication Pnnciples and Practice", Spnnger-Verlag, New York (1984)

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

To avoid autolysis of the protease dunng storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the puπfication procedure the punty of the stock protease solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsm inhibitor type II-T: turkey egg white (Sigma Chemical Company, St. Louis, Missouri).

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

Non-hmitmg examples of methods to prepare an mtramolecularly cross-linked protease of the present invention are set forth below wherein, in each example:

symbolizes a protease as descnbed herein above.

Example 1

A vanant of subtihsm BPN' with a substitution of leucme for tyrosme at position 217 and a substitution of cysteme for senne at position 78 is prepared. The vanant is dialyzed (100 mL at a concentration of approximately 2mg / mL) against 5 liters of 5 mM dithiothreitol (DTT; commercially available from Sigma-Aldnch Co., St. Louis, MO) m pH 5.5 buffer over four hours. The vaπant is transferred to fresh buffer without DTT and dialyzed for about 16 hours. The vaπant concentration is measured by absorbance at 280 nm. The thiol concentration is measured (see Deaken et al.. Biochemical Journal, Vol. 89, p. 263 (1963)) to ensure free thiol concentration is greater than the protease concentration. N-(gamma- maleιmιdobutyryloxy)succιnιmιde ester (commercially available from Pierce Chemical, Rockford, IL) is added at an excess of 1.2 times the free thiol concentration After five minutes, approximately 20 mL of 1M pH 6.8 buffer is added. After an additional 30 minutes, approximately 100 mL of 1M pH 5.5 buffer is added. The reaction mixture is then dialyzed against approximately 5 liters of 0.01 M pH 5.5 buffer. The mtramolecularly cross-linked protease compnsmg a covalent linkage between the cysteme of position 78 and the ammo terminus is puπfied over an ion-exchange column. Example 2

A vanant of subtihsm BPN' with a substitution of leucme for tyrosme at position 217, a substitution of cysteme for asparagme at position 240, and a substitution of lysine for asparagme at position 118 is prepared. All other lysine residues of the protease are substituted by an ammo acid other than lysine or cysteme. The vaπant is dialyzed (100 mL at a concentration of approximately 2mg / mL) against 5 liters of 5 mM dithiothreitol (DTT; commercially available from Sigma-Aldnch Co., St. Louis, MO) in pH 5.5 buffer over four hours. The vaπant is transferred to fresh buffer without DTT and dialyzed for about 16 hours. The vaπant concentration is measured by absorbance at 280 nm. The thiol concentration is measured to ensure free thiol concentration is greater than the protease concentration. Succιmmιdyl-4-(N- maleιmιdomethyl)-cyclohexane-l-carboxy(6-amιdocaproate) (commercially available from Pierce Chemical, Rockford, EL) is added at an excess of 1.2 times the free thiol concentration After five minutes, approximately 20 mL of 1M pH 6.8 buffer is added. After an additional 30 minutes, approximately 100 mL of 1M pH 5.5 buffer is added. The reaction mixture is then dialyzed against approximately 5 liters of 0.01 M pH 5.5 buffer. The mtramolecularly cross- linked protease comprising a covalent linkage between the cysteme of position 240 and the lysine of position 118 is puπfied over an ion-exchange column.

Example 3

The N-(gαmmα-maleιmιdobutyryloxy)succmιmιde ester utilized m Example 1 is substituted with any one of the following reagents to form the covalent linkage between the cysteme of position 78 and the ammo terminus: N-(α/p ztf-maleιmιdoacetoxy)succmιmιde ester, N-( ?eta-maleιmιdopropyloxy)succmιmιde ester, or m-maleimidobenzoyl-N-hydroxysuccmimide ester. Each of these reagents is commercially available from Pierce Chemical Company, Rockford, IL. Alternatively, any reagent containing a maleimide moiety and a succmimidyl ester moiety of similar size can be synthesized and substituted for the N-(gamma- maleιmιdobutyryloxy)succmιmιde ester (see Kalgutar et al„ Journal of Medicinal Chemistry, Vol. 39, pp. 1692 - 1703 (1996) and references therein).

