JP2002527059A - Modified polypeptides with reduced immune response - Google Patents

Abstract

  (57) [Summary] The present invention provides for reduced amino acids having one or more amino acid residues replaced by other amino acid residues and / or attaching one or more polymer molecules near a polypeptide metal binding site. Polypeptides having a reduced immune response including allergenicity, methods for preparing the modified polypeptides of the invention, use of said polypeptides to reduce immunogenicity and allergenicity, and said polypeptides A composition comprising the peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to polypeptides having one or more amino acid residues substituted and / or having a polymer molecule coupled on the surface of the three-dimensional structure of the polypeptide, The present invention relates to a method for preparing the modified polypeptide of the present invention, the use of said modified polypeptide to reduce immunogenicity and allergenicity, and to a composition comprising said polypeptide.

BACKGROUND OF THE INVENTION The use of polypeptides, such as enzymes, in the circulatory system to obtain certain physiological effects is well known in the medical arts. In addition, enzymes are used as functional ingredients in industrial applications such as laundry washing, fiber bleaching, personal care, contact lens washing, and food and feed preparation techniques. One of the important differences between pharmaceutical and industrial uses is that, for the latter type of use (ie, industrial use), polypeptides (often enzymes) do not enter the body's circulatory system. is there.

[0003] Certain polypeptides and enzymes have unsatisfactory stability and, under certain circulation (ie, depending on the challenge), produce an immune response, typically an IgG and / or IgE response. Can cause. It is now generally recognized that the stability of a polypeptide is improved and that the immune response is reduced when the polypeptide, eg, an enzyme, is attached to the polymer molecule. The reduced immune response appears to be the result of protection of epitopes on the surface of the polypeptide responsible for the immune response leading to antibody formation by the attached polymer molecule.

[0004] Techniques for conjugating polymer molecules to polypeptides are well known in the art. One of the first suitable commercial techniques was described in the early 1970's and is disclosed, for example, in U.S. Pat. No. 4,179,337. The patent relates to enzymes and peptide hormones conjugated to non-immunogenic polypeptides such as polyethylene glycol (PEG) or polypropylene glycol (PPG). At least 15
% Of the physiological activity of the polypeptide is maintained.

[0005] British Patent 1,183,257 (Crook et al.) Describes chemistry for conjugation of enzymes to polysaccharides through a triazine ring. Additionally, techniques for maintaining the enzymatic activity of an enzyme-polymer conjugate are also known in the art. WO 93/15189 (Veronese et al.) Refers to a method of maintaining activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a polymerized inhibitor. The conjugate is intended for medical use.

[0006] It has been found that attachment of a polymer molecule to a polypeptide often has the effect of reducing the activity of the polypeptide by preventing the interaction between the polypeptide and its substrate. European Patent No. 183503 (Beecham Group PLC
) Discloses the development of the above concept by providing a conjugate comprising a pharmaceutically useful protein linked to at least one water-soluble polymer by a reversible linking group.

[0007] EP 471,125 (Kanebo) discloses a parent protease (trade name Esperase) which is linked to a polysaccharide through a triazine ring to improve heat and storage stability.
(Trademark Bacillus Protease).
The coupling technique used is described in the above-mentioned British Patent No. 1,183,257 (Crook et al.
).

[0008] Japanese Patent No. 3083908 describes a skin cosmetic material comprising a transglutaminase from guinea pig liver modified with one or more water-soluble substances such as PEG, starch, cellulose and the like. The modification is performed by activating the polymer molecules and coupling them to enzymes. The composition is stated to be mild to the skin.

WO 98/35026 (Novo Nordisk A / S) discloses polypeptides having one or more attached and / or removed attachment groups for attaching polymer molecules on the surface of the polypeptide structure. A polymer conjugate is described. The conjugate has reduced immunogenicity and allergenicity.

SUMMARY OF THE INVENTION [0010] It is an object of the present invention to provide improved polypeptides suitable for industrial and pharmaceutical uses.
It is an object of the present invention. The term "improved polypeptide" means in the present invention polypeptides having a reduced immune response in humans and animals. As described further below, the immune response depends on the means of attack.

The inventor has recognized that a polypeptide, eg, an enzyme, may have one or more amino acid residues on the surface of the polypeptide replaced by other amino acid residues and / or may have a substantial effect on enzymatic activity. The enzyme-bound ligand, without any effect on the
For example, immunogenicity and / or allergenicity can be reduced by coupling the polymer molecule onto the surface of the enzyme near the metal ion.

When a pharmaceutical polypeptide is introduced directly into the circulatory system (ie the bloodstream), the potential risks are mainly due to the immunogenic response in the form of IgG, IgA and / or IgM antibodies. It is. In contrast, industrial polypeptides, such as enzymes used as functional components in detergents, do not enter the circulation. A potential danger for industrial polypeptides is inhalation, which causes an allergic response mainly in the form of IgE antibody formation. Thus, for industrial polypeptides, a potential risk is the respiratory allergenicity caused by inhalation, intratracheal and intranasal delivery of the polypeptide.

[0013] A major potential risk of a pharmaceutical polypeptide is the immunogenicity caused by the transdermal, intravenous or subcutaneous delivery of the polypeptide. The term "immunogenic" as used in the present invention is referred to in the clinical setting as allergic contact dermatitis and contacts the skin and cell-mediated delay to invading chemicals The resulting immune response. This cell-mediated response is also called delayed contact hypersensitivity (Gell of the immune system in tissue damage)
and Combs classification, type IV reaction).

