US20030087785A1

US20030087785A1 - Modified polypeptides

Info

Publication number: US20030087785A1
Application number: US10/209,812
Authority: US
Inventors: Arne Olsen; Claus Osten; Kim Andersen; Steffen Ernst; Erwin Roggen
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 1998-10-13
Filing date: 2002-08-01
Publication date: 2003-05-08
Also published as: US6461849B1

Abstract

The present invention relates to polypeptides with reduced immune response including reduced allergenicity having one or more amino acid residues being substituted with other amino acid residues and/or having coupled one or more polymeric molecules in the vicinity of the polypeptides metal binding site, a method for preparing modified polypeptides of the invention, the use of the polypeptide for reducing the immunogenicity and allergenicity and compositions comprising the polypeptide.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/417,359 filed Oct. 13, 1999 and claims priority under 35 U.S.C. 119 of U.S. provisional application Nos. 60/105,624 and 60/157,426 filed on Oct. 26, 1998 and Oct. 4, 1999, respectively, and of Danish application nos. PA 1998 01301 and PA 1999 01418 filed on Oct. 13, 1998 and Oct. 4, 1999, respectively, the contents of which are fully incorporated herein by reference.[0001]

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 subcutaneous 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 ^alpha-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 amino groups of Lysine residues and the N-terminal amino group. Polymeric molecules may also be coupled to the carboxylic acid groups (—COOH) of amino acid residues in the polypeptide chain located on the surface. Carboxylic acid attachment groups may be the carboxylic acid group of Aspartate or Glutamate and the C-terminal COOH-group. Another attachment group is SH-groups in Cysteine.

An “active site” means any amino acid residues and/or molecules which are known to be essential for the performance of the polypeptide, such as catalytic activity, e.g. the catalytic triad residues, Histidine, Aspartate and Serine in Serine proteases, or e.g. the heme group and the distal and proximal Histidines in a peroxidase such as the Arthromyces ramosus peroxidase.

A “ligand”, means in the context of the present invention a metal or metal ion or a cofactor.

In the context of the present invention “modification of amino acid residues” means that amino acid residues are substituted with other amino acid residues and/or a polymeric molecule is coupled to the amino acid residue. The polypeptide of the present invention may according to the invention be modified by substitution alone, by coupling of a polymeric molecule alone or by a combination of substitution and coupling.

In the context of the present invention “located” means the shortest distance from any atom in the ligand to the relevant C-atom in the amino acid residue.

Furthermore, in the context of the present invention, e.g. “R250K” means that the amino acid Arginine in position 250 of the polypeptide has been substituted with the amino acid Lysine according to the one-letter-code of amino acids.

In the second aspect the invention relates to a method for preparing polypeptides with reduced immune response comprising the steps of:

a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent polypeptide in question,

b) selecting target amino acid residues on the surface of the 3-dimensional structure of the parent polypeptide to be modified,

c) substituting one or more amino acid residues selected in step b) with other amino acid residue, and/or

d) coupling polymeric molecules to the amino acid residues in step b) and/or step c).

The invention also relates to the use of a modified polypeptide of the invention and the method of the invention for reducing the immunogenicity of pharmaceuticals and reducing the allergenicity of industrial products.

Finally the invention relates to compositions comprising a modified polypeptide of the invention and further ingredients used in industrial products or pharmaceuticals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows integrated IgE antibody levels in rats. [0042]
FIG. 2 shows integrated specific IgE levels in mice.[0043]