Example 4

The succmιmιdyl-4-(N-maleιmιdomethyl)-cyclohexane- 1 -carboxy(6-amιdocaproate) utilized in Example 2 is substituted with any one of the following reagents to form the covalent linkage between the cysteme of position 240 and the lysine of position 118: succmimidyl 4-(p- maleιmιdophenyl)-butyrate, sulfosuccimmidyl 4-(p-maleιmιdophenyl)-butyrate, or N-[kappa- maleimidoundecanoyloxy] sulfosuccmimide ester. Each of these reagents is commercially available from Pierce Chemical Company, Rockford, IL. Alternatively, any reagent containing a maleimide moiety and a succmimidyl ester moiety of similar size can be synthesized and substituted for the N-(gαmwα-maleιmιdobutyryloxy)succmιmιde ester (see Kalgutar et al. Journal of Medicinal Chemistry, Vol. 39, pp. 1692 - 1703 (1996) and references therein).

Example 5

A vanant of subtihsm BPN' with a substitution of leucme for tyrosme at position 217 and a substitution of cysteme for senne at position 78 is prepared All surface lysine residues of the protease are substituted by an amino acid other than lysme or cysteme. The vaπant is dialyzed (100 mL at a concentration of approximately 2mg / mL) against 5 liters of 5 mM dithiothreitol (DTT; commercially available from Sigma-Aldnch Co., St. Louis, MO) m pH 7 - 8 buffer over four hours The vanant is transferred to fresh buffer without DTT and dialyzed for about 4 hours The vaπant concentration is measured by absorbance at 280 nm. The thiol concentration is measured to ensure free thiol concentration is greater than the protease concentration N- succmimidyl (4-ιodoacetyl)ammobenzoate (commercially available from Pierce Chemical, Rockford, IL) is added at an excess of 1.2 times the free thiol concentration. After 30 minutes, approximately 100 mL of 1M pH 5.5 buffer is added The reaction mixture is then dialyzed against approximately 5 liters of 0.01 M pH 5.5 buffer. The mtramolecularly cross-linked protease compnsmg a covalent linkage between the cysteme of position 78 and the ammo terminus is puπfied over an ion-exchange column. Example 6

A vanant of subtihsm BPN' with a substitution of leucme for tyrosme at position 217 and a substitution of cysteme for seπne at position 78 is prepared. The variant is dialyzed (100 mL at a concentration of approximately 2mg / mL) against 5 liters of 5 mM dithiothreitol (PTT; commercially available from Sigma-Aldnch Co., St. Louis, MO) in pH 5.5 buffer over four hours. The vaπant is transferred to fresh buffer without PTT and dialyzed for about 16 hours. The vaπant concentration is measured by absorbance at 280 nm. The thiol concentration is measured to ensure free thiol concentration exceeds the concentration of the protease. N- maleιmιdo[4'-azιdo-2'-nιtrophenylammo]butyrate is added at an excess of 1.2 times the free thiol concentration. After five minutes the reaction is exposed to UV light (320 nm - 350 nm) for 10 minutes. Alternatively, the reaction is exposed to 10 flashes of a standard camera bulb The reaction mixture is then dialyzed against approximately 2 liters 0.01 M pH 5.5 buffer. The mtramolecularly cross-linked protease compnsmg a covalent linkage between the cysteme of posihon 78 and the ammo terminus is purified over an ion-exchange column.

Example 7

The GMAB utilized in Example 6 is substituted with any one of the following reagents to form the covalent linkage between a lysine substituted at position 78 and the ammo terminus (wherein all other lysine residues of the protease are substituted with another ammo acid): N-5- azιdo-2-mtrobenzoyloxysuccmιmιde, N-succmιmιdyl-6-(4'-azιdo-2'- nιtrophenylammo)hexanoate, N-hydroxysulfosuccmιmιdyl-4-azιdobenzoate, sulfosuccιmmιdyl(4- azιdosalιcylamιdo)hexanoate, sulfosuccimmidyl 7-azιdo-4-methylcoumann-3 -acetate, sulfosuccimmidyl 6-(4'-azιdo-2'-nιtrophenylamιno)hexanoate, and sulfosuccimmidyl 4-(p- azιdophenyl)butyrate. Each of these reagents is commercially available from Pierce Chemical Company, Rockford, IL.