The term “allergenic” or “respiratory allergenic” is an immediate hypersensitivity reaction, initially following, for example, inhalation of the polypeptide (according to Gell and Combs, type I antibody-mediated reactions). ). According to the present invention, it is possible to provide a polypeptide having a reduced immune response with substantially retained residual activity. Allergenic and immune responses are referred to in one term, at least in the present invention, as an "immune response".

In a first aspect, the invention relates to a polypeptide having a reduced immune response, wherein one or more of the amino acid residues has been modified, wherein the Cα-atom of said amino acid residue is Located less than 15 ° from the ligand bound to the polypeptide. The reduced immune response is preferably reduced allergenic. Modification of a polypeptide can be by substituting one or more amino acid residues in the parent polypeptide with other amino acid residues, and / or selecting variants from various libraries of variants of the parent polypeptide. And / or by coupling the polymer molecule to the surface of the parent polypeptide.

The term “parent polypeptide” refers to a polypeptide that is modified by coupling to a polymer molecule or by replacing an amino acid residue. The parent polypeptide can be a naturally occurring (or wild type) polypeptide, or a variant thereof prepared by any suitable means. For example, a parent polypeptide can be 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 variant of a naturally occurring polypeptide.

“Suitable linking group” means in the context of the present invention any amino acid residue on the surface of a polypeptide which can be coupled to the polymer molecule in question. Preferred linking groups are the amino acid and the N-terminal amino group of a lysine residue. Polymer molecules can also be attached to the carboxylic acid group (-COOH) of an amino acid residue in a polypeptide chain located on the surface. The carboxylic acid linking group can be an asparagine or glutamine carboxylic acid group, and a C-terminal COOH-group. Another linking group is the SH-group on cysteine.

“Active site” refers to any amino acid residue and / or molecule known to be essential for the performance of a polypeptide, eg, catalytic activity, eg, a catalytic triad, ie, Histidine, asparagine and serine in serine proteases or, for example, peroxidases such as Arthromyces ramosus
ces ramosus) means the heme group in peroxidase, and distal and proximal histidine. “Ligand” means, in the present invention, a metal or metal ion, or cofactor.

In the context of the present invention, “modification of an amino acid residue” means that an amino acid residue is replaced by another amino acid residue and / or a polymer molecule is attached to an amino acid residue. The polypeptides of the invention can be modified according to the invention by substitution only, by conjugation of polymer molecules only, or by a combination of substitution and conjugation.

In the context of the present invention, “located” means the shortest distance from any atom in the ligand to the appropriate C-atom in the amino acid residue. Furthermore, in the present invention, for example, “R250K” means that the amino acid arginine at position 250 of the polypeptide has been replaced by the amino acid lysine according to the one-letter code amino acid.

In a second aspect, the present invention provides a method comprising: a) identifying amino acids located on the surface of the three-dimensional structure of the parent polypeptide of interest; b) the tertiary amino acid of said parent polypeptide to be modified Selecting a target amino acid residue on the surface of the original structure; c) replacing one or more amino acid residues selected in step b) with another amino acid residue; and / or d) steps b) and And / or coupling a polymer molecule to the amino acid residue in step c). Comprising a step of preparing a polypeptide having a reduced immune response.

The present invention also relates to the use of the modified polypeptides of the invention and the methods of the invention for reducing the immunogenicity of a medicament and reducing the allergenicity of an industrial product. Finally, the present invention relates to a composition comprising the modified polypeptide of the present invention and additional components used in an industrial product or medicine.

DETAILED DESCRIPTION OF THE INVENTION: Providing improved polypeptides suitable for industrial and pharmaceutical use
It is an object of the present invention. Where the polypeptides used for pharmaceutical and industrial use can vary considerably, the principles of the present invention can be tailored to a particular type of parent polypeptide (ie, enzymes, hormonal peptides, etc.).

The inventor has determined that a polypeptide, eg, an enzyme, may replace one or more amino acid residues near a ligand, eg, a metal ion, at a metal ion binding site and / or on the surface of the parent polypeptide by one or more amino acids. It has been found that by coupling multiple polymer molecules, it can be rendered less immunogenic and / or less allergenic.
In addition, the inventors have found that a high percentage of the retained residual catalytic activity can be maintained for those modified polypeptides.

In a first aspect, the invention relates to an improved polypeptide wherein one or more amino acid groups have been modified, wherein the Cα-atom of said amino acid residue is a ligand bound to said polypeptide. Located less than 15mm from Substitution of amino acid residues and coupling of polymer molecules can be performed according to the following conventional aspects.

Reduced Immune Response vs. Residual Enzyme Activity Maintained : For enzymes, there is a conflict between reduced immune response and substantial residual enzyme activity. Without being limited to any theory, the loss of enzymatic activity of the enzyme-polymer conjugate may be due to the impeded access of the substrate to the active site, especially in the form of spatial interference of the substrate by macro and / or heavy polymer molecules. It appears to be the result of gender. It can also be caused, at least in part, by adverse minor structural changes in the three-dimensional structure of the enzyme due to the stresses produced by the coupling of the polymer molecules. In addition, polypeptides modified by substituting one or more amino acid residues have reduced enzymatic activity.