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide improved polypeptides suitable for industrial and pharmaceutical applications. [0044]
Even though polypeptides used for pharmaceutical applications and industrial applications can be quite different the principle of the present invention may be tailored to the specific type of parent polypeptide (i.e. enzyme, hormone peptides, etc.). [0045]
The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or less allergenic by substituting amino acid residues in the vicinity of the ligand e.g. metal ion at the metal ion binding site and/or by coupling one or more polymeric molecules on the surface of the parent polypeptide. In addition thereto the inventors have found that a high percentage of maintained residual catalytic activity may be maintained in these modified polypeptides. [0046]
In the first aspect the invention relates to an improved polypeptide having one or more amino acid residues modified, wherein the C[0047] ^alpha-atom of said amino acid residues is located less than 15 Å from the ligand bound to said polypeptide.
The substitution of amino acid residues and coupling of polymeric molecule may be carried out in a conventional manner as described below. [0048]
Reduced Immune Response vs. Maintained Residual Enzymatic Activity [0049]
For enzymes, there is a conflict between reducing the immune response and maintaining a substantial residual enzymatic activity. [0050]
Without being limited to any theory it is believed that the loss of enzymatic activity of enzyme-polymer conjugates might be a consequence of impeded access of the substrate to the active site in the form of spatial hindrance of the substrate by especially bulky and/or heavy polymeric molecules to the catalytic cleft. It might also, at least partly, be caused by disadvantageous minor structural changes of the 3-dimensional structure of the enzyme due to the stress made by the coupling of the polymeric molecules. [0051]
Also, polypeptides modified by substituting one or more amino acid residues may have reduced enzymatic activity. [0052]
Maintained Residual Activity [0053]
A modified polypeptide of the invention has a substantially maintained catalytic activity. [0054]
A “substantially” maintained catalytic activity is in the context of the present invention defined as an activity which is above 20%, at least between 20% and 30%, preferably between 30% and 40%, more preferably between 40% and 60%, better from 60% up to 80%, even better from 80% up to about 100%, in comparison to the activity of the modified polypeptide prepared on the basis of corresponding parent polypeptides. [0055]
In the case of polypeptide-polymer conjugates of the invention where no polymeric molecules are coupled at or close to the active site(s) the residual activity may even be up to 100% or very close thereto. If attachment group(s) of the parent polypeptide is(are) removed from the active site the activity might even be more than 100% in comparison to modified (i.e. polymer coupled) parent polypeptide conjugate. [0056]
The Attachment Group [0057]
Virtually all ionized groups, such as the amino groups of Lysine residues, are located on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, (1993), “Proteins”, W. H. Freeman and Company, New York). [0058]
Therefore, the number of readily accessible attachment groups (e.g. amino groups) on a modified or parent polypeptide equals generally the number of Lysine residues in the primary structure of the polypeptide plus the N-terminus amino group. [0059]
The chemistry of coupling polymeric molecules to amino groups is quite simple and well established in the art. Therefore, it is preferred to add Lysine residues (i.e. attachment groups) to the parent polypeptide in question to obtain improved conjugates with reduced immunogenicity and/or allergenicity and/or improved stability and/or high percentage maintained catalytic activity. [0060]
Polymeric molecules may also be coupled to the carboxylic groups (—COOH) of amino acid residues on the surface of the polypeptide. Therefore, if using carboxylic groups (including the C-terminal group) as attachment groups addition and/or removal of Aspartate and Glutamate residues may also be suitable according to the invention. [0061]
If using other attachment groups, such as —SH groups, they may be added and/or removed analogously. [0062]
Substitution of the amino acid residues is preferred over insertion, as the impact on the 3-dimensional structure of the polypeptide normally will be less pronounced. [0063]
The Parent Polypeptide [0064]
In the context of the present invention, the term “polypeptides” includes proteins, peptides and/or enzymes for pharmaceutical or industrial applications. Typically the polypeptides in question have a molecular weight in the range between about 1 to 1000 kDa, preferred 4 to 100 kDa, more preferred 12 to 60 kDa. [0065]
Pharmaceutical Polypeptides [0066]
The term “pharmaceutical polypeptides” is defined as polypeptides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body of humans and/or animals. [0067]
Pharmaceutical polypeptides are potentially immunogenic as they are introduced into the circulatory system. [0068]
Examples of “pharmaceutical polypeptides” contemplated according to the invention include insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO) and prolactin. [0069]
Industrial Polypeptides [0070]
Polypeptides used for industrial applications often have an enzymatic activity. Industrial polypeptides (e.g. enzymes) are (in contrast to pharmaceutical polypeptides) not intended to be introduced into the circulatory system of the body. [0071]
It is not very like that industrial polypeptides, such as enzymes used as ingredients in industrial compositions and/or products, such as detergents and personal care products, including cosmetics, come into direct contact with the circulatory system of the body of humans or animals, as such enzymes (or products comprising such enzymes) are not injected (or the like) into the bloodstream. [0072]
Therefore, in the case of the industrial polypeptide the potential risk is respiratory allergy (i.e. IgE response) as a consequence of inhalation of polypeptides through the respiratory passage. [0073]
In the context of the present invention “industrial polypeptides” are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be administered to humans and/or animals. [0074]
Examples of such polypeptides are polypeptides, especially enzymes, used in products such as detergents, household article products, agrochemicals, personal care products, such as skin care products, including cosmetics and toiletries, oral and dermal pharmaceuticals, composition use for processing textiles, compositions for hard surface cleaning, and compositions used for manufacturing food and feed etc. [0075]
Enzymatic Activity [0076]
Pharmaceutical or industrial polypeptides exhibiting enzymatic activity will often belong to one of the following groups of enzymes including Oxidoreductases (E.C. 1, “Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Transferases, (E.C. 2), such as transglutaminases (TGases); Hydrolases (E.C. 3), including proteases, especially subtilisins, and lipolytic enzymes; Isomerases (E.C. 5), such as Protein disulfide Isomerases (PDI). [0077]
Hydrolases [0078]
Proteolytic Enzymes [0079]
Contemplated proteolytic enzymes include proteases selected from the group of Aspartic proteases, such as pepsins, Cysteine proteases, such as Papain, Serine proteases, such as subtilisins, or metallo proteases, such as Neutrase®. [0080]
Specific examples of parent proteases include PD498 (WO 93/24623 and SEQ ID NO: 2), Savinase® (von der Osten et al., (1993), Journal of Biotechnology, 28, p. 55+, SEQ ID NO: 3), Proteinase K (Gunkel et al., (1989), Eur. J. Biochem, 179, p. 185-194), Proteinase R (Samal et al, (1990), Mol. Microbiol, 4, p. 1789-1792), Proteinase T (Samal et al., (1989), Gene, 85, p. 329-333), Subtilisin DY (Betzel et al. (1993), Arch. Biophys, 302, no. 2, p. 499-502), Lion Y (JP 04197182-A), Rennilase® (Available from Novo Nordisk A/S), JA16 (WO 92/17576), Alcalase® (a natural subtilisin Carlberg variant) (von der Osten et al., (1993), Journal of Biotechnology, 28, p. 55+), Subtilisin BPN′ J. Mol. Biol. 178:389-413 (1984); Hirono S., Akagawa H., Mitsui Y., litaka Y. (Available from Novo Nordisk A/S). [0081]
Carbohydrases [0082]
Parent carbohydrases may be defined as all enzymes capable of hydrolyzing carbohydrate chains (e.g. starches) of especially five- and six-membered ring structures (i.e. enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)). [0083]
Examples include carbohydrases selected from those classified under the Enzyme Classification (E.C.) numbers: alpha-amylase (3.2.1.1) beta-amylase (3.2.1.2), glucan 1,4-alpha-glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1,3(4)-beta-glucanase (3.2.1.6), endo-1,4-beta-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17), beta-glucosidase (3.2.1.21), alpha-galactosidase (3.2.1.22), beta-galactosidase (3.2.1.23), amylo-1,6-glucosidase (3.2.1.33), xylan 1,4-beta-xylosidase (3.2.1.37), glucan endo-1,3-b-D-glucosidase (3.2.1.39), alpha-dextrin endo-1,6-glucosidase (3.2.1.41), sucrose alpha-glucosidase (3.2.1.48), glucan endo-1,3-alpha-glucosidase (3.2.1.59), glucan 1,4-beta-glucosidase (3.2.1.74), glucan endo-1,6-beta-glucosidase (3.2.1.75), arabinan endo-1,5-alpha-arabinosidase (3.2.1.99), lactase (3.2.1.108), chitonanase (3.2.1.132). [0084]
Examples of relevant carbohydrases include alpha-1,3-glucanases derived from [0085] Trichoderma harzianum; alpha-1,6-glucanases derived from a strain of Paecilomyces; beta-glucanases derived from Bacillus subtilis; beta-glucanases derived from Humicola insolens; beta-glucanases derived from Aspergillus niger; beta-glucanases derived from a strain of Trichoderma; beta-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1,4-alpha-D-glucosidases (glucoamylases) derived from Aspergillus niger; alpha-amylases derived from Bacillus subtilis; alpha-amylases derived from Bacillus amyloliquefaciens; alpha-amylases derived from Bacillus stearothermophilus; alpha-amylases derived from Aspergillus oryzae; alpha-amylases derived from non-pathogenic microorganisms; alpha-galactosidases derived from Aspergillus niger; Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulases derived from Humicola insolens; cellulases derived from Trichoderma reesei; cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger; dextranases derived from Penicillium lilacinum; endo-glucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus; beta-galactosidases derived from Kluyveromyces fragilis; xylanases derived from Trichoderma reesei.
Specific examples of readily available commercial carbohydrases include Alpha-Gal®, Bio-Feed® Alpha, Bio-Feed® Beta, Bio-Feed® Plus, Bio-Feed® Plus, Novozyme® 188, Carezyme®, Celluclast®, Cellusoft®, Ceremyl®, Citrozym®, Denimax®, Dezyme®, Dextrozyme®, Finizym®, Fungamyl®, Gamanase®, Glucanex®, Lactozym®, Maltogenase®, Pentopan®, Pectinex®, Promozyme®, Pulpzyme®, Novamyl®, Termamyl®, AMG (Amyloglucosidase Novo), Maltogenase®, Aquazym®, Natalase® (all enzymes available from Novo Nordisk A/S). Other carbohydrases are available from other companies. [0086]
It is to be understood that also carbohydrase variants are contemplated as the parent enzyme. [0087]
The activity of carbohydrases can be determined as described in “Methods of Enzymatic Analysis”, third edition, 1984, Verlag Chemie, Weinheim, vol. 4. [0088]
Oxidoreductases [0089]
Laccases [0090]
Contemplated laccases include [0091] Polyporus pinisitus laccase (WO 96/00290), Myceliophthora laccase (WO 95/33836), Scytalidium laccase (WO 95/338337), and Pyricularia oryzae laccase (Available from Sigma).
Peroxidases [0092]
Contemplated peroxidases include [0093] B. pumilus peroxidases (WO 91/05858), Myxococcaceae peroxidase (WO 95/11964), Coprinus cinereus (WO 95/10602) and Arthromyces ramosus peroxidase (Kunishima et al. (1994), J. Mol. Biol. 235, p. 331-344).
Transferases [0094]
Transglutaminases [0095]
Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S). [0096]
Isomerases [0097]
Protein Disulfide Isomerase [0098]
Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S). [0099]
Contemplated isomerases include xylose/glucose Isomerase (5.3.1.5) including Sweetzyme®. [0100]
Lyases [0101]
Suitable lyases include Polysaccharide lyases: Pectate lyases (4.2.2.2) and pectin lyases (4.2.2.10), such as those from [0102] Bacillus licheniformis disclosed in WO 99/27083.
The Polymeric Molecule [0103]
The polymeric molecules coupled to the polypeptide may be any suitable polymeric molecule, including natural and synthetic homo-polymers, such as polyols (i.e. poly-OH), polyamines (i.e. poly-NH[0104] ₂) and polycarboxyl acids (i.e. poly-COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
Examples of suitable polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylen glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), Branced PEGs, poly-vinyl alcohol (PVA), poly-carboxylates, poly-(vinylpyrolidone), poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starches and hydroxy propyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acid hydrolysates and bio-polymers. [0105]
Preferred polymeric molecules are non-toxic polymeric molecules such as (m)polyethylene glycol ((m)PEG) which further requires a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface. [0106]
Generally seen polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG and especially mPEG, are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the like, have few reactive groups capable of cross-linking. [0107]
Even though all of the above mentioned polymeric molecules may be used according to the invention the methoxypolyethylene glycols (mPEG) may advantageously be used. This arises from the fact that methoxyethylene glycols have only one reactive end capable of conjugating with the enzyme. Consequently, the risk of cross-linking is less pronounced. Further, it makes the product more homogeneous and the reaction of the polymeric molecules with the enzyme easier to control. [0108]
An example of a branched PEG conjugate is Branched PEG2-NHS-ester of Lysine (available from Shearwater). [0109]
Activation and Coupling of Polymers to Polypeptides [0110]
If the polymeric molecules to be conjugated with the polypeptide in question are not active, they must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person. [0111]
Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), “Protein immobilisation. Fundamental and applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups. [0112]
In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used. [0113]
Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press). [0114]
Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups. [0115]
Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho/pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned. [0116]
Accessible arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups. [0117]
Techniques involving coupling electrophilically activated PEGs to the amino groups of lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used. [0118]
Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide. [0119]
Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups. [0120]
Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide. [0121]
Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively. [0122]
Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (U.S. Pat. No. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds are very high but may make the hydrolysis too fast. [0123]
PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group makes the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide. [0124]
Furthermore, a special linker can be introduced. The most commonly used is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378. [0125]
Coupling of PEG to an aromatic amine followed by diazotation yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage. [0126]
As some peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol. [0127]
PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone. [0128]
The coupling technique used in the examples is the N-succinimidyl carbonate conjugation technique described in WO 90/13590 (Enzon). [0129]
Method for Preparing Improved Polypeptides [0130]
It is also an object of the invention to provide a method for preparing improved polypeptides comprising the steps of: [0131]
a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent polypeptide in question, [0132]
b) selecting target amino acid residues on the surface of the 3-dimensional structure of the parent polypeptide to be modified, [0133]
c) substituting one or more amino acid residues selected in step b) with other amino is acid residue, and/or [0134]
d) coupling polymeric molecules to the amino acid residues in step b) and/or step c). [0135]
Step a) Identifying Amino Acid Residues Located on the Surface of the Parent Polypeptide 3-dimensional Structure [0136]
To perform the method of the invention a 3-dimensional structure of the parent polypeptide in question is required. This structure may for example be an X-ray structure, an NMR structure or a model-built structure. The Brookhaven Databank is a source of X-ray- and NMR-structures. [0137]
A model-built structure may be produced by the person skilled in the art if one or more 3-dimensional structure(s) exist(s) of homologous polypeptide(s) sharing at least 30% sequence identity with the polypeptide in question. Several software packages exist which may be employed to construct a model structure. One example is the Homology 95.0 package from MSI Inc. [0138]
Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3-dimensional structures exist, definition of Structurally Conserved Regions (SCRs), assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (≧3 residues) relative to the known 3-dimensional structures are known to be quite difficult to model, and structural predictions must be considered with care. [0139]
Having obtained the 3-dimensional structure of the polypeptide in question, or a model of the structure based on homology to known structures, this structure serves as an essential prerequisite for the fulfillment of the method described below. [0140]
Step b) Selection of Target Amino Acid Residues [0141]
Target amino acid residues to be modified are according to the invention selected from those amino acid residues, wherein the C[0142] ^alpha-atom is located less than 15 Å from a ligand. In a preferred embodiment a possible C^beta-atom should be closer to the ligand than the C^alpha-atom. In a more preferred embodiment the C^alpha-atom of the amino acid residue is located less than 10 Å from the ligand and the amino acid residues have an accessibility of at least 15%, preferably at least 20% and more preferably at least 30%.
Step c) Substitution [0143]
Conservative Substitution [0144]
It is preferred to make conservative substitutions in the polypeptide when the polypeptide has to be conjugated, as conservative substitutions secure that the impact of the substitution on the polypeptide structure is limited. [0145]
In the case of providing additional amino groups this may be done by substitution of arginine to lysine, both residues being positively charged, but only the lysine having a free amino group suitable as an attachment group. [0146]
In the case of providing additional carboxylic acid groups the conservative substitution may for instance be an Asparagine to Aspartic acid or Glutamine to Glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups being present on the acidic residues. [0147]
In the case of providing SH-groups the conservative substitution may be done by substitution of threonine or serine to cysteine. [0148]
Which amino acids to substitute depends in principle on the coupling chemistry to be applied. [0149]
When no coupling is performed after substitution there is in general no limit on the selection of amino acids for substitution. However, preferred amino acids for substitutions are substitutions to polar residues e.g. K, R, D, E, H, Q, N, S, T, C. Also, substitutions to residues with short side chains G and A are preferred. [0150]
Further, when no coupling is to be performed, the changes may be in the form of addition or deletion of at least one amino acid for which the C[0151] ^alphaatom is located within 15 Å from the bound ligand, preferably deleting an amino acid. Furthermore, the parent protein may be changed by substituting some amino acids and deleting/adding other.
Only substitutions which provide polypeptides with reduced immune response when evaluated in animal models are within the concept of the present invention. [0152]
The mutation(s) performed in step c) may be performed by standard techniques well known in the art, such as site-directed mutagenesis (see, e.g., Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y. [0153]