Example 8

A vaπant of subtihsm BPN' with a substitution of leucme for tyrosme at position 217 and a substitution of cysteme for seπne at position 78 and a substitution of cysteme for alanme at position 1 is prepared. The vaπant is dialyzed (100 mL at a concentration of approximately 2mg / mL) against 5 liters of 5 mM dithiothreitol (DTT; commercially available from Sigma-Aldnch Co., St. Louis, MO) in pH 5.5 buffer over four hours. The vanant is transferred to fresh buffer without PTT and dialyzed for about 16 hours. The vanant concentration is measured by absorbance at 280 nm. The thiol concentration is measured to ensure free thiol concentration is greater than twice the protease concentration. 10 mL 1M (N-(2-hydroxyethyl)pιperazme-N'-(2- ethanesulfo c acid)) (HEPES) / NaOH buffer pH 7.0 is added to the protease solution Bismaleimidohexane (commercially available from Pierce Chemical, Rockford, IL) is added at an excess of 1.2 times the protease concentration. After one hour, the reaction mixture is then dialyzed twice against twenty volumes of 0.01 M pH 5.5 buffer. The mtramolecularly cross- linked protease comprising a covalent linkage between the cysteme of position 78 and the cysteme at the amino terminus is purified over an ion-exchange column.

Analytical Methods

The present mtramolecularly cross-linked proteases may be tested for enzymatic activity and immunogenic response using the following methods, both of which are known to one skilled in the art. Other methods well-known in the art may alternatively be used. Protease Activity

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

Using Scotch #3750G tape, human skm flakes are stripped from the legs of a subject repeatedly until the tape is substantially opaque with flakes. The tape is then cut into 1 inch by 1 inch squares and set aside. In a 10 mm by 35 mm pern dish, 2 mL of 0.75 mg / mL of a control enzyme (for example, subtihsm BPN') or the protease to be tested is added in 0.01 M KH₂P0₄ pH 5.5 buffer. To this solution 1 mL of 2.5% sodium laurate pH 8.6 solution is added The solution is gently mixed on a platform shaker. The previously prepared tape square is soaked in the solution (flake side up) for ten minutes continuing gentle mixing. The tape square is then nnsed gently in tap water for fifteen seconds. Stevenel Blue Stam (3 mL, commercially available from Sigma Chemical Co., St. Louis, MO) is pipetted into a clean pern dish. The nnsed tape square is placed into the stam for three minutes (flake side up) with gentle mixing. The tape square is removed from the stam and rinsed consecutively in two beakers of 300 mL distilled water, for fifteen seconds per πnse. The tape square is allowed to air-dry. The color intensity between the tape square obtained from the control enzyme and the tape square obtained from the protease is compared visually or by using a chromameter. Relative to the control enzyme tape square, a protease tape square showing less color intensity is indicative of a protease having higher activity. Pved Collagen Activity Method

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

Mouse Intranasal Test for Immunogenicity

The immunogenic potential of the mtramolecularly cross-linked proteases of the present invention may be determined using a methods known in the art or by the Mouse Intranasal Test for lmmunogemcity presented herein below. This test is similar to the assays descnbed in Robinson et al , "Specific Antibody Responses to Subtihsm Carlsberg (Alcalase) m Mice Pevelopment of an Intranasal Exposure Model", Fundamental and Applied Toxicology, Vol. 34, pp. 15 - 24 (1996) and Robmson et al.. "Use of the Mouse Intranasal Test (MINT) to Petermme the Allergemc Potency of Petergent Enzymes: Companson to the Guinea Pig Intratracheal (GPIT) Test", Toxicological Science, Vol. 43, pp. 39 - 46 (1998), both of which assays may be utilized in place of the test set forth herein below.

Female BPF1 mice (Charles River Laboratoπes, Portage, MI) weighing from about 18 to about 20 grams are utilized in the test. The mice are quarantined one week pnor to dosmg. The mice are housed in cages with wood chip bedding in rooms controlled for humidity (30 - 70%), temperature (67 - 77 °F) and 12 hour light and dark cycles. The mice are fed Punna^® mouse chow (Punna Mills, Richmond, IN) and water ad libitum.

The potential antigen to be tested (either subtihsm BPN' as positive control or an mtramolecularly cross-linked protease of the present invention) is dosed to a group of five mice Pnor to dosmg, each mouse is anesthetized by an mtrapentoneal (l.p.) injection of a mixture of Ketaset (88.8 mg/kg) and Rompun (6.67 mg/kg). The anesthetized animal is held m the palm of the hand, back down, and dosed mtranasally with 5 mL protease in buffer solution (0.01 M KH₂P0 , pH 5.5). While each group receives the same dosage, vaπous dosages may be tested Dosmg solutions are gently placed on the outside of each nostril and inhaled by the mouse Dosmg is repeated on days 3, 10, 17, and 24.

Serum samples are collected on day 29. Enzyme-specific IgGl antibody m mouse serum is measured by an antigen capture ELISA method Immunogenicities of the mtramolecularly cross-lmked protease may be compared against those of subtihsm BPN' using standard ED₃₀ values.