Retained residual activity : The modified polypeptides of the present invention have substantially retained catalytic activity. “Substantially” maintained catalytic activity is defined herein as 20% of the activity of a modified polypeptide prepared based on the corresponding parent polypeptide.
More than at least 20% to 30%, preferably 30% to 40%, more preferably 40% to 60%
%, Better defined as an activity that is between 60% and 80%, and even better between 80% and 100%.

For polypeptide-polymer conjugates of the invention in which the polymer molecule is not attached at or near the active site, the residual activity is up to or very close to 100%. When the binding group of the new polypeptide is removed from the active site, the activity is greater than 100% as compared to the modified (ie, attached polymer) parent polypeptide conjugate.

Binding group : Virtually all ionized groups, such as the amino group of a lysine residue, are located on the surface of a polypeptide molecule (eg, Thomas E. Creighton, (1993), “Pro
teins ", WH Freeman and Company, Now York). Thus, modified or readily accessible linking groups on the parent polypeptide (
For example, the number of amino groups) is determined by the number of lysine residues and N in the primary structure of the polypeptide.
Generally equal to the number of terminal amino groups.

The chemistry of coupling a polymer molecule to an amino group is very simple and is well established in the art. Thus, in order to obtain an improved conjugate with reduced immunogenicity and / or allergenicity and / or improved stability and / or catalytic activity maintained at a high percentage, the parent polymer in question is It is preferred to add a lysine residue (ie, a linking group) to the peptide.

[0031] The polymer molecule can also be attached to the carboxyl group (-COOH) of an amino acid residue on the surface of the polypeptide. Thus, when a carboxyl group (including the C-terminal group) is used as the linking group, the addition of asparagine and glutamine residues and / or
Or removal is also appropriate according to the invention. If other linking groups are used, such as the -SH group, they are similarly added, and / or
Or it can be removed. Substitution of amino acid residues is preferred over insertion when the impact on the three-dimensional structure of the polypeptide is less apparent.

Parent polypeptide : As used herein, the term "polypeptide" includes proteins, peptides and / or enzymes for pharmaceutical or industrial use. Typically, the polypeptide in question has a molecular weight in the range of about 1-1000 kDa, preferably 4-100 kDa, more preferably 2-60 kDa. Pharmaceutical polypeptides:

The term “pharmaceutical polypeptide” refers to a physiologically active polypeptide, such as a peptide, such as a peptide hormone, a protein, and / or when introduced into the circulatory system of the human and / or animal body. Defined as an enzyme. Pharmaceutical polypeptides are potentially immunogenic when they are introduced into the circulation. Examples of “pharmaceutical polypeptides” designed according to the invention include insulin, ACTH
, Glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigment hormone, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic opening factor, antidiuretic hormone, thyroid stimulating hormone,
Including relaxin, interferon, thrombopoietin (TPO) and prolactin.

Industrial Polypeptides: Polypeptides used for industrial applications often have enzymatic activity. Industrial polypeptides (eg, enzymes) are not introduced into the body's circulatory system (compared to pharmaceutical polypeptides).

Industrial polypeptides, such as enzymes used as components in industrial compositions and / or products, such as detergents and personal care products, such as cosmetics, comprise such enzymes (or comprise such enzymes) Product) is not infused (or similar) into the bloodstream, and therefore has little direct contact with the circulatory system of the human or animal body. (Thus, for industrial polypeptides, a potential risk is respiratory allergy (ie, an IgE response) as a result of inhalation of the polypeptide through the respiratory route.

As used herein, “industrial polypeptide” is defined as a polypeptide that is not administered to humans and / or animals, for example, peptides, proteins and / or enzymes. Examples of such polypeptides are products, such as detergents, household goods, agricultural chemicals, personal care products, such as skin care products, such as cosmetics and toiletries, oral and dermatological drugs, and for processing fibers. Polypeptides, especially enzymes, used in compositions, compositions for cleaning hard surfaces, and compositions used for the production of food and feedstuffs, and the like.

Enzyme activity: Pharmaceutical or industrial polypeptides exhibiting enzymatic activity are often of the following enzyme group:
It would belong to one: oxidoreductase (EC1, “Enzyme Nomenclature,
(1992), Academic Press Inc.), such as laccase and superoxide dismutase (SOD); transferase (EC2), such as transglutaminase (TGase); hydrolase (EC3), such as proteases, especially subtilisins and lipolytic enzymes; EC5), for example, protein disulfide isomerase (PDI).

The hydrolase proteolytic enzymes: Proteolytic enzymes are contemplated are, aspartic proteases, for example pepsin, cysteine proteases such as papain, serine proteases, such as subtilisin, or metalloprotease selected from the group of for example Neutrase (TM) Proteases.

Specific examples of parent proteases are PD498 (WO93 / 24623 and SEQ ID NO: 2), Savin
ase ™ (von der Osten et al., (1993), Journal of Biotechnology, 28, p.
.55+, SEQ ID NO: 3), proteinase K (Gunkel et al., (1989), Eur. J. Bioche
m, 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 ™ (Novo Nordisk A / S
, JA16 (WO92 / 17576), Alcalase ™ (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); Hiron
o S., Akagawa H., Mitsui Y., Iitake Y.) (available from Novo Nordisk A / S)
Is included.

Carbohydrases: Parent carbohydrases are hydrocarbon chains, especially of 5- and 6-membered ring structures (eg starch)
All enzymes that can hydrolyze (i.e., Recommendations (1992) of the In
It is defined as an enzyme classified under the enzyme classification number EC3.2 (glycosidase) according to the ternational Union of Biochemistry and Molecular Biology (IUBMB).