A general description of nucleotide substitution can be found in e.g. Ford et al., 1991, [0154] Protein Expression and Purification 2, p. 95-107.
In a preferred embodiment of the invention, more than one amino acid residue is substituted, added or deleted, these amino acids possibly being located close to different bound ligands. In that case, it may be difficult to assess a priori how well the functionality of the protein is maintained while antigenicity, immunogenicity and/or allergenicity is reduced. This can be achieved by establishing a library of diversified mutants each having one or more changed amino acids introduced and selecting those variants which show good retention of function and at the same time a good reduction in antigenicity. In the case of protease, this can be tested by assaying the secreted variants for enzyme activity (as described below in the experimental section) and for antigen binding (e.g. by competitive ELISA using methods known in the art. (see e.g. J. Clausen, Immunochemical Techniques For The Identification and Estimation of Macromolecules, Elsevier, Amsterdam, 1988 pp. 187-188). Specifically, the competivity ELISA can be performed with the wild-type protease coated on ELISA plates, and incubated with specific polyclonal anti-protease antiserum from rabbits in the presence of protease variant. The scope of these embodiments of the invention is by no means limited to protease, which serves only to provide an example. A diversified library can be established by a range of techniques known to the person skilled in the art (Reetz M T; Jaeger K E, in Biocatalysis—from Discovery to Application edited by Fessner W D, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p.389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998). In a more preferable embodiment, substitutions are found by a method comprising the following steps: 1) a range of substitutions, additions, and/or deletions are listed, 2) a library is designed which introduces a randomized subset of these changes in the amino acid sequence into the target gene, e.g. by random mutagenesis, 3) the library is expressed, and preferred variants are selected. In a most preferred embodiment, this method is supplemented with additional rounds of screening and/or family shuffling of hits from the first round of screening (J. E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896, 1999) and/or combination with other methods of reducing allergenicity by genetic means (such as that disclosed in WO 92/10755). [0155]
Generation of Site Directed Mutations [0156]
Prior to mutagenesis the gene encoding the polypeptide of interest must be cloned in a suitable vector. Methods for generating mutations in specific sites is described below. [0157]
Once the polypeptide-encoding gene has been cloned, desirable sites for mutation identified, and the residue(s) to substitute for the original one(s) have been decided, these mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a preferred method, Site-directed mutagenesis is carried out by SOE-PCR mutagenesis technique described by Kammann et al. (1989) Nucleic Acids Research 17(13), 5404, and by Sarkar G. and Sommer, S. S. (1990); Biotechniques 8, 404-407. [0158]
Step d) Coupling Polymeric Molecules to the Optionally Modified Parent Enzyme [0159]
Polypeptide-polymer conjugates of the invention may be prepared by any coupling method known in the art including the above mentioned techniques. [0160]
Preparation of Enzyme Variants [0161]
Enzyme variants to be conjugated may be constructed by any suitable method. A number of methods are well established in the art. For instance enzyme variants according to the invention may be generated using the same materials and methods described in e.g. WO 89/06279 (Novo Nordisk A/S), EP 130,756 (Genentech), EP 479,870 (Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP application no. 87303761 (Genentech), EP 260,105 (Genencor), WO 88/06624 (Gist-Brocades NV), WO 88/07578 (Genentech), WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 88/08164 (Genex), Thomas et al. (1985) Nature, 318 375-376; Thomas et al. (1987) J. Mol. Biol., 193, 803-813; Russel and Fersht (1987) Nature 328 496-500. [0162]
Coupling of Polymeric Molecules to the Polypeptide in Question [0163]
See previous paragraphs [0164]
Immunogenicity and Allergenicity [0165]
“Immunogenicity” is a wider term than “antigenicity” and “allergenicity”, and expresses the immune system's response to the presence of foreign substances. Said foreign substances are called immunogens, antigens and allergens depending of the type of immune response the elicit. [0166]
An “immunogen” may be defined as a substance which, when introduced into circulatory system of animals and humans, is capable of stimulating an immunologic response resulting in formation of immunoglobulin. [0167]
The term “antigen” refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule. [0168]
Further, an “allergen” may be defined as an antigen which may give rise to allergic sensitization or an allergic response by IgE antibodies (in humans, and molecules with comparable effects in animals). [0169]
Assessment of Immunogencity [0170]
Assessment of the immunogenicity may be made by injecting an animal subcutaneously to enter the immunogen into the circulation system and comparing the response with the response of the corresponding parent polypeptide. [0171]
The “circulatory system” of the body of humans and animals means, in the context of the present invention, the system which mainly consists of the heart and blood vessels. The heart delivers the necessary energy for maintaining blood circulation in the vascular system. The circulation system functions as the organism's transportation system, when the blood transports O[0172] ₂, nutritious matter, hormones, and other substances of importance for the cell regulation into the tissue. Further the blood removes CO₂from the tissue to the lungs and residual substances to e.g. the kidneys. Furthermore, the blood is of importance for the temperature regulation and the defense mechanisms of the body, which include the immune system.
A number of in vivo animal models exist for assessment of the immunogenic potential of polypeptides. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a mice model. [0173]
This model seeks to identify the immunogenic response in the form of the IgG response in Balb/C mice being injected subcutaneously with modified and unmodified polypeptides. [0174]
Also other animal models can be used for assessment of the immunogenic potential. [0175]
A polypeptide having “reduced immunogenicity” according to the invention indicates that the amount of produced antibodies, e.g. immunoglobulin in humans, and molecules with comparable effects in specific animals, which can lead to an immune response, is significantly decreased, when introduced into the circulatory system, in comparison to the corresponding parent polypeptide. [0176]
For Balb/C mice the IgG response gives a good indication of the immunogenic potential of polypeptides. [0177]
Assessment of Allergenicity [0178]
Assessment of allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent enzymes with the corresponding modified is enzymes according to the invention. [0179]
A number of in vivo animal models exist for assessment of the allegenicity of enzymes. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitized animals. According to these models the alleged allergens are introduced intratracheally into the animals. [0180]
A suitable strain of guinea pigs, the Dunkin Hartley strain, do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG1A and IgG1B (see e.g. Prentø, ATLA, 19, p. 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B is a measure of the allergenicity level. [0181]
The Balb/C mice strain is suitable for intratracheal, intradermal or subcutaneous exposure. Balb/C mice produce IgE as the allergic response. [0182]
More details on assessing respiratory allergens in guinea pigs and mice is described by Kimber et al., 1996, Fundamental and Applied Toxicology, 33, p. 1-10. [0183]
Other animals such as rats, rabbits etc. may also be used for comparable studies. [0184]
Composition [0185]
The invention relates to a composition comprising a modified polypeptide of the invention. [0186]
The composition may be a pharmaceutical or industrial composition. [0187]
The composition may further comprise other polypeptides, proteins or enzymes and/or ingredients normally used in e.g. detergents, including soap bars, household articles, agrochemicals, personal care products, including skin care compositions, cleaning compositions for e.g. contact lenses, oral and dermal pharmaceuticals, composition use for treating textiles, compositions used for manufacturing food, e.g. baking, and food/feed etc. [0188]
Use of the Polypeptide [0189]
The invention also relates to the use of the method of the invention for reducing the immune response of polypeptides. [0190]
It is also an object of the invention to use the polypeptide-polymer conjugate or the polypeptide otherwise modified according to the invention to reduce the allergenicity of industrial products, such as detergents, such as laundry, disk wash and hard surface cleaning detergents, food or feed products, personal care products and textile products. [0191]
Material and Methods [0192]
Materials [0193]
Enzymes: [0194]
PD498: Protease of subtilisin type shown in WO 93/24623. The sequence of PD498 is shown in SEQ ID NOS: 1 and 2. [0195]
Savinase®: The sequence is shown in SEQ ID NO: 3 (Available from Novo Nordisk A/S). [0196]
Subtilisin BPN′: The sequence can be found in the SWISS-PROT database. The sequence is also disclosed in: GALLAGHER T., OLIVER J., BOTT R., BETZEL C., GILLILAND G. L.; “Subtilisin BPN′ at 1.6-A resolution: analysis for discrete disorder and comparison of crystal forms.”; Acta Crystallogr. D 52:1125-1135(1996). The enzyme is available from Novo Nordisk A/S. [0197]
Amylase AA560: The alkaline alpha-amylase may be derived from a strain of Bacillus sp. DSM 12649. The strain was deposited on Jan. 25, 1999 by the inventors under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen und Zelikulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig D E. The sequence is shown in SEQ ID NO: 4. [0198]
Strains: [0199]
[0200] B. subtilis 309 and 147 are variants of Bacillus lentus, deposited with the NCIB and accorded the accession numbers NCIB 10309 and 10147, and described in U.S. Pat. No. 3,723,250 incorporated by reference herein.
[0201] E. coli MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol. 138 179-207), was made r⁻,m⁺ by conventional methods and is also described in U.S. patent application Ser. No. 039,298.
Vectors: [0202]
pPD498: [0203] E. coli—B. subtilis shuttle vector (described in U.S. Pat. No. 5,621,089 under section 6.2.1.6) containing the wild-type gene encoding for PD498 protease (SEQ ID NO: 2). The same vector is use for mutagenesis in E. coli as well as for expression in B. subtilis.
Materials, Chemicals and Solutions: [0204]
Horse Radish Peroxidase labeled anti-rat-Ig (Dako, DK, P162, #031; dilution 1:1000). [0205]
Mouse anti-rat IgE (Serotec MCA193; dilution 1:200). [0206]
Rat anti-mouse IgE (Serotec MCA419; dilution 1:100). [0207]
Biotin-labeled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140; dilution 1:1000). [0208]
Biotin-labeled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; dilution 1:1000). [0209]
Streptavidin-horse radish peroxidase (Kirkegard & Perry 14-30-00; dilution 1:1000). [0210]
CovaLink NH[0211] ₂plates (Nunc, Cat #459439)
Cyanuric chloride (Aldrich) [0212]
Acetone (Merck) [0213]
Rat anti-Mouse IgG1, biotin (SeroTec, Cat #MCA336B) [0214]
Streptavidin, peroxidase (KPL) [0215]
Ortho-Phenylene-diamine (OPD) (Kem-en-Tec, Cat #4260) [0216]
H[0217] ₂O₂, 30% (Merck)
Tween 20 (Merck) [0218]
Skim Milk powder (Difco) [0219]
H[0220] ₂SO₄(Merck)
Buffers and Solutions: [0221]
Carbonate buffer (0.1 M, pH 10 (1 liter)) Na[0222] ₂CO₃10.60 g
PBS (pH 7.2 (1 liter)) NaCl 8.00 g KCl 0.20 g K[0223] ₂HPO₄1.04 g KH₂PO₄0.32 g
Washing buffer PBS, 0.05% (v/v) Tween 20 [0224]
Blocking buffer PBS, 2% (wt/v) Skim Milk powder [0225]
Dilution buffer PBS, 0.05% (v/v) Tween 20, 0.5% (wt/v) Skim Milk powder [0226]
Citrate buffer (0.1 M, pH 5.0-5.2 (1 liter)) Na Citrate 20.60 g Citric acid 6.30 g [0227]
Sodium Borate, borax (Sigma) [0228]
3,3-Dimethyl glutaric acid (Sigma) [0229]
CaCl[0230] ₂(Sigma)
Tresyl chloride (2,2,2-triflouroethansulfonyl chloride) (Fluka) [0231]
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fluka) [0232]
N-Hydroxy succinimide (Fluka art. 56480)) [0233]
Phosgene (Fluka art. 79380) [0234]
Lactose (Merck 7656) [0235]
PMSF (phenyl methyl sulfonyl flouride) from Sigma [0236]
Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitroanilide (Suc-MPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole. [0237]
Activation of CovaLink Plates: [0238]
Make a fresh stock solution of 10 mg cyanuric chloride per ml acetone. [0239]
Just before use, dilute the cyanuric chloride stock solution into PBS, while stirring, to a final concentration of 1 mg/ml. [0240]
Add 100 ml of the dilution to each well of the CovaLink NH2 plates, and incubate for 5 minutes at room temperature. [0241]
Wash 3 times with PBS. [0242]
Dry the freshly prepared activated plates at 50° C. for 30 minutes. [0243]
Immediately seal each plate with sealing tape. [0244]
Preactivated plates can be stored at room temperature for 3 weeks when kept in a plastic bag. [0245]
Test Animals: [0246]
Female Balb/C mice (about 20 grams) purchased from Bomholdtgaard, Ry, Denmark. [0247]
Female Brown-Norway rats, weighing on the average 180 g [0248]
Equipment: [0249]
XCEL II (Novex) [0250]
ELISA reader (UVmax, Molecular Devices) [0251]
HPLC (Waters) [0252]
PFLC (Pharmacia) [0253]
Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW. [0254]
SLT: Fotometer from SLT LabInstruments [0255]
Size-exclusion chromatograph (Spherogel TSK-G2000 SW). [0256]
Size-exclusion chromatograph (Superdex 200, Pharmacia, SW) [0257]
Amicon Cell [0258]
Enzymes for DNA Manipulations [0259]
Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restriction endonucleases, ligases etc., are obtained from New England Biolabs. Inc. [0260]
Media: [0261]
BPX: Composition (per liter) [0262]
Potato starch 100 g [0263]
Ground barley 50 g [0264]
Soybean flour 20 g [0265]
Na[0266] ₂HPO₄×12 H₂O 9 g
Pluronic 0.1 g [0267]
Sodium caseinate 10 g [0268]
The starch in the medium is liquefied with alpha-amylase and the medium is sterilized by heating at 120° C. for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition of NaHCO[0269] ₃to 0.1 M.
Methods [0270]
General Molecular Biology Methods: [0271]
Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990). [0272]
Enzymes for DNA manipulations were used according to the specifications of the suppliers. [0273]
Fermentation of PD498 Variants [0274]
Fermentation of PD498 variants in [0275] B. subtilis are performed at 30° C. on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days. In order to make an e.g. 2 liter broth 20 Erlenmeyer flasks are fermented simultaneously.
Purification of PD498 Variants [0276]
Approximately 1.6 liters of PD498 variant fermentation broth are centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The supernatants are adjusted to pH 7.0 using 10% acetic acid and filtered on Seitz Supra S100 filter plates. [0277]
The filtrates are concentrated to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate is centrifuged and filtered prior to absorption at room temperature on a Bacitracin affinity column at pH 7. The PD498 variant is eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethyl-glutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7. [0278]
The fractions with protease activity from the Bacitracin purification step are combined and applied to a 750 ml Sephadex G25 column (5 cm diameter) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 6.0. [0279]
Fractions with proteolytic activity from the Sephadex G25 column are combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.1 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.0. [0280]
The protease is eluted using a linear gradient of 0-0.5 M sodium chloride in 1 liter of the same buffer. [0281]
Protease containing fractions from the CM Sepharose column are combined and filtered through a 2 micron filter. [0282]
Determination of the Molecular Weight [0283]
Electrophoretic separation of proteins was performed by standard methods using 4-20% gradient SDS polyacrylamide gels (Novex). Proteins were detected by silver staining. The molecular weight was measured relative to the mobility of Mark-12® wide range molecular weight standards from Novex. [0284]
Protease Activity [0285]
Analysis with Suc-Ala-Ala-Pro-Phe-pNa: [0286]
Proteases cleave the bond between the peptide and p-nitroaniline to give a visible yellow color absorbing at 405 nm. [0287]
Buffer: e.g. Britton and Robinson buffer pH 8.3 [0288]
Substrate: 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO). 100 ml of this is diluted into 10 ml with Britton and Robinson buffer. [0289]
The substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS[0290] _{405 nm}/min. The temperature should be controlled (20-50° C. depending on protease). This is a measure of the protease activity in the sample.
Proteolytic Activity [0291]
In the context of this invention proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU). The activity is determined relatively to an enzyme standard (SAVINASE_), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference. [0292]
A GU is a Glycine Unit, defined as the proteolytic enzyme activity which, under standard conditions, during a 15 minute incubation at 40° C., with N-acetyl casein as substrate, produces an amount of NH[0293] ₂-group equivalent to 1 mmole of glycine.
Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of American Oil Chemists Society, Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988). [0294]
ELISA IgE Test System (for Brown Norway Rats): [0295]
A three layer sandwich ELISA is used to determine relative concentrations of specific antibodies. [0296]
The immunizing molecule is used as coating antigen with 10 mg per ml and 50 ml per well, in neutral phosphate buffer, incubated overnight at 4° C. All remaining binding spots on the well surface are blocked in 2% skim milk, 200 ml per well in phosphate buffer for at least 30 minutes at room temperature (RT). All seras to be tested with this antigen are added at 50 ml per well to this plate using a 8-channel pipette in dilution series from 10× diluted followed by 3-fold dilutions. Dilutions are made in phosphate buffer with 0.5% skim milk and 0.05% Tween 20, incubated 2 hours on agitation platform at RT. The “tracer” molecule is biotinylated Mouse [0297] anti Rat IgE 50 ml per well and diluted 2000× in phosphate buffer with 0.5% skim milk and 0.05% Tween 20, incubated 2 hours on an agitation platform at RT. Control (blank) was identical sequence but without rat sera. 50 ml per well streptavidin horse raddish peroxidase, diluted 2000× was incubated 1 hour on an agitation platform. Colouring substrate at 50 ml per well is OPD (6 mg) and H₂O₂(4 ml of a 30% solution) per 10 ml citrate buffer pH 5.2. The reaction is stopped using 100 ml per well 2 N H₂SO₄. All readings on SLT at 486 nm and 620 nm as reference. Data is calculated and presented in Lotus.
ELISA Procedure to Determine Relative Concentrations of IgE Antibodies in BALB/C Mice [0298]
A three layer sandwich ELISA is used to determine relative concentrations of specific IgE serum antibodies. [0299]
1) Coat the ELISA-plate with 10 mg rat anti-mouse IgE or mouse anti-rat IgE/ml buffer 1. [0300]
[0301] 50 ml/well. Incubate over night at 4° C.
2) Empty the plates and block with Blocking buffer at least Y2 hour at room temperature. [0302]
200 ml/well. Shake gently. Wash the plates 3 times with Washing Buffer. [0303]
3) Incubate with mouse/rat sera, starting from undiluted and continue with 2-fold dilutions. Keep some wells free for buffer 4 only (blanks). 50 ml/well. Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer. [0304]
4) Dilute the enzyme in Dilution buffer to the appropriate protein concentration. 50 ml/well. [0305]
Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer. [0306]
5) Dilute specific polyclonal anti-enzyme antiserum serum (plg) for detecting bound antibody in Dilution buffer. 50 ml/well. Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer. [0307]
6) Dilute Horseradish Peroxidase-conjugated anti-plg-antibody in Dilution buffer. 50 ml/well. [0308]
Incubate at room temperature for 30 minutes. Shake gently. Wash the plates 3 times in Washing Buffer. [0309]
7) Mix 0.6 mg ODP/ml+0.4 microliter H[0310] ₂O₂/ml in substrate Buffer. Make the solution just before use. Incubate for 10 minutes. 50 microliters/well.
8) To stop the reaction: add Stop Solution. 50 microliters/well. [0311]
9) Read the plates at 492 nm with 620 nm as reference. [0312]
Data is calculated and presented in Lotus. [0313]

