Protease conjugates having sterically protected clip sites

Details Protease conjugates having sterically protected clip sites Protease conjugates having sterically protected clip sites

2000-07-18

2003-05-20

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S222000, C435S252300, C435S320100, C435S471000, C510S350000, C524S267000, C536S023200

Reexamination Certificate

active

06566115

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to chemically modified subtilisin proteases which are useful in compositions such as, for example, personal care compositions, laundry compositions, hard surface cleansing compositions, and light duty cleaning compositions.
BACKGROUND OF THE INVENTION
Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has typically been exploited by incorporating naturally occurring and genetically engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the 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 Modelling 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 efficacy of the wild-type proteases in their natural environment is frequently not optimized for the artificial environment of a cleaning composition. Specifically, protease characteristics 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 amino acid sequence of serine 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 allergenic response (herein collectively described 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 primary 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 risks associated with extended tissue contact with the finished composition.
It has also been proposed that reduction in immunogenicity of a protease may be achieved by attaching polymers to the protease. See. e.g., U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979; U.S. Pat. No. 5,856,451, Olsen et al., assigned to Novo Nordisk, issued Jan. 5, 1999; WO 99/00489, Olsen et al., assigned to Novo Nordisk, published Jan. 7, 1999; WO 98/30682, Olsen et al., assigned to Novo Nordisk, published Jul. 16, 1998; and WO 98/35026, Von Der Osten et al., published Aug. 13, 1998. However, such proposals have not suggested the importance of attaching polymers to particular amino acid regions of the protease in order to most effectively decrease the immunogenic response.
It has recently been discovered that the subtilisin protease comprises three epitope regions and that conjugation of one or more polymers, polypeptides, or other groups should be attached at one or more of these regions to effect significant reduction in immunogenicity of the protease. See, e.g., U.S. patent application Ser. No. 09/088,912, Weisgerber et al., assigned to The Procter & Gamble Co., filed Jun. 2, 1998.
As an alternative to protection of the epitope regions of the subtilisin protease, the present inventors have discovered that steric protection of one or more “clip sites” (i.e., locations of the protease where hydrolysis occurs in vivo) of the protease may be utilized to prevent or impede presentation of an epitope and decrease the immunogenicity of the protease. Accordingly, the present inventors provide chemically modified subtilisins wherein the chemical modification is at a region in steric proximity to one or more of the clip sites. The present inventors have therefore discovered subtilisin proteases which evoke a decreased immunogenic response yet maintain their activity as an efficient and active protease. Accordingly, the present protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.
SUMMARY OF THE INVENTION
The present invention relates to protease conjugates comprising a protease moiety and one or more addition moieties wherein each addition moiety is covalently attached to an amino acid of the protease moiety at a position selected from the group consisting of 13, 14, 15, 16, 18, 19, 20, 21, 84, 85, 88, 158, 159, 160, 161, 162, 163, 164, 165, 170, 186, 191, 192, 193, 194, 196, 259, 260, 261, 262, and 274 corresponding to subtilisin BPN′; wherein the addition moieties each, independently, have the structure:
wherein X is selected from the group consisting of nil and a linking moiety; R
1
is selected from the group consisting of nil, a first polypeptide, and a first polymer; and R
2
is selected from the group consisting of nil, a second polypeptide, and a second polymer; wherein at least one of X, R
1
, and R
2
is not nil.
The protease conjugates of the present invention have decreased immunogenicity relative to the parent protease. Accordingly, such protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.
DETAILED DESCRIPTION OF THE INVENTION
The essential components of the present invention are herein described below. Also included are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.
The present invention can comprise, consist of, or consist essentially of any of the required or optional components and/or limitations described herein.
All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated.
All component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, 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 publications, are hereby incorporated by reference in their entirety.
Referred to herein are trade names for materials including, but not limited to, enzymes. The inventors herein do not intend to be limited by materials under a certain trade name. Equivalent materials (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 in the protease conjugates and compositions herein.
As used herein, abbreviations will be used to describe amino acids. Table I provides a list of abbreviations used herein:
TABLE I
Amino Acid
Three-letter Abbreviation
One-letter Abbreviation
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic A

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