Examples include carbohydrases selected from those classified under the Enzyme Classification (EC) number: α-amylase (3.2.1.1), β-amylase (3.2). 1.2), glucan 1,4-α-glucosidase (3.2.1.
3), cellulase (3.2.1.4), endo-1,3 (4) -β-glucanase (3.2.1.6), endo-1,4-β-xylanase (3.2.1.4). 1.8
), Dextranase (3.2.1.11), chitinase (3.2.1.14)
, Polygalacturonase (3.2.1.15), lysozyme (3.2.1.17)
), Β-glucosidase (3.2.1.21), α-galactosidase (3.2
. 1.22), β-galactosidase (3.2.1.23),

Amylo-1,6-glucosidase (3.2.1.33), xylan 1,4-β
-Xylosidase (3.2.1.37), glucanendo-1,3-β-D-glucosidase (3.2.1.39), α-dextrin endo-1,6-glucosidase (3.2.1). .41), sucrose α-glucosidase (3.2.1.
48), glucan endo-1,3-α-glucosidase (3.2.1.59),
Glucan 1,4-β-glucosidase (3.2.1.74), glucan end-
1,6-β-glucosidase (3.2.1.75), arabinan end-1,5
-Α-arabinosidase (3.2.1.19), lactase (3.2.1.10)
8), chitonanases (3.2.1.132).

Examples of suitable carbohydrases also include: α-1,3-glucanase from Trichoderma hazianum; α-1,6-glucanase from Paecilomyces strain; Β-glucanase from Bacillus subtilis; Humicola
Β-glucanase derived from Humicola insolens; β-glucanase derived from Aspergillus niger; β-glucanase derived from Trichoderma strain; derived from Oerskovia xanthinoeolytica Β-glucanase;

Exo-1,4-α-D-glucosidase (glucoamylase) derived from Aspergillus niger; α-amylase derived from Bacillus subtilis; α-amylase derived from Bacillus amyloliquefaciens
Amylase; Bacillus stearothermophilu
α-amylase derived from s); Aspergillus oriyz
α-amylase derived from ae); α-amylase derived from a non-pathogenic microorganism; α-galactosidase derived from Aspergillus niger; pentosase, xylanase, cellobinase, cellulase, hemi-cellulase derived from Humicola insolens;

Cellulase derived from Trichoderma reesei; cellulase derived from non-pathogenic fungus; pectinase, cellulase, arabinase, hemi-cellulose derived from Aspergillus niger; Penicillium relasinum (
Dextranase derived from Penicillium lilacinum; Endoglucanase derived from non-pathogenic mold; Bacillus acidopullytic
us); Kluyveromyces pullulanase; Kluyveromyces
β-galactosidase from C. fragillis; xylanase from Trichoderma reesei.

Specific examples of readily available commercially available carbohydrases include: Alpha-GalO, Bio-feedO alpha, Bio-FeedO Beta, Bio-FeedO plus, Bio-FeedO.
plus, Novozyme ™ 188, Carezyme ™, Celluclast ™, Cellus
oft ™, Ceremyl ™, CitrozymO, DenimaxO, DezymeO, DextrozymeO,
MaltogenaseO, PentopanO, PectinexO, Promozyme (TM), PulpzymeO, Novam
ylO, Termamyl ™, AMG (Amyloglucosidase Novo), Maltogenase ™,
Aquazym ™, Natalase ™ (all enzymes are available from Novo Nordisk A / S). Other carbohydrases are available from other companies.

It should also be understood that carbohydrase variants are designed as parent enzymes. The activity of carbohydrase is measured in “Methods of Enzymatic Analysis”, third edi
tion, 1984, Verlag Chemie, Weinheim, vol.

[0048] oxidoreductase laccase: laccase to be planning is, Polyporus, Pinishitasu (Polyporus ponisitus)
Laccase (WO96 / 00290), Myceliophthora laccase (WO95 / 33836), Schytalidium laccase (WO95 / 338337) and Pyricularia oryzae ligase (available from Sigma).

Peroxidases: The proposed peroxidases are B. pumilus peroxidase (WO91 / 05858), Myxococcaceae peroxidase (WO9).
No. 5/11964), Coprinus sinereus (WO95 / 10602) and Arthromyces ramosus peroxidase (kunis)
(1994), J. Mol. Biol. 235, p. 331-344).

[0050] Transferase Transglutaminase: Suitable transferases No. WO96 / 06931 (Novo Nordisk A / S) and WO96 / 2
Includes any transglutaminase disclosed in No. 2366 (Nove Nordisk A / S).

Isomerase Protein Disulfide Isomerase: Suitable protein disulfide isomerases include, but are not limited to, WO
No. 95/01425 (Novo Nordisk A / S). The proposed isomerase is xylose / glucose isomerase (5.3
. 1.5), for example, Sweetzyme ™.

Lyases Suitable lyases are polysaccharide lyases, namely pectate lyase (4.2.2).
. 2) and pectin lyases (4.2.2.10), such as those lyases from Bacillus licheniformis disclosed in WO 99/27083.

Polymer molecule The polymer molecule attached to the polypeptide can be any suitable polymer molecule,
For example, natural and synthetic homopolymers, such as polyols (i.e., poly-OH),
Polyamines (i.e., poly -NH ₂₎ and polycarboxylic acid (i.e., poly -COO
H), and also heteropolymers, ie polymers comprising one or more different coupling groups, such as hydroxyl and amine groups.