EXAMPLES

Example 1

Subtilisin BPN′[0314]
In order to identify the residues to be modified, a distance and a directional criteria are applied. [0315]
As disclosed earlier residues having their C[0316] ^alpha-atom closer than 15 Å to a ligand are targets for modification. Preferably, residues having their C^beta-atom closer to the ligand bound than the C^alpha-atom, thereby allowing a potential side chain to point in the direction of the ligand, are targets for modification.
The relevant distance can easily be measured using e.g. molecular graphics programs like InsightII from Molecular Simulations INC. [0317]
Especially surface exposed residues, defined as having ACC>0 when applying the DSSP program to the relevant protein part of the structure, are targets for modification. The DSSP program is disclosed in W. Kabsch and C. Sander, BIOPOLYMERS 22 (1983) pp. 2577-2637. [0318]

In Thomas E. Creighton, PROTEINS; Structure and Molecular Priciples, W H Freeman and Company, NY, ISBN: 0-7167-1566-X (1984) is disclosed a table listing the accessible surface areas of individual amino acid residues. In the table below 15% and 20% accessibility has been determined.



Total ACC	20% of Total	15% of Total

AA	Å × Å	Å × Å	Å × Å
Ala	115	23.0	17.3
Arg	225	45.0	33.8
Asn	160	32.0	24.0
Asp	150	30.0	22.5
Cys	135	27.0	20.3
Gln	180	36.0	27.0
Glu	190	38.0	28.5
Gly	75	15.0	11.3
His	195	39.0	29.3
Ile	175	35.0	26.3
Leu	170	34.0	25.5
Lys	200	40.0	30.0
Met	185	37.0	27.8
Phe	210	42.0	31.5
Pro	145	29.0	21.8
Ser	115	23.0	17.3
Thr	140	28.0	21.0
Trp	255	51.0	38.3
Tyr	230	46.0	34.5
Val	155	31.0	23.3

When dividing the found accessible surface area (ACC) for each amino acid of the protein with the accessible surface area for that individual amino acid (found in the Creighton table) the accessibility value in percent is obtained. [0320]
In order to find residues to modify, the method described above was applied to the X-ray structure of Subtilisin BPN′ in complex with the inhibitor CI-2 (entry 2SNI in the Brookhaven Protein Data Bank). [0321]
Only the Subtilisin BPN′ and the two metal ions in the structure was used for the analysis. Both ions are calcium ions. They are found in site 1 and site 2. [0322]

The results of the analysis are seen in the tables below. The columns shows the distance in Å from the metal ion to the C ^alphaand C^betaas well as the accessibility as determined by DSSP for each residue to modify.