Compositions of the Present Invention

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

Cleaning Compositions

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

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

As used herein, "effective amount of protease", or the like, refers to the quantity of mtramolecularly cross-lmked protease necessary to achieve the proteolytic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g , granular, bar) composition is required, and the like. Preferably, the cleaning compositions compπse from about 0.0001% to about 10%, more preferably from about 0.001% to about 1%, and most preferably from about 0.01% to about 0.1 % of one or more proteases of the present invention. Several examples of vaπous cleaning compositions wherein the proteases may be employed are discussed m further detail below

In addition to the present proteases, the present cleaning compositions further compπse a cleaning composition earner compnsmg one or more cleaning composition mateπals compatible with the protease. The term "cleaning composition mateπal", as used herein, means any matenal selected for the particular type of cleaning composition desired and the form of the product (e g , liquid, granule, bar, spray, stick, paste, gel), which matenals are also compatible with the protease used in the composition. The specific selection of cleaning composition mateπals is readily made by considering the surface matenal to be cleaned, the desired form of the composition for the cleaning condition during use (e g , through the wash detergent use). The term "compatible", as used herein, means the cleaning composition mateπals do not reduce the proteolytic activity of the protease to such an extent that the protease is not effective as desired dunng normal use situations Specific cleaning composition matenals are exemplified in detail hereinafter The proteases of the present mvention may be used in a vaπety of detergent compositions wherein high sudsing and good cleansing is desired. Thus the proteases can be used with vanous conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabnc laundeπng compositions, and the like. Such compositions can be m the form of liquids, granules, bars, and the like. Such compositions can be formulated as "concentrated" detergents which contain as much as from about 30% to about 60% by weight of surfactants

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

Nonhmitmg examples of surfactants useful herein include the conventional C_{j ]}-Cι g alkyl benzene sulfonates and pnmary and random alkyl sulfates, the Ciø-Cig secondary (2,3) alkyl sulfates of the formulas CH3(CH₂)_x(CHOS03)-M⁺)CH₃ and CH3(CH₂)_y(CHOS0₃-M⁺)

CH2CH3 wherein x and (y+1) are integers of at least about 7, preferably at least about 9, and M is a water-solubihzmg cation, especially sodium, the Cι _υ-Cι g alkyl alkoxy sulfates (especially

EO 1-5 ethoxy sulfates), Cjo-Cjg alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the Ci rj-Ci g alkyl polyglycosides, and their corresponding sulfated polyglycosides, C^-CJ S a-sulfonated fatty acid esters, Ci 2-Cι alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), Ci 2-Cι g betames and sulfobetaines ("sultames"), Cjo-Cj amme oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. The use of such surfactants in combination with the amme oxide and / or betame or sultame surfactants is also preferred, depending on the desires of the formulator. Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the Ci _Q-Cig N-methyl glucamides disclosed in U.S. Pat No. 5, 194.639, Connor et al.. issued March 16, 1993.

A wide vaπety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein including, for example, other active ingredients, earners, hydrotropes, processing aids, dyes or pigments, and solvents for liquid formulations If an additional increment of sudsmg is desired, suds boosters such as the Ci g-Ci g alkolamides can be incorporated into the compositions, typically at about 1% to about 10% levels. The C10-C14 monoethanol and diethanol amides illustrate a typical class of such suds boosters Use of such suds boosters with high sudsmg adjunct surfactants such as the amme oxides, betames and sultames noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2,

MgS04, and the like, can be added at levels of, typically, from about 0.1% to about 2%, to provide additional sudsmg.

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

The detergent compositions herein will preferably be formulated such that dunng use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of, for example, buffers, alkalis, and acids. Such techniques are well known to those skilled in the art.

When formulating the hard surface cleaning compositions and fabnc cleaning compositions of the present invention, the formulator may wish to employ vanous builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccmates, layered silicates, phosphates, and the like. Other conventional builders are listed m standard formulaπes.

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

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

Soil release agents, especially of the anionic ohgoester type, chelatmg agents, especially the ammophosphonates and ethylenediammedisuccmates, clay soil removal agents, especially ethoxylated tetraethylene pentamine, dispersing agents, especially polyacrylates and polyasparatates, bnghteners, especially anionic bnghteners, suds suppressors, especially sihcones and secondary alcohols, fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formulaπes and published patents contain multiple, detailed descnptions of such conventional mateπals.

Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0 1% to about 1%)

The present vaπants are useful m hard surface cleaning compositions As used herein "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like Hard surface cleaning compositions of the present invention compπse an effective amount of one or more proteases of the present invention, preferably from about 0.001%) to about 10%, more preferably from about 0.01%) to about 5%, and more preferably still from about 0.05% to about 1% by weight of protease of the composition. In addition to compnsmg one or more of the proteases, such hard surface cleaning compositions typically compπse a surfactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy and / or streaky residue on the glass surface

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

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

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

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

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

Examples 7 - 12 Liquid Hard Surface Cleaning Compositions

All formulas are adjusted to pH 7.

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

Examples 13 - 16 Liquid Dish Detergent

All formulas are adjusted to pH 7.

Examples 17 - 19 Liquid Fabnc Cleaning Compositions

Personal Care Compositions

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

To illustrate, the present proteases are suitable for inclusion in the compositions descnbed in the following references: U.S. Pat. No. 5,641,479, Lmares et al., issued June 24, 1997 (skm cleansers); U.S. Pat. No. 5,599,549, Wivell et al.. issued February 4, 1997 (skm cleansers); U.S. Pat. No. 5,585,104, Ha et al., issued Pecember 17, 1996 (skm cleansers); U.S. Pat. No. 5,540,852, Kefauver et al. issued July 30, 1996 (skm cleansers); U.S. Pat. No 5,510,050, Punbar et al. issued April 23, 1996 (skm cleansers); U.S. Pat. No. 5,612,324, Guang Lin et al., issued March 18, 1997 (anti-acne preparations); U.S. Pat. No. 5,587,176, Warren et al.. issued Pecember 24, 1996 (anti-acne preparations); U.S. Pat. No. 5,549,888, Venkateswaran, issued August 27, 1996 (anti-acne preparations); U.S. Pat. No. 5,470,884, Corless et al., issued November 28, 1995 (anti-acne preparations); U.S. Pat. No. 5,650,384, Gordon et al., issued July 22, 1997 (shower gels); U.S. Pat. No. 5,607,678, Moore et al.. issued March 4, 1997 (shower gels); U.S. Pat. No. 5,624,666, Coffindaffer et al.. issued April 29, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,618,524, Bohch et al.. issued Apπl 8, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,612,301, Inman, issued March 18, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,573,709, Wells, issued November 12, 1996 (hair conditioners and / or shampoos); U.S. Pat. No. 5,482,703, Pmgs, issued January 9, 1996 (hair conditioners and / or shampoos); U.S. Pat. No. Re. 34,584, Grote et al.. Reissued April 12, 1994 (hair conditioners and / or shampoos); U.S. Pat. No. 5,641,493, Date et al., issued June 24, 1997 (cosmetics); U.S. Pat No. 5,605,894, Blank et al., issued February 25, 1997 (cosmetics); U.S. Pat. No. 5,585,090, Yoshioka et al., issued December 17, 1996 (cosmetics); U.S. Pat. No. 4,939,179, Cheney et al.. issued July 3, 1990 (hand, face, and / or body lotions); U.S. Pat. No. 5.607,980, McAtee et al, issued March 4, 1997 (hand, face, and / or body lotions); U.S. Pat. No 4,045,364, Richter et al., issued August 30, 1977 (cosmetic and cleansing wipes); European Patent Application, EP 0 619 074, Touchet et al„ published October 12, 1994 (cosmetic and cleansing wipes); U.S. Pat. No 4,975,217, Brown-Skrobot et al., issued December 4, 1990 (cosmetic and cleansing wipes); U.S Pat. No. 5,096,700, Seibel, issued March 17, 1992 (oral cleaning compositions); U.S. Pat. No

5.028.414, Sampathkumar, issued July 2, 1991 (oral cleaning compositions); U.S. Pat. No

5.028.415. Benedict et al., issued July 2, 1991 (oral cleaning compositions); U.S. Pat No 5,028,415, Benedict et al., issued July 2, 1991 (oral cleaning compositions); U.S. Pat No 4,863,627, Davies et al., September 5, 1989 (contact lens cleaning solutions); U.S. Pat. No. Re. 32,672, Huth et al, reissued May 24, 1988 (contact lens cleaning solutions); and U.S. Pat No 4,609,493, Schafer, issued September 2, 1986 (contact lens cleaning solutions).

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

Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally compπse from about 50% to about 99 99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99%, by weight of the composition.