Examples of suitable polymer molecules are polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), such as polyethylene glycol (PEG), methoxy polyethylene glycol (mPEG) and polypropylene glycol, PEG-glycidyl ether (Epox- PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEG, poly-vinyl alcohol (PVA), poly-carboxylate, poly-
(Vinylpyrrolidone), poly-D, L-amino acid, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride,

Dextran such as carboxymethyl-dextran, heparin, homologous albumin, cellulose such as methylcellulose, carboxymethylcellulose,
Ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starch and propyl-starch, glycogen, agarose and their derivatives, guar gum, pullulan, inulin, xantham, carragenyl, pectin Polymer molecules selected from the group comprising alginic acid hydrolysates and biological polymers.

Preferred polymer molecules are non-toxic polymer molecules that further require relatively simple chemistry for their covalent attachment to a linking group on the surface of the enzyme, such as (m) polyethylene glycol ((m) PEG ). Generally, polyalkylene oxide (PAO), such as polyethylene oxide, such as PEG
And especially mPEG, because their polymer molecules have few reactive groups that can be crosslinked, compared to polysaccharides such as dextran, pullulan and the like.
Preferred polymer molecules.

Even if all of the above polymer molecules can be used according to the present invention, methoxy polyethylene glycol (mPEG) can be conveniently used. This arises from the fact that methoxyethylene glycol has only one reactive end that can be conjugated to the enzyme. Thus, the danger of crosslinking is less obvious. In addition, it makes the product more homogeneous and makes the reaction of the enzyme with the polymer molecule easier to control. An example of a branched PEG conjugate is a branched PEG2-NHS-ester of lysine (Shearwa
ter).

Activation and Coupling of the Polymer to the Polypeptide : If the polymer molecules conjugated by the polypeptide in question are not active, they should be activated by using appropriate techniques. Attachment of the polymer molecule to the polypeptide through a linker is contemplated according to the invention. Suitable linkers are well known to those skilled in the art.

Methods and chemistry for activation of polymer molecules and conjugation of polypeptides have been extensively described in the literature. Commonly used methods for activation of insoluble polymers include the functionalization of cyano bromide, periodate, glutaraldehyde, biepoxide, epichlorohydrin, divinyl sulfone, carbodiimide, sulfonyl halide, trichlorotriazine, etc. Activation (RF Taylor, (1991
), “Protein Immobilisation. Fundamental and Applications”, Mercel Dekk
er, NY ,; SS Wong, (1992), “Chemistry of Protein Conjugation and Cro
sslinking ”, CRC Press, Boca Raton; GT Hermanson et al., (1993),“ Immo
bilized Affinity Ligand Techniques ", Academic Press, NY).

Some of the above methods relate to the activation of insoluble polymers, but are also applicable to the activation of soluble polymers such as periodate, trichlorotriazine, sulfonyl halide, divinyl sulfone, carbodiimide, and the like. The amino, hydroxyl, triol, carboxyl, aldehyde, or sulfhydryl functional groups on the polymer and the selected linking groups on the protein are usually from i) activation of the polymer, ii) conjugation, and iii) blocking of residual active groups. Should be considered in the choice of activation and conjugation chemistry.

In the following, a number of suitable polymer activation methods will be briefly described.
However, it should be understood that other methods may also be used. Coupling of the polymer molecule to the free acid groups of the polypeptide is accomplished by diimide and, for example, amino-PEG or hydrazino-PEG (Pollak et al., (1996), J. Amr. Chem. S.
oc., 98, 289-291) or diazoacetate / amide (Wong et al., (1992), “Che
mistry of Protein Conjugation and Crosslinking ", CRC Press).

The coupling of a polymer molecule to a hydroxy group is generally very difficult as it must be done in water. Usually, hydrolysis is more prominent than reaction with hydroxyl groups. Coupling of the polymer molecule to free sulfhydryl groups can be reacted with certain groups, such as maleimide or orthopyridyl disulfide. Also, vinyl sulfones (US Pat. No. 5,415,135, (1995), Snow et al.) Are preferred for sulfhydryl groups, but are less selective than others mentioned. Accessible arginine residues in the polypeptide chain can be targeted by a group comprising two vicinal carbonyl groups.

Techniques involving the coupling of electrophilically activated PEG to the amino group of lysine are also useful. Many of the usual leaving groups for markol give rise to amine linkages. For example, alkyl sulfonates such as tresylate (Nilsson
, (1984), Methods in enzymology vol. 104, Jacoby, WB, Ed., Academ
ic Press: Orlando, P.56-66; Nilsson et al., (1987), Method in Enzymology.
vol. 135: Mosbach, K., Ed., Academic Press: Orlando, p. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic.
Press: Orlando, 1987, p. 79-84; Crossland et al., (1971), J. Amr. Chem. So
c. 1971, 93, p. 4217-4219), mesylate (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, p. 341-352. ), Arylsulfonates such as tosylate and para-nitrobenzenesulfonate can be used.

Organic sulfonyl chlorides, such as tresyl chloride, are stable between polymers and polypeptides when hydroxy groups in many polymers, such as PEG, can be reacted with nucleophilic groups, such as amino groups in polypeptides. Effectively converts to good leaving groups (sulfonates) which allow the formation of chains. In addition to high joining yield,
The reaction conditions are generally mild (neutral to slightly alkaline pH to avoid denaturation or little or no disruption of activity), and the non-destructive requirements for the polypeptide are Fulfill.