Site 1:

resid	res.no	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

GLY	80	4.40		14	18.67
ASN	77	4.68	4.57	62	38.75
ASP	41	5.14	4.36	0
GLN	2	5.46	4.64	47	26.11
ALA	74	5.57	5.12	0
GLY	83	7.80		0
PRO	86	8.44	7.42	8
GLY	70	9.04		1
THR	208	9.38	8.66	0
HIS	39	10.41	9.97	3
PRO	5	10.46	10.17	18	12.41
LYS	43	10.62	10.53	137	68.50
TYR	214	10.68	9.62	75	32.61
GLN	206	11.79	11.27	88	48.89
VAL	8	12.42	10.89	2
THR	22	13.14	12.12	22	15.71
GLY	215	13.52		14	18.67
PRO	14	13.53	13.29	45	31.03
HIS	17	13.64	12.25	28	14.36
THR	66	13.80	13.76	0
SER	9	14.40	14.22	58	50.43
ALA	13	14.66	13.53	0
GLY	7	14.74		0
LEU	90	14.79	13.38	1
ASP	36	14.87	14.57	20	13.33
GLY	211	14.88		45	60.00

Site 2:

Resid	resno	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

GLU	195	4.44	4.28	48	25.26
ALA	176	4.67	3.85	0
GLY	169	5.16		0
ASP	197	5.90	5.14	21	14.00
VAL	165	8.35	6.96	6
ALA	151	8.54	8.04	0
GLY	166	9.43		14	18.67
GLY	193	9.46		0
GLY	264	9.63		7
VAL	149	9.85	9.50	3
GLY	178	10.74		0
VAL	139	10.95	9.63	0
GLY	154	11.31		17	22.67
SER	163	11.34	10.12	29	25.22
ARG	247	11.35	10.32	47	20.89
LYS	265	11.66	11.35	76	38.00
GLN	251	11.74	10.57	26	14.44
SER	191	11.83	11.04	0
SER	224	12.34	12.02	0
VAL	143	12.36	10.91	41	26.45
MET	124	12.43	11.71	0
GLY	127	12.44		61	81.33
SER	260	12.47	12.12	72	62.61
GLY	131	12.69		29	38.67
VAL	227	13.37	11.90	0
THR	220	13.55	12.34	3
LEU	250	13.58	12.73	3
LEU	135	13.60	13.21	6
GLY	266	13.93		0
GLY	128	14.04		16	21.33
SER	190	14.12	14.09	0
ALA	142	14.13	13.36	0
ILE	122	14.17	13.65	0
ALA	223	14.44	13.70	0
ASN	243	14.50	13.94	21	13.13
ALA	200	14.63	14.15	0

The table below shows functional preferred substitutions in sites 1 and 2 of subtilisin BPN′. For Gly 80 the substitution G to S/T, G to N/Q and G to K/D means that glycine in position 80 may preferably be substituted with serine/threonine or asparagine/glutamine or lysine/aspartic acid.



	Subtilisin BPN'

SITE 1
Gly-80	G to S/T	G to N/Q	G to K/D
Asn-77	N to D/E	N to K/R	N to A/C
Gln-2	Q to D/E	Q to K/R	Q to A/C
Pro-5	P to G/A	P to C/S	P to K/D
Lys-43	K to S/T/C	K to D/E/R	K to Q/N
Tyr-214	Y to N/Q	Y to A/G/G	Y to K/H
Gln-206	Q to D/E	Q to K/R	Q to A/C
Thr-22	T to K/R	T to Q/N/A	T to D/E/C
Gly-215	G to S/T	G to N/Q	G to K/D
Pro-14	P to G/A	P to C/S	P to K/D
Ser-9	S to K/R	S to Q/N/A	S to D/E/C
Gly-211	G to S/T	G to N/Q	G to K/D
SITE 2
Glu-195	G to S/T	G to N/Q	G to K/D
Gly-166	G to S/T	G to N/Q	G to K/D
Gly-154	G to S/T	G to N/Q	G to K/D
Ser-163	S to K/R	S to Q/N/A	S to D/E/C
Arg-247	R to K/H	R to Q/N	R to A/C/E
Lys-265	K to S/T/C	K to D/E/R	K to Q/N
Val-143	V to A/G/H	V to Q/E/C	V to T/S/K
Gly-127	G to S/T	G to N/Q	G to K/D
Ser-260	S to K/R	S to Q/N/A	S to D/E/C
Gly-131	G to S/T	G to N/Q	G to K/D
Gly-128	G to S/T	G to N/Q	G to K/D

Example 2

PD498 [0325]
The 3-dimensional Structure of PD 498 as Determined by X-ray Crystallography in Brookhaven Protein Data Bank (PDB) Format [0326]
The sequence which was used to elucidate the three-dimensional structure forming the basis for the present invention consists of the 280 amino acids derived from Bacillus sp. PD498, NCIMB No. 40484 as disclosed in SEQ ID NO: 2. [0327]
The structure of PD498 was solved in accordance with the principle for X-ray crystallographic methods given in “X-Ray Structure Determination”, Stout, G. K. and Jensen, L. H., John Wiley & Sons, inc. NY, 1989 and “Protein Crystallography” by Blundell, T. L. and Johnson, L. N., Academic Press, London, 1990. The structural coordinates for the solved crystal structure of PD 498 at 2.2 Å resolution using the isomorphous replacement method are given in a standard PDB format (Brookhaven Protein Data Base) in Appendix 1. It is to be understood that Appendix 1 forms part of the present application. [0328]
In Appendix 1 the amino acid residues of the enzyme are identified by three-letter amino acid code (capitalized letters). [0329]
PD498 has three bound metal ions. Site 1 is equivalent to site 1 in subtilisin BPN′ and contains a calcium ion. Site 2 does not have an equivalent in subtilisin BPN′ and contains a calcium ion. Site 3 is in the same region as the 2nd site in subtilisin BPN′ and does here contain a sodium ion and a monopropylene glycol ligand. [0330]

Applying the above method disclosed in example 1 results in:



Site 1:

residue	resno	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

GLY	89	4.26		4
ASP	5	5.02	3.92	0
ASP	48	5.10	4.36	0
ASN	86	5.15	4.73	33	20.63
ALA	82	5.84	4.97	0
GLY	87	6.05		41	54.67
GLY	92	7.33		0
TYR	8	7.87	7.12	12
TYR	7	8.01	7.63	89	38.70
PRO	47	8.13	8.09	59	40.69
PRO	3	8.61	7.55	9
GLY	78	8.69		0
THR	213	9.19	8.55	0
ARG	51	10.39	9.61	162	72.00
HIS	46	10.41	9.93	1
LYS	52	10.56	9.41	10
TYR	219	10.74	9.79	56	24.35
ALA	211	11.55	11.03	9
GLN	12	11.67	10.44	22	12.22
GLY	218	12.00		18	24.00
ALA	10	12.35	12.15	65	56.52
TYR	11	12.46	12.00	121	47.45
VAL	53	13.30	13.18	18	11.61
PRO	15	13.52	12.10	0
ARG	28	13.77	12.93	103	45.78
ILE	99	14.16	13.16	0
ASP	43	14.36	14.04	8
TRP	1	14.43	13.90	71	27.84
GLY	14	14.60		1
GLY	234	14.85		0
GLY	29	14.97		13	17.33

Site 2:

resid	resno	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

ASN	65	4.25	4.04	65	40.63
ASP	61	4.98	3.62	88	58.67
ASP	63	5.30	4.43	46	30.67
ASP	58	5.39	3.87	0
MET	67	5.53	5.42	42	22.70
ILE	60	7.09	6.76	48	27.43
ARG	103	7.67	6.23	4
GLY	41	8.03		1
LEU	69	8.99	8.35	114	67.06
GLY	56	10.02		2
LYS	55	10.15	9.43	115	57.50
ALA	101	11.02	10.20	0
TYR	44	11.83	11.14	35	15.22
GLY	73	13.18		0
ASN	45	13.57	13.14	114	71.25
GLY	119	13.62		0
GLY	111	13.75		36	48.00
GLY	71	13.78		4
SER	115	13.82	12.77	24	20.87
GLY	109	13.90		32	42.67
THR	74	13.96	13.69	0
PRO	215	14.41	13.20	30	20.69
VAL	53	14.70	13.64	18	11.61
VAL	37	14.80	14.62	1

Site 3:

resid	resno	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

ALA	179	4.07	4.05	0
ALA	181	4.65	4.11	0
TRP	200	6.65	6.57	46	18.04
ASP	202	6.86	6.02	19	12.67
ALA	160	7.85	7.10	0
VAL	158	8.84	8.28	0
THR	170	9.23	8.58	65	46.43
VAL	148	10.12	8.77	0
LYS	268	10.74	9.64	108	54.00
ARG	250	11.05	10.04	30	13.33
GLY	183	11.15		2
GLY	198	11.37		8
TRP	152	11.64	10.35	35	13.73
LEU	133	11.65	10.63	0
GLU	254	11.66	10.63	15	7.89
GLY	136	11.84		39	52.00
TYR	269	12.12	11.37	45	19.57
GLY	163	12.15		11	14.67
SER	229	12.16	11.65	0
LEU	144	13.01	12.62	2
ASN	196	13.01	12.00	1
VAL	232	13.12	11.71	0
LEU	131	13.25	12.69	0
ILE	253	13.27	12.22	1
ALA	151	13.59	12.87	0
THR	225	13.88	12.66	1
ASN	246	14.04	13.33	17	10.63
GLY	270	14.22		0
ILE	249	14.51	14.36	4
ALA	228	14.65	14.00	0
SER	141	14.78	14.70	21	18.26
ALA	236	14.93	13.63	0

The table below shows the preferred functional substitutions in sites 1, 2 and 3 of PD498.



	PD498

SITE 1
Asn-86	N to D/E	N to K/R	N to A/C
Gly-87	G to S/T	G to N/Q	G to K/D
Tyr-7	Y to N/Q	Y to A/G/C	Y to K/H
Pro-47	P to G/A	P to C/S	P to K/D
Arg-51	R to K/H	R to Q/N	R to A/C/E
Tyr-219	Y to N/Q	Y to A/G/C	Y to K/H
Gly-218	G to S/T	G to N/Q	G to K/D
Ala-10	A to N/Q	A to K/R	A to D/E
Tyr-11	Y to N/Q	Y to A/G/C	Y to K/H
Arg-28	R to K/H	R to Q/N	R to A/C/E
Trp-1	W to N/Q	W to A/G/C	W to K/H
Gly-29	G to S/T	G to N/Q	G to K/D
SITE 2
Asn-65	N to D/E	N to K/R	N to A/C
Asp-61	D to N/Q	D to K/H	D to A/G/C
Asp-63	D to N/Q	D to K/H	D to A/G/C
Met-67	M to A/G/H	M to Q/E/C	M to T/S/K
Ile-60	I to A/G/H	I to Q/E/C	I to T/S/K
Leu-69	L to A/G/H	L to Q/E/C	L to T/S/K
Lys-55	K to S/T/C	K to D/E/R	K to Q/N
Tyr-44	Y to N/Q	Y to A/G/C	Y to K/H
Asn-45	N to D/E	N to K/R	N to A/C
Gly-111	G to S/T	G to N/Q	G to K/D
Ser-115	S to K/R	S to Q/N/A	S to D/E/C
Gly-109	G to S/T	G to N/Q	G to K/D
Pro-215	P to G/A	P to C/S	P to K/D
SITE 3
Trp-200	W to N/Q	W to A/G/C	W to K/H
Thr-170	T to K/R	T to Q/N/A	T to D/E/C
Lys-268	K to S/T/C	K to D/E/R	K to Q/N
Gly-136	G to S/T	G to N/Q	G to K/D
Tyr-269	Y to N/Q	Y to A/G/C	Y to K/H
Ser-141	S to K/R	S to Q/N/A	S to D/E/C

Example 3

Savinase [0333]

For this example the X-ray structure entry 1SVN in the Brookhaven Protein Data Bank was used. This structure contains two metal ions. Site 1 contains a calcium ion and is at a position equivalent to site 1 in subtilisin BPN′. Site 2 contains a calcium ion at a position equivalent to site 2 in subtilisin BPN′. In the following list a SEQUENTIAL numbering have been applied and NOT the numbering system used in the structure file.



