Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1998-06-02
2002-12-17
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C424S401000, C424S070100, C424S094630, C435S177000, C435S178000, C435S180000, C435S188000, C435S219000, C435S221000, C510S392000
Reexamination Certificate
active
06495136
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to subtilisin protease conjugates and compositions comprising the conjugates which have decreased immunogenicity relative to their corresponding parent proteases.
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 protein 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 frequently does not translate into the unnatural cleaning composition environment. 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 proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause immunogenic and/or allergenic responses (herein collectively described as immunogenic responses) in mammals. In fact, sensitization to serine proteases has been observed in environments wherein humans are regularly exposed to the proteases. Such environments include manufacturing facilities, wherein workers are exposed to the proteases through such vehicles as uncontrolled dust or aerosolization. Aerosolization can result by the introduction of the protease into the lung, which is the route of protease exposure which causes the most dangerous response. Protease sensitization can also occur in the marketplace, wherein consumers' repeated use of products containing proteases may cause an immunogenic response.
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 been minimally 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, and shampoos, is due to the aforementioned problem of human sensitization leading to undesirable immunogenic responses. It would therefore be highly advantageous to provide a personal care composition which provides the cleansing properties of proteases without the provocation of an immunogenic response.
Presently, immunogenic responses 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 and worker exposure to protease-containing dust or aerosol during manufacturing.
In the medical field, suggestions have been made to diminish the immunogenicity of enzymes through yet another method. This method involves attaching polymers to enzymes. See. e.g., U.S. Pat. No. 4,179,337, Davis, et al., issued Dec. 18, 1979 and PCT Application WO 96/17929, Olsen, et al., published Jun. 13, 1996.
One approach toward decreasing the immunogenic activity of a protease is through alleviation of the immunogenic properties of epitopes. Epitopes are those amino acid regions of an antigen which evoke an immune response through the binding of antibodies or the presentation of processed antigens to T cells via a major histocompatibility complex protein (MHC). Changes in the epitopes can affect their 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 present inventors have discovered that those serine proteases commonly known as subtilisins, including subtilisin BPN′, have prominent epitope regions at amino acid positions 70-84, 103-126, and 217-252 corresponding to subtilisin BPN′. The present inventors have herein chemically modified such subtilisins at one or more of these epitope regions to alleviate the immunogenic properties of the protease. In so doing, the active site of the protease is minimally affected. The present inventors have therefore discovered subtilisin-like 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 subtilisin protease conjugates comprising a protease moiety and one or more addition moieties wherein:
(a) 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 wherein:
(i) when a substitution occurs in the first epitope region, the substitution occurs at one or more positions corresponding to positions 70-84 of subtilisin BPN′;
(ii) when a substitution occurs in the second epitope region, the substitution occurs at one or more positions corresponding to positions 103-126 of subtilisin BPN′; and
(iii) when a substitution occurs in the third epitope region, the substitution occurs at one or more positions corresponding to positions 217-252 of subtilisin BPN′; and
(b) wherein each of the addition moieties is covalently attached to one of the substituting amino acids present on the protease moiety and has 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 present invention further relates to cleaning and personal care compositions comprising such protease conjugates.
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 do
Correa Paul Elliott
Rubingh Donn Nelton
Weisgerber David John
Kendall Dara M.
McDow-Dunham Kelly L.
Naff David M.
The Procter & Gamble & Company
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