Tosylate is more reactive than mesylate, but also more unstable, degrading PEG, dioxane and sulfonic acid (Zalipsky, (1995), Bioconj.
ugate Chem., 6, 150-165). Epoxides have also been used to create amine linkages, but are less reactive than the groups mentioned above.

Conversion of PEG to chloroformate by phosgene results in a carbamate linkage to lysine. This theme covers N-hydroxysuccinimide (U.S. Patent No. 5,122,614 (1992); Zalipsky et al., (1992), Biotechnol. Appl. Bioche.
m., 15, p.100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-6.
9), imidazole (Allen et al., (1991), Carbohydr. Res., 213, p.309-319)
, Para-nitrophenol, DMAP (European Patent No. 632082A1, (1993), Lo
oze, Y.), etc., can play a role in many variants that replace chlorine. Derivatives are usually prepared by reacting the desired leaving group with chloroformate. All those groups give rise to cardmate linkages to the peptide.

In addition, isocyanates and isothiocyanates are used to produce urea and thiourea, respectively. Amides can be obtained from PEG acids using the same leaving groups and cyclic imidotrons as described above (US Pat. No. 5,349,001, (1994), Greenwald, etc.). The reactivity of these compounds is very high, but speeds up the hydrolysis. PEG succinate prepared from reaction with succinic anhydride may also be used. The ester groups included thereby make the conjugate more susceptible to hydrolysis (US Pat. No. 5,122,614, (1992), Zalipsky). This group can be activated by N-hydroxysuccinimide.

Further, certain linkers can be introduced. Cyanuric chloride is most commonly used (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Patent No. 4,179,337, (1979), Davis et al.,; Shafer Etc., (1986), J. Pol
ym. Sci. Polym. Chem. Ed., 24, 375-378). Coupling of PEG to the aromatic amine, followed by diazotization, produces a highly reactive diazonium salt that can be reacted in situ with the peptide. Amide linkages can also be obtained by reacting azlactone derivatives of PEG (US Pat.
No., 321,095, (1994), Greenwald, RB), thus introducing an additional amide linkage.

Since some peptides do not contain many lysines, it is advantageous to attach more than one PEG to the same lysine. This is for example 1,3-diamino-2
-Can be carried out by using propanol. PEG can also be linked to the amino group of the enzyme by a carbamate linkage (WO95 / 11
No. 924, Greenwald, etc.). Lysine residues can also be used as a backbone. The coupling technique used in the examples is the N-type described in WO 90/13590 (Enzon).
This is a succinimidyl carbonate bonding technique.

Method for Preparing the Improved Polypeptides a) Identifying the amino acids located on the surface of the three-dimensional structure of the parent polypeptide in question; b) Identifying the three-dimensional structure of the parent polypeptide to be modified Selecting a target amino acid residue on the surface; c) replacing one or more amino acid residues selected in step b) with another amino acid residue; and / or d) step b) and / or step It is also an object of the present invention to provide a method for preparing an improved polypeptide comprising the step of coupling a polymer molecule to the amino acid residue in c).

Step a) Identification of the amino acid residues located on the surface of the parent polypeptide: three-dimensional structure: In order to carry out the method of the invention, the three-dimensional structure of the parent polypeptide in question is required. This structure can be, for example, an X-ray structure, an NMR structure or a model-constructed structure. Brookhaven Databank is the source of X-ray and NMR-structures. Model-constructed structures can be constructed by one of skill in the art where one or more three-dimensional structures of homologous polypeptides that share at least 30% sequence identity with the polypeptide in question exist. There are several software packages that can be used to construct the model structure. One example is the Homology 95.0 package from MSI Inc.

The typical actions required for the construction of a model structure are as follows: alignment of homologous sequences where a three-dimensional structure exists, definition of a structurally conserved region (SCR), SCR
Assignment of coordinates to, search for structural fragments / loops in the structure database to replace variable regions, assign coordinates to those regions, and improve structure by minimizing energy. Regions containing large inserts (three or more residues) for a known three-dimensional structure are known to be very difficult for the model, and structure prediction should be carefully considered It is. Having obtained a model of the structure of the polypeptide in question based on its three-dimensional structure, or homology to a known structure, this structure serves as an essential prerequisite for the realization of the method described below.

Step b) Selection of target amino acid residues: The target amino acid residues to be modified are selected according to the invention from those amino acid residues whose Cα-atom is located less than 15 ° from the ligand. You. In a preferred embodiment, the potential Cβ-atom should be closer to the ligand than the Cα-atom. In a more preferred embodiment, the Cα-atom of the amino acid residue is located less than 10 ° from the ligand, and said amino acid residue is at least 15%, preferably at least 20% and more preferably at least 30% closer. Has accessibility.

Step c) Substitutions: Conservative substitutions: Conservative substitutions ensure that the impact of substitution on the polypeptide structure is limited, so that conservative substitutions in the polypeptide are to be made if the polypeptide is to be conjugated. It is preferred to make substitutions. If an additional amino group is provided, this is done by substitution of arginine for lysine, both residues being positively charged, but only lysine has a suitable free amino group as a linking group.