Site 1:

Resid	resno	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

GLY	78	4.28		14	18.67
ASN	75	4.74	4.64	61	38.13
ASP	40	5.08	4.34	0
GLN	2	5.39	4.59	45	25.0
ALA	72	5.49	4.99	0
GLY	81	7.68		0
PRO	84	8.28	7.29	5
GLY	68	8.88		1
THR	202	9.19	8.67	0
HIS	38	10.40	9.89	13
PRO	5	10.47	10.26	14	9.66
ASN	42	10.55	10.50	94	58.75
TYR	208	10.72	9.76	65	28.26
GLN	200	11.75	11.39	82	45.56
ILE	8	12.10	10.58	3
PRO	14	12.91	12.63	49	33.79
THR	22	13.01	12.24	29	20.71
HIS	17	13.44	12.07	29	14.87
ALA	13	13.78	12.63	0
GLY	7	14.60		2
LEU	88	14.86	13.68	0
GLY	223	14.89		0
GLY	23	14.93		0

Site 2:

resid	resno	dist(C^alpha)	dist(C^beta)	ACC (Å × Å)	ACC (%)

ALA	170	4.88	4.24	0
GLY	189	5.10		46	61.33
ASP	191	7.22	6.52	6
ALA	149	7.79	7.05	0
ILE	159	8.29	6.89	1
VAL	147	8.98	8.40	0
VAL	137	9.81	8.44	0
GLY	187	10.71		3
GLY	258	10.85		3
ARG	241	10.90	9.77	39	17.33
GLY	172	11.27		0
GLY	125	11.66		46	61.33
THR	141	11.72	10.47	20	14.29
LEU	122	11.73	10.70	0
GLY	152	11.96		8
LEU	133	12.29	11.70	3
GLN	185	12.41	11.63	14	7.74
THR	218	12.51	11.95	0
LYS	245	12.79	11.71	48	24.00
SER	259	12.93	12.67	35	30.43
ASN	237	13.34	12.53	22	13.75
ALA	120	13.49	13.00	0
THR	254	13.53	13.19	100	71.43
VAL	221	13.62	12.14	0
ALA	140	13.65	13.13	0
VAL	145	13.91	13.88	0
THR	214	14.00	12.84	2
GLY	157	14.11		42	56.00
LEU	244	14.27	13.26	0
ALA	217	14.97	14.17	0

The table below shows the preferred functional substitutions in sites 1 and 2 of Savinase.



	Savinase

SITE 1
Gly-78	G to S/T	G to N/Q	G to K/D
Asn-75	N to D/E	N to K/R	N to A/C
Gln-2	Q to D/E	Q to K/R	Q to A/C
Asn-42	N to D/E	N to K/R	N to A/C
Tyr-208	Y to N/Q	Y to A/G/C	Y to K/H
Gln-200	Q to D/E	Q to K/R	Q to A/C
Pro-14	P to G/A	P to C/S	P to K/D
Thr-22	T to K/R	T to Q/N/A	T to D/E/C
His-17	H to S/T/C	H to D/E	H to Q/N
SITE 2
Gly-189	G to S/T	G to N/Q	G to K/D
Arg-241	R to K/H	R to Q/N	R to A/C/E
Gly-125	G to S/T	G to N/Q	G to K/D
Lys-245	K to S/T/C	K to D/E/R	K to Q/N
Ser-259	S to K/R	S to Q/N/A	S to D/E/C
Thr-254	T to K/R	T to Q/N/A	T to D/E/C
Gly-157	G to S/T	G to N/Q	G to K/D

Example 4

Amylase (AA560) [0336]
For this example the structure of M560 has been found by homology modelling using the BAN/Termamyl alpha-amylase structure disclosed in WO 96/23874 which is hereby incorporated by reference. This structure contains two metal ions. Both sites 1 and 2 contain a calcium ion. [0337]
The example shows how a 3-dimensional structure determined by model building using coordinates from a homologous structure, can be used to identify residues of the ligand binding site, which may be modified in order to reduce the immune response. [0338]

Applying the method disclosed above results in:



Res	ACC (Å × Å)	ACC ()

Site 1
TYR 58: CA	23	10.00
GLY 59: CA	4
ALA 60: CA	0
VAL 103: CA	0
VAL 104: CA	1
MET 105: CA	6
ASN 106: CA	1
HIS 107: CA	6
LYS 108: CA	14
GLY 109: CA	2
VAL 122: CA	3
PRO 124: CA	27	18.62
ASN 126: CA	28	17.50
ARG 127: CA	17
ASN 128: CA	107	66.88
THR 141: CA	0
TRP 159: CA	75	29.41
TYR 160: CA	96	41.74
HIS 161: CA	2
PHE 162: CA	0
ASP 163: CA	1
GLY 164: CA	0
VAL 165: CA	6
ASP 166: CA5	64	42.67
ILE 177: CA	12
TYR 178: CA	0
LYS 179: CA	27	13.50
PHE 180: CA	0
LYS 185: CA	36	18.00
GLY 186: CA	24	32.00
TRP 187: CA	27	10.59
ASP 188: CA	0
TRP 189: CA	136	53.33
GLU 190: CA	39	20.53
VAL 191: CA	0
ASP 192: CA	11
THR 193: CA	84	60.00
GLU 194: CA	88	46.32
ASN 195: CA	36	22.50
GLY 196: CA	27	36.00
ASN 197: CA	8
TYR 198: CA	41	17.83
ASP 199: CA	1
TYR 200: CA	2
LEU 201: CA	50	29.41
MET 202: CA	72	38.92
TYR 203: CA	93	40.43
ALA 204: CA	2
ASP 205: CA	0
ILE 206: CA	4
ASP 207: CA	6
MET 208: CA	5
ASP 209: CA	74	49.33
HIS 210: CA	39	20.00
VAL 213: CA	0
VAL 214: CA	26	16.77
LEU 217: CA	4
ILE 235: CA	0
ASP 236: CA	15
ALA 237: CA	5
VAL 238: CA	0
LY5 239: CA	14
HIS 240: CA	13
ILE 241: CA	1
LY5 242: CA	44	22.00
TYR 243: CA	5
SER 244: CA	40	34.78
PHE 245: CA	10
THR 246: CA	0
ARG 247: CA	60	26.67
TRP 249: CA	0
ALA 265: CA	0
GLU 266: CA	17	8.95
PHE 267: CA	2
TRP 268: CA	27	10.59
Site 2:
ASN 296: CA	25	15.63
LEU 297: CA	1
TYR 298: CA	68	29.57
ASN 299: CA	72	45.00
ALA 300: CA	0
SER 301: CA	0
LYS 302: CA	117	58.50
SER 303: CA	43	37.39
GLY 304: CA	70	93.33
GLY 305: CA	8	10.67
ASN 306: CA	149	93.13
TYR 307: CA	49	21.30
ASP 308: CA	59	39.33
MET 309: CA	0
ARG 310: CA	143	63.56
GLN 311: CA	99	55.00
ILE 312: CA	3
PHE 313: CA	17	8.10
ASN 314: CA	76	47.50
GLU 345: CA	73	38.42
TRP 347: CA	89	38.70
PHE 348: CA	2
LEU 351: CA	2
ALA 352: CA	0
TYR 404: CA	32	13.91
LEU 405: CA	35	20.59
ASP 406: CA	78	52.00
HIS 407: CA	69	35.38
HIS 408: CA	100	51.28
ASN 409: CA	31	19.38
ILE 410: CA	19	10.86
ILE 411: CA	0
GLY 412: CA	0
ILE 429: CA	0
MET 430: CA	5
SER 431: CA	0
ASP 432: CA	5
GLY 433: CA	19	25.33
ALA 434: CA	73	63.48
GLY 435: CA	35	46.67
GLY 436: CA	21	28.00
ASN 437: CA	86	53.75
VAL 474: CA	0
ASN 475: CA	53	33.13
GLY 476: CA	41	54.67
GLY 477: CA	29	38.67
SER 478: CA	18	15.65
VAL 479: CA	2

The table below shows functional preferred substitutions in site 1 and 2 of the amylase AA560. For ASN 126 the substitution N to D/E means that asparagine in position 126 may preferably be substituted with aspartic acid or glutamic acid, lysine or arginine, or alanine or cysteine.



Functional preferred substitutions

Site 1
Asn 126	N to D/E	N to K/R	N to A/C
Asn 128	N to D/E	N to K/R	N to A/C
Trp 159	W to N/Q	W to A/G/C	W to K/H
Tyr 160	Y to N/Q	Y to A/G/C	Y to K/H
Asp 166	D to N/Q	D to K/H	D to A/G/C
Lys 185	K to S/T/C	K to D/E	K to Q/N
Trp 189	W to N/Q	W to A/G/C	W to K/H
Glu 190	E to N/Q	E to K/H	E to A/G/C
Asp 209	D to N/Q	D to K/H	D to A/G/C
His 210	H to S/T/C	H to D/E	H to Q/N
Val 214	V to Q/N	V to G/A/C	V to K/H/D
Lys 242	K to S/T/C	K to D/E	K to Q/N
Ser 244	S to K/R	S to Q/N/A	S to D/E/C
Arg 247	R to K/H	R to Q/N	R to A/C/E
Site 2
Lys 302	K to S/T/C	K to D/E	K to Q/N
Ser 303	S to K/R	S to Q/N/A	S to D/E/C
Asn 306	N to D/E	N to K/R	N to A/C
Tyr 307	Y to N/Q	Y to A/G/C	Y to K/H
Asp 308	D to N/Q	D to K/H	D to A/G/C
Arg 310	R to K/H	R to Q/N	R to A/C/E
Gln 311	Q to D/E	Q to K/R	Q to A/C
Asn 314	N to D/E	N to K/R	N to A/C
Glu 345	E to N/Q	E to K/H	E to A/G/C
Trp 347	W to N/Q	W to A/G/C	W to K/H
Asp 406	D to N/Q	D to K/H	D to A/G/C
His 407	H to S/T/C	H to D/E	H to Q/N
His 408	H to S/T/C	H to D/E	H to Q/N
Ala 434	A to N/Q	A to K/R	A to D/E
Asn 437	N to D/E	N to K/R	N to A/C
Asn 475	N to D/E	N to K/R	N to A/C
Gly 476	G to S/T	G to N/Q	G to K/D
Ser 478	S to K/R	S to Q/N/A	S to D/E/C

Example 5

Conjugation of Savinase Variant R241K with Activated bis-PEG-1000 [0341]
228 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 510 mg of N-succinimidyl carbonate activated bis-PEG 1000 in a reaction volume of approximately 30 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours. The reaction was stopped by adding 1 M HCl to a final pH of 6.0. [0342]
Reagent excess was removed by ultra filtration using a Filtron-Ultrasette and the final product stored at −20° C., in 50 mM Sodium Borate, 150 mM NaCl, 1 mM CaCl2, 50% mono propylene glycol at H 6.0. [0343]
Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide (SEQ ID NO: 6)). [0344]

