Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-10-13
2002-10-08
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S220000, C435S221000, C435S222000, C435S263000, C424S094640, C426S063000, C510S392000
Reexamination Certificate
active
06461849
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polypeptides having substituted one or more amino acid residues to the polypeptide and/or having coupled polymeric molecules on the surface of the 3-dimensional structure of the polypeptide, a method for preparing modified polypeptides of the invention, the use of the modified polypeptides for reducing immunogenicity and allergenicity, and compositions comprising the polypeptide.
DESCRIPTION OF THE RELATED ART
The use of polypeptides, including enzymes, in the circulatory system to obtain a particular physiological effect is well-known in the medical arts. Further, within the arts of industrial applications, such as laundry washing, textile bleaching, personal care, contact lens cleaning, and food and feed preparation enzymes are used as a functional ingredient. One of the important differences between pharmaceutical and industrial application is that for industrial applications the polypeptides (often enzymes) are not intended to enter into the circulatory system of the body.
Certain polypeptides and enzymes have an unsatisfactory stability and may under certain circumstances—dependent on the way of challenge—cause an immune response, typically an IgG and/or IgE response.
It is today generally recognized that the stability of polypeptides is improved and the immune response is reduced when polypeptides, such as enzymes, are coupled to polymeric molecules. It is believed that the reduced immune response is a result of the shielding of (the) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation by the coupled polymeric molecules.
Techniques for conjugating polymeric molecules to polypeptides are well-known in the art.
One of the first suitable commercial techniques was described in the early 1970's and disclosed in e.g. U.S. Pat. No. 4,179,337. This patent concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG). At least 15% of the polypeptides' physiological activity is maintained.
GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.
Further, techniques for maintaining the enzymatic activity of enzyme-polymer conjugates are also known in the art.
WO 93/15189 (Veronese et al.) concerns a method for maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.
It has been found that the attachment of polymeric molecules to a polypeptide often has the effect of reducing the activity of the polypeptide by interfering with the interaction between the polypeptide and its substrate. EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.
EP 471,125 (Kanebo) discloses skin care products comprising a parent protease (Bacillus protease with the trade name Esperase®) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability. The coupling technique used is also described in the above mentioned GB patent no. 1,183,257 (Crook et al.).
JP 3083908 describes a skin cosmetic material which contains a transglutaminase from guinea pig liver modified with one or more water-soluble substances such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme. The composition is stated to be mild to the skin.
WO 98/35026 (Novo Nordisk A/S) describes polypeptide-polymer conjugates having added and/or removed one or more attachment groups for coupling polymeric molecules on the surface of the polypeptide structure. The conjugates have reduced immunogenicity and allergenicity.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide improved polypeptides suitable for industrial and pharmaceutical applications.
The term “improved polypeptides” means in the context of the present invention polypeptides having a reduced immune response in humans and animals. As will be described further below the immune response is dependent on the way of challenge. The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or allergenic by substituting one or more amino acid residues on the surface of the polypeptide with other amino acid residues and/or by coupling polymeric molecules on the surface of the enzyme in the vicinity of a bound ligand of the enzyme e.g. a metal ion substantially without affecting the enzymatic activity.
When introducing pharmaceutical polypeptide directly into the circulatory system (i.e. bloodstream) the potential risk is an immunogenic response in the form of mainly IgG, IgA and/or IgM antibodies. In contrast hereto, industrial polypeptides, such as enzymes used as a functional ingredient in e.g. detergents, are not intended to enter the circulatory system. The potential risk in connection with industrial polypeptides is inhalation causing an allergenic response in the form of mainly IgE antibody formation.
Therefore, in connection with industrial polypeptides the potential risk is respiratory allergenicity caused by inhalation, intratracheal and intranasal presentation of polypeptides.
The main potential risk of pharmaceutical polypeptides is immunogenicity caused by intradermal, intravenous or subcuaneous presentation of the polypeptide.
The term “immunogenicity” used in connection with the present invention may be referred to as allergic contact dermatitis in a clinical setting and is a cell mediated delayed immune response to chemicals that contact and penetrate the skin. This cell mediated reaction is also termed delayed contact hypersensitivity (type IV reaction according to Gell and Combs classification of immune mechanisms in tissue damage).
The term “allergenicity” or “respiratory allergenicity” is initially an immediate anaphylactic reaction (type I antibody-mediated reaction according to Gell and Combs) following inhalation of e.g. polypeptides.
According to the present invention it is possible to provide polypeptides with a reduced immune response, which has a substantially retained residual activity.
The allergic and the immunogenic response are in one term, at least in the context of the present invention called the “immune response”.
In the first aspect the invention relates to a polypeptide with reduced immune response, having one or more amino acid residues modified, wherein the C
&agr;
-atoms of the amino acid residues are located less than 15 Å from the ligand bound to the polypeptide.
The reduced immune response is preferably reduced allergenicity.
The modification of the polypeptide is conducted by substituting one or more amino acid residues in the parent polypeptide with other amino acid residues to the polypeptide, and/or by selecting variants from a diverse library of variants of the parent polypeptide and/or by coupling a polymeric molecule to the surface of the parent polypeptide.
The term “parent polypeptide” refers to the polypeptide to be modified by coupling to polymeric molecules or by substituting amino acid residues. The parent polypeptide may be a naturally-occurring (or wild-type) polypeptide or may be a variant thereof prepared by any suitable means. For instance, the parent polypeptide may be a variant of a naturally-occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.
A “suitable attachment group” means in the context of the present invention any amino acid residue group on the surface of the polypeptide capable of coupling to the polymeric molecule in question.
Preferred attachment groups are ami
Andersen Kim Vilbour
Ernst Steffen
Olsen Arne Agerlin
Osten Claus von der
Roggen Erwin Ludo
Garbell Jason I.
Lambiris Elias J.
Nashed Nashaat T.
Novozymes A/S
LandOfFree
Modified polypeptide does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Modified polypeptide, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Modified polypeptide will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2945391