When providing an additional carboxylic acid group, for example, a conservative substitution can be asparagine for aspartic acid or glutamine for glutamic acid. The residues are similar in size and shape to one another, except that the carboxylic acid group is on an acidic residue. When providing an SH-group, conservative substitutions may be made by substitution of threonine or serine for cysteine. The amino acid to be replaced mainly depends on the coupling chemistry applied.

If no coupling is performed after the substitution, there are generally no restrictions on the choice of amino acids for the substitution. However, preferred amino acids for substitution are the polar residues K, R, D, E, H, Q, N, S, T, C. In addition, if no coupling takes place, the change is in the form of an addition or deletion of at least one amino acid located within 15 ° of the ligand to which the Cα atom is bound, preferably one amino acid is deleted. can do. In addition, the parent protein may be altered by substituting some amino acids and deleting / adding others.

Only those substitutions that provide a polypeptide with a reduced immune response when evaluated in an animal model are within the concept of the invention. Mutations made in step c) can be made by standard techniques well known in the art,
For example, it can be performed for site-directed mutagenesis. (For example, Sambrook et al. (1989)
, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, NY). General descriptions of nucleotide substitutions can be found, for example, in Ford et al., 1991, Protein Expre.
ssion and Purification 2, p.95-107.

In a preferred embodiment of the invention, more than one amino acid residue is substituted, added or deleted, and those amino acids are probably located close to different supplied ligands . In this case, it is difficult to assess how well the functionality of the protein is maintained while reducing antigenicity, immunogenicity, and / or allergenicity. This establishes a library of diversified variants, each having one or more modified amino acids introduced, and those those exhibiting good retention of function and, at the same time, good reduction of antigenicity This can be achieved by selecting mutants.

In the case of a protease, this is due to its enzymatic activity (as described in the experimental section below) and its secretion for antigen binding (eg, by competitive ELISA using methods known in the art). (See, eg, J. Clausen, Immunochemical Techniques For Th
e Identification and Estimation of Macromolecules, Elsevier, Amsterdam,
1988 p.187-188). In particular, competitive ELISAs are performed with wild-type proteases displayed on ELISA plates and can be incubated with specific polyclonal anti-protease antisera from rabbits in the presence of protease variants.

The scope of those aspects of the invention is illustrative only and is not limited to proteases. Diversified libraries can be established by a wide variety of techniques known to those skilled in the art (Reetz MT; Jaeger KE, in Biocatalysis-from Discovery to A
pplication edited by Fessner WD, vol. 200, pp. 31-57 (1999); Stemmer, Na
ture, 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. S
ci., USA, vol. 95, pp 5511-5515, 1998).

In a more preferred embodiment, the substitutions are found by a method comprising the following steps: 1) enumerate the range of substitutions, additions and / or deletions, and 2) those randomized in the amino acid sequence. A library is designed that introduces a subset of the changes into the target gene, for example, by random mutagenesis, 3) the library is expressed, and preferred variants are selected. In a most preferred embodiment,
This method is based on additional screening steps and / or family shuffling from initial screening (jR Ness, et al., Nature Biotechnol
ogy, vol. 17, p.893-896, 1999), and / or in combination with other methods of reducing allergenicity by genetic means (eg, as disclosed in WO 92/10755).

Site-Directed Mutagenesis Generation: Prior to mutagenesis, the gene encoding the polypeptide of interest should be cloned by a suitable vector. Methods for inducing mutations at specific sites are described below.

Once the gene encoding the polypeptide has been cloned, the desired sites for mutation have been identified, and the residues to replace the original residue determined, the mutations are synthesized. It can be introduced using an oligonucleotide. These oligonucleotides comprise a nucleotide sequence flanked by the desired mutation site; variant nucleotides are introduced during oligonucleotide synthesis. In a preferred method, site-directed mutagenesis is performed by Kammann et al. (1989) Nucleic Acids R
esearch 17 (13), 5404, and Sarker G. and Sommer, SS (1990) Biotechnique
s 8, 404-407 by the SOE-PCR mutagenesis technique.

Step d) Coupling of the polymer molecule to an optionally modified parent enzyme: The polynucleotide-polymer conjugate of the present invention may be any coupling known in the art, including the techniques described above. It can be prepared by a method.

Preparation of Enzyme Variants: The enzyme variants to be conjugated can be constructed by any suitable method. Many methods are well established in the art. For example, the enzyme variant of the present invention is, for example, WO89 / 06279 (Novo Nordisk A / S), EP 130,756 (Genentech), EP
479,870 (Novo Nordisk A / S), EP 214,435 (Henkel), WO87 / 04461 (Amge
n), WO87 / 05050 (Genex), EP Patent Application No. 87303761 (Genentech), EP 26
No. 0,105 (Genencor), WO88 / 08028 (Genex), WO88 / 08033 (Amgen), WO88 / 081
No. 64 (Genex), Thomas et al. (1985) Nature, 318 375-376, Thomas et al. (198
7) J. Mol. Biol., 193, 803-813; Russel and Fersht (1987) Nature 328 496-
It can be produced using the same materials and methods described in 500.

Coupling of polymer molecules to the polypeptide of interest: see previous paragraph. Immunogenicity and allergenicity: "Immunogenic" is a broader term than "antigenic" and "allergenic" and describes the response of the immune system to the presence of exogenous substances. Said exogenous substances are called immunogens, antigens and allergens, depending on the type of immune response elicited. An "immunogen" may be defined as a substance that, when introduced into the circulation of animals and humans, can stimulate an immune response that results in the formation of immunoglobulins.