Example 6

Conjugation of Savinase Variant R241K with Activated bis-PEG-2000 [0345]
353 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 1621 mg of N-succinimidyl carbonate activated bis-PEG 2000 in a reaction volume of approximately 35 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours. The reaction was stopped by adding 1 M HCl to a final pH of 6.0. [0346]
Reagent excess was removed by ultra filtration using a Filtron-Ultrasette and the final product stored at −20° C., in 50 mM Sodium Borate, 150 mM NaCl, 1 mM CaCl2, 50% mono propylene glycol at pH 6.0. [0347]
Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide (SEQ ID NO: 6)). [0348]

Example 7

Determination of IgE Levels in Rats of R241KbPEG1000 and R241KbPEG2000 [0349]
Methods: [0350]
Sample Management: Each sample was diluted to 0.075 mg protein/ml, and aliquoted in 1.5 ml. These fractions were sent to the stables for storage at −20° C. until use. Additionally, 100 microliters of the respective fractions were stored in the lab-freezer at −20° C. for immunochemical analysis at the beginning, halfway and at the end of the study. For each immunization and each analysis a new fraction was taken. [0351]
Immunization: Twenty intratracheal immunizations were performed weekly with 100 microliters 0.9% (wt/vol) NaCl (control group), or 100 microliters of the protein dilution mentioned before. ([0352] group 5 unmodified R241K variant of Savinase, group 6 R241K-bis-S-PEG1000, and group 7 R241K-bis-S-PEG2000. Each group contained 10 rats. Blood samples (2 ml) were collected from the eye one week after every second immunization. Serum was obtained by blood clothing, and centrifugation.
ELISA: Specific IgE levels were determined using the ELISA's specific for rat IgE. The sera were titrated at ½ dilutions, starting from undiluted. Optical densities were measured at 492/620 nm. [0353]
The results are shown in FIG. 1. As can be seen the IgE levels of the conjugated savinase variants R241K are reduced compared to the savinase variant R241K. [0354]

Example 8

Determination of IgE Levels in Mice of Savinase Variants R241Q, R241E, R241H and R241K. [0355]
Female Balb/c mice, 9 weeks of age were immunised subcutaneously for 20 consecutive weeks, with wild type savinase, and with variants having single mutations in position R241 (R241Q, R241E, R241H, R241K). Every other week, IgG1 and IgE serum levels were determined by ELISA. [0356]
Sample Management: Each sample was diluted to 0.010 mg protein/ml, and aliquoted in 1.5 ml. These fractions were sent to the stables for storage at −20° C. until use. Additionally, 100 microliters of the respective fractions were stored in the lab-freezer at −20° C. for immunochemical analysis at the beginning, halfway and at the end of the study. For each immunization and each analysis a new fraction was taken. [0357]
Immunization: Twenty subcutanuous immunizations were performed weekly with 100 microliters 0.9% (wt/vol) NaCl (control group), or 100 microliters of the protein dilution mentioned before. Thus, group 1 received wild type Savinase, group 2 (R241Q), group 3 (R241H), group 4 (R241E), and group 5 (R241K). Each group contained 10 mice. Blood samples (100 microliters) were collected from the eye one week after every second immunization. Serum was obtained by blood clotting, and centrifugation. [0358]
ELISA: Specific IgG1 levels were determined using the ELISA specific for mouse IgG1. The sera were titrated at ½ dilutions, starting from 1:160. [0359]
Specific IgE levels were determined using the ELISAs specific for mouse IgE. The sera were titrated at ½ dilutions, starting from undiluted. Optical densities were measured at 492/620 nm. [0360]
Statistical analysis: Differences between data sets were analysed by using nonparametric methods: the Kruskal-Wallis Test and the Dunn's Multiple Comparison Test. [0361]
The results are shown in FIG. 2. As can be seen the IgE levels of the Savinase variants are significantly reduced. [0362]
1 6 1 840 DNA Bacillius sp. CDS (1)...(840) 1 tgg tca ccg aat gac cct tac tat tct gct tac cag tat gga cca caa 48 Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 1 5 10 15 aac acc tca acc cct gct gcc tgg gat gta acc cgt gga agc agc act 96 Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr 20 25 30 caa acg gtg gcg gtc ctt gat tcc gga gtg gat tat aac cac cct gat 144 Gln Thr Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp 35 40 45 ctt gca aga aaa gta ata aaa ggg tac gac ttt atc gac agg gac aat 192 Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe Ile Asp Arg Asp Asn 50 55 60 aac cca atg gat ctt aac gga cat ggt acc cat gtt gcc ggt act gtt 240 Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 gct gct gat acg aac aat gga att ggc gta gcc ggt atg gca cca gat 288 Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp 85 90 95 acg aag atc ctt gcc gta cgg gtc ctt gat gcc aat gga agt ggc tca 336 Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser 100 105 110 ctt gac agc att gcc tca ggt atc cgc tat gct gct gat caa ggg gca 384 Leu Asp Ser Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gln Gly Ala 115 120 125 aag gta ctc aac ctc tcc ctt ggt tgc gaa tgc aac tcc aca act ctt 432 Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser Thr Thr Leu 130 135 140 aag agt gcc gtc gac tat gca tgg aac aaa gga gct gta gtc gtt gct 480 Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala 145 150 155 160 gct gca ggg aat gac aat gta tcc cgt aca ttc caa cca gct tct tac 528 Ala Ala Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr 165 170 175 cct aat gcc att gca gta ggt gcc att gac tcc aat gat cga aaa gca 576 Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala 180 185 190 tca ttc tcc aat tac gga acg tgg gtg gat gtc act gct cca ggt gtg 624 Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val 195 200 205 aac ata gca tca acc gtt ccg aat aat ggc tac tcc tac atg tct ggt 672 Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly 210 215 220 acg tcc atg gca tcc cct cac gtg gcc ggt ttg gct gct ttg ttg gca 720 Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Ala Ala Leu Leu Ala 225 230 235 240 agt caa ggt aag aat aac gta caa atc cgc cag gcc att gag caa acc 768 Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu Gln Thr 245 250 255 gcc gat aag atc tct ggc act gga aca aac ttc aag tat ggt aaa atc 816 Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile 260 265 270 aac tca aac aaa gct gta aga tac 840 Asn Ser Asn Lys Ala Val Arg Tyr 275 280 2 280 PRT Bacillius sp. 2 Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 1 5 10 15 Asn Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr 20 25 30 Gln Thr Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp 35 40 45 Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe Ile Asp Arg Asp Asn 50 55 60 Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp 85 90 95 Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser 100 105 110 Leu Asp Ser Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gln Gly Ala 115 120 125 Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser Thr Thr Leu 130 135 140 Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala 145 150 155 160 Ala Ala Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr 165 170 175 Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala 180 185 190 Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val 195 200 205 Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly 210 215 220 Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Ala Ala Leu Leu Ala 225 230 235 240 Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu Gln Thr 245 250 255 Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile 260 265 270 Asn Ser Asn Lys Ala Val Arg Tyr 275 280 3 269 PRT Bacillius lentus 3 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 4 1458 DNA Bacillius sp. CDS (1)...(1458) 4 cac cat aat ggt acg aac ggc aca atg atg cag tac ttt gaa tgg tat 48 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 cta cca aat gac gga aac cat tgg aat aga tta agg tct gat gca agt 96 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 aac cta aaa gat aaa ggg atc tca gcg gtt tgg att cct cct gca tgg 144 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 aag ggt gcc tct caa aat gat gtg ggg tat ggt gct tat gat ctg tat 192 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gat tta gga gaa ttc aat caa aaa gga acc att cgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 acg cgc aat cag tta caa gct gca gtt aac gcc ttg aaa agt aat gga 288 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 att caa gtg tat ggc gat gtt gta atg aat cat aaa ggg gga gca gac 336 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 gct acc gaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga aat 384 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 caa gaa gtg tcc ggt gaa tat aca att gag gct tgg aca aag ttt gac 432 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 ttt cca gga cga ggt aat act cat tca aac ttc aaa tgg aga tgg tat 480 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 cac ttt gat gga gta gat tgg gat cag tca cgt aag ctg aac aat cga 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 att tat aaa ttt aga ggt gat gga aaa ggg tgg gat tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 aca gaa aac ggt aac tat gat tac cta atg tat gca gat att gac atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 gat cac cca gag gta gtg aat gag cta aga aat tgg ggt gtt tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 acg aat aca tta ggc ctt gat ggt ttt aga ata gat gca gta aaa cat 720 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 ata aaa tac agc ttt act cgt gat tgg att aat cat gtt aga agt gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 act ggc aaa aat atg ttt gcg gtt gcg gaa ttt tgg aaa aat gat tta 816 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 ggt gct att gaa aac tat tta aac aaa aca aac tgg aac cat tca gtc 864 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 ttt gat gtt ccg ctg cac tat aac ctc tat aat gct tca aaa agc gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 ggg aat tat gat atg agg caa ata ttt aat ggt aca gtc gtg caa aga 960 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 cat cca atg cat gct gtt aca ttt gtt gat aat cat gat tcg caa cct 1008 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 gaa gaa gct tta gag tct ttt gtt gaa gaa tgg ttc aaa cca tta gcg 1056 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 tat gct ttg aca tta aca cgt gaa caa ggc tac cct tct gta ttt tat 1104 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 gga gat tat tat ggc att cca acg cat ggt gta cca gcg atg aaa tcg 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 aaa att gac ccg att cta gaa gcg cgt caa aag tat gca tat gga aga 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 caa aat gac tac tta gac cat cat aat atc atc ggt tgg aca cgt gaa 1248 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 ggg aat aca gca cac ccc aac tcc ggt tta gct act atc atg tcc gat 1296 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 ggg gca gga gga aat aag tgg atg ttt gtt ggg cgt aat aaa gct ggt 1344 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 caa gtt tgg acc gat atc act gga aat cgt gca ggt act gtt acg att 1392 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 aat gct gat gga tgg ggt aat ttt tct gta aat gga gga tca gtt tct 1440 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg gta aac aaa taa 1458 Ile Trp Val Asn Lys * 485 5 485 PRT Bacillius sp. 5 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 6 4 PRT Bacillius sp. 6 Ala Ala Pro Phe 1

Claims

What is claimed is:

1. A polypeptide with reduced immune response, having one or more amino acid residues modified, wherein the C^alpha-atoms of the amino acid residues are located less than 15 Å from a ligand bound to the polypeptide.

2. The polypeptide of claim 1, wherein the polypeptide has reduced allergenicity.

3. The polypeptide of claim 1, wherein the C^beta-atom of the amino acid residues is located closer to the ligand than the C^alpha-atom.

4. The polypeptide of claim 1, wherein the C^alpha-atoms of the amino acid residues are located less than 10 Å from the ligand and the amino acid residues have an accessibility of at least 15%.