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

In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity compπse one or more proteases of the present invention Such denture cleaning compositions compπse an effective amount of one or more of the proteases, preferably from about 0.0001% to about 50%, more preferably from about 0.001% to about 35%, more preferably still from about 0.01% to about 20%, by weight of the composition, and a denture cleansing carrier Vanous denture cleansing composition formats 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 appropnate for incorporation of one or more of the proteases for removing protemaceous stams from dentures. In another embodiment of the present mvention, contact lens cleaning compositions compπse one or more proteases of the present invention. Such contact lens cleaning compositions compπse an effective amount of one or more of the proteases, preferably from about 0.01% to about 50% of one or more of the proteases, more preferably from about 0.01% to about 20%, more preferably still from about 1% to about 5%, by weight of the composition, and a contact lens cleaning carrier. Vanous contact lens cleaning composition formats such as tablets, liquids, and the like are well known in the art and are generally appropriate for incorporation of one or more proteases of the present invention for removing protemaceous stams from contact lens

Examples 20 - 23 Contact Lens Cleaning Solution

Examples 32-33 Leave-on Skm Moisturizing Composition

Example 34 Cleansing Wipe Composition

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

Claims

What is claimed is:

1. A subtilisin-hke protease characterized in that said protease compnses an intramolecular cross-link, wherein the intramolecular cross-lmk comprises a linking moiety between an ammo acid of a first residue of the protease and an ammo acid of a second residue of the protease.

2. A protease according to Claim 1 wherein at least one of the residues occurs withm a region selected from the group consisting of:

(a) an ammo terminus region corresponding to position 1 of subtihsm BPN';

(b) a first epitope region corresponding to positions 70 - 84 of subtihsm BPN';

(c) a second epitope region corresponding to positions 103 - 126 of subtihsm BPN',

(d) a third epitope region coπespondmg to positions 220 - 246 of subtihsm BPN';

(e) a first clip site region corresponding to positions 156 - 165, 170, 186, 191 - 196, and 259 - 262 of subtihsm BPN';

(f) a second clip site region corresponding to positions 12 - 24, 27, 84 - 88, 271, and 274 of subtihsm BPN';

(g) a first proximal epitope region corresponding to positions 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 of subtihsm BPN';

(h) a second proximal epitope region corresponding to positions 25, 26, 27, 46, 47, 48,

49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133. 134,

136, 137, 138, 140, 141, 144, and 145 of subtihsm BPN'; and (i) a third proximal epitope region corresponding to positions 9, 10, 22, 23, 24, 62, 63,

143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256,

265, 267, 269, 271, 272, and 275 of subtihsm BPN'.

3. A protease according to Claim 2 wherein the first proximal epitope region corresponds to positions 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86, 87, 89, 206, 209, 214, and 215 of subuhsm BPN'; the second proximal epitope region coπesponds to positions 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138, and 141 of subtihsm BPN'; the third proximal epitope region corresponds to positions 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254, 265. and 275 of subtihsm BPN'.

4. A protease according to Claim 3 wherein the second clip site region corresponds to positions 13 - 24 of subtihsm BPN'.

5. A protease according to Claim 4 wherein the first clip site region corresponds to positions 156 - 165, 170, 191 - 195, 261, and 262 of subtihsm BPN'.

6. A protease according to Claim 1 wherein the first epitope region corresponds to positions 75 - 83 of subtihsm BPN'.

7. A protease according to Claim 6 wherein the amino acid of the first residue is lysine or wherein the first residue corresponds to position 1 of subtihsm BPN'; and wherein the ammo acid of the second residue is cysteme.

8. A protease according to Claim 6 wherein each of the first and second residues occur within a region selected from the group consisting of the ammo terminus region, the first epitope region, the second epitope region, the third epitope region, the first clip site region, the second clip site region, the first proximal epitope region, the second proximal epitope region, and the third proximal epitope region.

9. A protease according to Claim 8 wherein the first residue corresponds to position 1 of subtihsm BPN' and the second residue occurs withm a region selected from the group consisting of the first epitope region, the second epitope region, the third epitope region, the first clip site region, the second clip site region, the first proximal epitope region, the second proximal epitope region, and the third proximal epitope region.

10. A protease according to Claim 9 wherein the linking moiety comprises an alkyl succmimide; the second residue occurs withm the first epitope region; and wherein the second residue corresponds to position 78 of subtihsm BPN'.

11. A cleaning composition compnsmg a protease according to Claim 1 and a cleaning composition earner. A personal care composition comprising a protease according to Claim 1 and a personal care carrier.