5. The polypeptide of claim 1, wherein the ligand is a metal or metal ion.

6. The polypeptide of claim 1, wherein the polypeptide is modified by substitution of amino acid residues.

7. The polypeptide of claim 1, wherein the modified polypeptide is selected from a diverse library of variants.

8. The polypeptide of claim 6, wherein the substituting amino acids contain amino groups in the form of Lysine residues(s), or carboxylic groups in the form of Aspartic acid or Glutamic acid residues, or SH-groups in the form of Cysteine residues.

9. The polypeptide of claim 6, wherein the modification(s) is(are) prepared by a conservative substitution of an amino acid residue, such as an Arginine to Lysine substitution or Aspargine to Aspartate/Glutamate or a Glutamine to Aspartate/Glutamate substitution or Threonine/Serine to Cysteine.

10. The polypeptide of claim 1, wherein the polypeptide is modified by coupling one or more polymeric molecules to the polypeptide, thereby providing a polypeptide-polymer conjugate.

11. The polypeptide of claim 10, wherein the parent polypeptide moiety of the conjugate has a molecular weight from 1 to 1000 kDa.

12. The polypeptide of claim 10, wherein the polymeric molecules coupled to the polypeptide have a molecular weight from 0.1 to 100 kDa.

13. The polypeptide of claim 1, wherein the polypeptide or parent polypeptide is an enzyme selected from the group of Oxidoreductases, including laccases and Superoxide dismutase (SOD); Hydrolases, including carbohydrases, amylases, proteases, especially subtilisins; Transferases, including Transglutaminases (TGases); Isomerases, including Protein disulfide Isomerases (PDI); Lyases, including Pectate lyases.

14. The polypeptide of claim 13, wherein the polypeptide or parent polypeptide is PD498, Savinase®, BPN′, Amylase, Proteinase K, Proteinase R, Subtilisin DY, Lion Y, Rennilase®, JA16, Alcalase®.

15. The polypeptide of claim 14, wherein the polypeptide or parent polypeptide of the conjugate is a PD498 variant with one or more of the following substitutions: The amino acid residues in position 86, 87, 7, 47, 51, 219, 12, 218, 10, 11, 53, 28, 1, 65, 61, 63, 67, 60, 69, 55, 44, 45, 111, 115, 109, 215, 200, 202, 170, 268, 250, 152, 254, 136, 269, 246, 141 is substituted with K, D, E, or C, preferably R250K, R250D, R250E, R250C.

16. The polypeptide of claim 14, wherein the polypeptide or parent polypeptide is a BPN′ variant with one or more of the following substitutions: The amino acid residues in position 77, 2, 5, 43, 214, 206, 22, 215, 14, 17, 9, 36, 211, 195, 197, 154, 163, 247, 265, 251, 143, 127, 260, 131, 128, 243 is substituted with K, D, E, or C, preferably R247K, R247D, R247E, R247C.

17. The polypeptide of claim 14, wherein the polypeptide or parent polypeptide is a Savinase® variant with one or more of the following substitutions: The amino acid residues in position 75, 2, 42, 208, 200, 14, 22, 17, 189, 241, 125, 125, 141, 245, 259, 237, 254, 157 is substituted with K, D, E, or C, preferably R241K, R241D, R241E, R241C.

18. The polypeptide of claim 14, wherein the polypeptide or parent polypeptide is an amylase variant with one or more of the following substitutions: The amino acid residue in position 124, 126, 128, 159, 160, 166, 185, 186, 189, 190, 193, 194, 195, 196, 198, 201, 202, 203, 209, 210, 214, 242, 244, 247, 296, 298, 299, 302, 303, 304, 306, 307, 308, 310, 311, 314, 345, 347, 405, 406, 407, 408, 409, 433, 434, 435, 436, 437, 475, 476, 477, 478 is substituted with K, D, E, or C.

19. The polypeptide of claim 10, wherein the polymeric molecule is selected from natural or synthetic homo- and heteropolymers, selected from the group of the synthetic polymeric molecules including Branched PEGs, poly-vinyl alcohol (PVA), poly-carboxyl acids, poly-(vinylpyrolidone) and poly-D,L-amino acids, or natural occurring polymeric molecules including dextrans, including carboxymethyl-dextrans, and celluloses such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, pullulans, xanthan gums, carrageenin, pectin and alginic acid.

20. The polypeptide of claim 19, wherein the modified polypeptide is savinase variant R241KbPEG1000 or R241KbPEG2000.

21. The polypeptide of claim 1, wherein the modified polypeptide is savinase variants R241Q, R241E, R241H or R241K.

22. A method for preparing polypeptides with reduced immune response comprising the steps of:

(a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent polypeptide in question,

(b) selecting target amino acid residues on the surface of the 3-dimensional structure of the parent polypeptide to be modified,

(c) substituting one or more amino acid residues selected in step b) with other amino acid residues, and/or

(d) coupling polymeric molecules to the amino acid residues in step b)and/or step c).

23. The method of claim 22, wherein the C^alpha-atoms of the amino acid residues are located less than 15 Å from the ligand bound to said polypeptide.

24. The method of claim 22, wherein the C^beta-atoms of the amino acid residues are located closer to the ligand than the C^alpha-atom.

25. The method of claim 22, wherein the C^alpha-atoms of the amino acid residues are located less than 10 Å from the ligand and the amino acid residues have an accessibility of at least 15%.

26. The method of claim 22, wherein the identification of amino acid residues located on the surface on the polypeptide referred to in step (a) are performed by a computer program analyzing the 3-dimensional structure of the parent polypeptide in question.

27. The method of claim 22, wherein step (b) comprises selecting arginine or lysine residues on the surface of the parent polypeptide.

28. The method of claim 27, wherein one or more arginine residues identified in step b) is (are) substituted with a lysine residue(s) in step (c).

29. A composition comprising a modified polypeptide of claim 1 and further comprising ingredients used in industrial products.

30. The composition of claim 29, wherein the industrial product is a detergent, such as a laundry, dish wash or hard surface cleaning product, including bio-film products or a food or feed product or a textile product.

31. An enzyme selected from the group consisting of carbohydrases, lipases, oxidoreductases, transferases, isomerases and lyases with reduced immune response, said enzyme having one or more amino acid residues modified, wherein the C^alpha-atoms of said amino acid residues are located less than 15 Å from a ligand bound to said enzyme.

32. The enzyme of claim 31, wherein the enzyme has reduced allergenicity.

33. The enzyme of claim 31, wherein the C^beta-atom of the amino acid residues is located closer to the ligand than the C^alpha-atom.

34. The enzyme of claim 31, wherein the C^alpha-atoms of the amino acid residues are located less than 10 Å from the ligand and said amino acid residues have an accessibility of at least 15%.

35. The enzyme of claim 31, wherein the ligand is a metal or metal ion.

36. The enzyme of claim 31, wherein the enzyme is modified by substitution of amino acid residues.

37. The enzyme of claim 31, wherein the modified enzyme has been selected from a diverse library of variants.

38. The enzyme of claim 36, wherein the substituting amino acids contain amino groups in the form of lysine residues(s), carboxylic groups in the form of aspartic acid or glutamic acid residues, or SH-groups in the form of cysteine residues.

39. The enzyme of claim 36, wherein the modification(s) is (are) a conservative substitution of an amino acid residue.

40. The enzyme of claim 39, wherein the conservative substitution is a substitution of arginine to lysine, aspargine to aspartate/glutamate, glutamine to aspartate/glutamate or threonine/serine to cysteine.

41. The enzyme of claim 31, wherein the enzyme is modified by coupling one or more polymeric molecules to said enzyme, thereby providing an enzyme-polymer conjugate.

42. The enzyme of claim 41, wherein the parent enzyme moiety of the conjugate has a molecular weight from 1 to 1000 kDa, preferred 4 to 100 kDa, more preferred 12 to 60 kDa.

43. The enzyme of claim 41, wherein the polymeric molecules coupled to the enzyme have a molecular weight from 0.1 to 100, preferably 0.1 to 60 kDa, more preferably 0.3-5 kDa, most preferably 1 to 2 kDa.

44. The enzyme of claim 41, wherein the polymeric molecule is selected from the group comprising a natural or synthetic homo- and heteropolymers, selected from the group of the synthetic polymeric molecules including Branched PEGs, polyvinyl alcohol (PVA), poly-carboxyl acids, poly-(vinylpyrolidone) and poly-D,L-amino acids, or natural occurring polymeric molecules including dextrans, including carboxymethyl-dextrans, and celluloses such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, pullulans, xanthan gums, carrageenin, pectin and alginic acid.

45. The enzyme of claim 31, wherein the enzyme or parent enzyme is an amylase variant comprising one or more substitutions at positions 124, 126, 128, 159, 160, 166, 185, 186, 189, 190, 193, 194, 195, 196, 198, 201, 202, 203, 209, 210, 214, 242, 244, 247, 296, 298, 299, 302, 303, 304, 306, 307, 308, 310, 311, 314, 345, 347, 405, 406, 407, 408, 409, 433, 434, 435, 436, 437, 475, 476, 477, 478 with C, D, E, or K.

46. A composition, comprising an enzyme of claim 31 and ingredients used in industrial products.

47. The composition of claim 46, wherein the industrial product is a detergent, such as a laundry, dish wash or hard surface cleaning product, including bio-film products or a food or feed product or a textile product.

48. The composition of claim 47, further comprising ingredients used in personal care products, especially skin care products.

48. A pharmaceutical composition, comprising an enzyme of claim 31 ingredients used in pharmaceuticals.

49. A method for preparing enzymes with reduced immune response comprising the steps of:

(a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent enzyme in question,

(b) selecting target amino acid residues on the surface of said 3-dimensional structure of said parent enzyme to be modified,

(c) substituting one or more amino acid residues selected in step (b) with other amino acid residues, and/or

(d) coupling polymeric molecules to the amino acid residues in step (b)and/or step (c).

50. The method of claim 49, wherein the C^alpha-atoms of the amino acid residues are located less than 15 Å from the ligand bound to said enzyme.

51. The method of claim 49 or 50, wherein the C^beta-atoms of the amino acid residues are located closer to the ligand than the C^alpha-atom.

52. The method of any of claims 49-51, wherein the C^alpha-atoms of the amino acid residues are located less than 10 Å from the ligand and said amino acid residues have an accessibility of at least 15%, preferable at least 20%, more preferably at least 30%.

53. The method of any of claims 49-52, wherein the identification of amino acid residues located on the surface on the enzyme referred to in step (a) are performed by a computer program analyzing the 3-dimensional structure of the parent enzyme in question.

54. The method of any of claims 49-53, wherein step (b) comprises selecting arginine or lysine residues on the surface of the parent enzyme.

55. The method of claim 54, wherein one or more arginine residues identified in step (b) is (are) substituted with a lysine residue(s) in step (c).

56. Use of the enzyme of any of claims 31-45 for reducing the allergenicity of industrial products.

57. Use of the enzyme of any of claims 31-45 for reducing the immunogenicity of pharmaceuticals.