The term “antigen” refers to a substance capable of producing antibodies alone when recognized as a non-self molecule. In addition, an "allergen" may be defined as an antigen capable of eliciting an allergic sensitization or response by an IgE antibody (a molecule that has considerable effects in humans and in animals).

Assessment of immunogenicity : Assessment of immunogenicity is determined by injecting an animal subcutaneously to put the immunogen into the circulation and comparing the response to that of the corresponding parent polypeptide. Done. The "circulatory system" of the human and animal body means, in the context of the present invention, a system consisting mainly of the heart and blood vessels. The heart supplies the necessary energy to maintain blood circulation in the vasculature. The circulatory system functions as an organism's transport system when the blood transports enzymes, nutrients, hormones, and other important substances for cell regulation into tissues. In addition, blood carries carbon dioxide from tissues to the lungs and residuals to the kidneys, for example. In addition, blood includes temperature regulation, and the immune system,
It is important for the body's defense mechanism.

[0089] Many in vivo animal models exist for assessing the immunogenic potential of a polypeptide. Some of these models provide a suitable basis for risk assessment in humans. Suitable models include mouse models. This model allows the identification of immunogenic responses in the form of IgG responses in Balb / C mice injected subcutaneously with modified and unmodified polypeptides. Other animal models can also be used for assessing immunogenic potential.

The polypeptide having “reduced immunogenicity” of the present invention may comprise an antibody,
For example, the amount of an immunoglobulin in a human and a molecule that has a significant effect in a particular animal that can elicit an immune response when introduced into the circulatory system is significantly lower than its corresponding parent polypeptide. Indicates that For Balb / C mice, an IgG response confers a good indication of the immunogenic potential of the polypeptide.

Evaluation of allergenicity : Allergenicity is evaluated by an absorption test comparing the effect of the parent enzyme administered intratracheally (into the trachea) with the effect of the corresponding modified enzyme of the invention. Can be done. Many in vivo animal models exist for evaluating the allergenicity of enzymes.
Some of these models provide a suitable basis for risk assessment in humans. Suitable models include the guinea pig model and the mouse model.
These models allow the identification of respiratory allergens as a function of the evoked response elicited in previously sensitized animals. According to those models, the tonic allergen is introduced into the trachea of the animal.

A suitable strain of guinea pig, the Dunkin Hartley strain, does not produce IgE antibodies in the context of allergic responses, as in humans. However, they are another type of antibody that is responsible for their allergen response to inhaled polypeptides, such as enzymes, namely IgG1A and IgG1B (eg, Prento, ATLA, 19,
p.8-14, 1991). Thus, when using the Dunkin Hartley animal model, the relative amounts of IgG1A and IgG1B are a measure of the allergenic level.

The Balb / C mouse strain is suitable for intratracheal, transdermal or subcutaneous exposure. Balb / C
Mice produce IgE as an allergic response. For more information on the assessment of respiratory allergens in guinea pigs and mice, see
Kimber et al., (1996), Fundamental and Applied Toxicology, 33, p. 1-10. Other animals, such as rats, rabbits, etc., can also be used for comparative studies.

Compositions: The present invention relates to compositions comprising the modified polypeptides of the present invention. The composition can be a pharmaceutical or an industrial composition. The composition may further comprise other polypeptides, proteins or enzymes, and / or, for example, detergents, such as soap bars, household items, agricultural chemicals, personal care products, such as skin care compositions, contact lenses, oral and parenteral products. It comprises ingredients commonly used in cleaning compositions for dermatological drugs, compositions for treating fibers, compositions used for the production of food / feed, and the like.

Use of a Polypeptide: The present invention also relates to the use of the method of the present invention to reduce the immune response of a polypeptide. Industrial products such as detergents, such as laundry, dishwashing and hard surface detergents, foodstuffs are used to reduce the allergenicity of feed products, personal care products and textile products.
It is also an object of the present invention to use a polypeptide-polymer conjugate or polymer modified according to the invention.

Materials and Methods: Materials: Enzymes: PD498: A protease of the subtilisin type shown in WO93 / 24623. The sequence of PD498 is shown in SEQ ID NOs: 1 and 2. Savinase ™: This sequence is shown in SEQ ID NO: 3 (available from Novo Nordisk A / S).

Subtilisin BPN ⁻ : This sequence can be found in the SWISS-PROT database.
This sequence also includes GALLAGHER T., OLIVER J., BOTT R., BETZEL C., GILLILAND G
.L .; “Subtilisin BPN 'at 1.6-A resolution: Analysis for Discrete Disor
der and Comparison of Crystal Forms ”; Acta Crystallogr. D 52: 1125-1135
(1996). Enzymes are available from Novo Nordisk A / S.

Amylase AA560: Alkaline α-amylase can be derived from a strain of Bacillus sp. DSM 12649. This strain is Deutshe Sammlung von Microorganismen und Zellk
ulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig DE, based on the international recognition of the deposit of microorganisms for the purposes of patent proceedings, under the conditions of Budapest, by the inventor on January 25, 1999. Has been deposited. The sequence is shown in SEQ ID NO: 4.

Strain: B. subtilis 309 and 147 are variants of Bacillus lentus, deposited in the NCIB under accession numbers NCIB 10309 and 10147, and are incorporated herein by reference. It is described in Japanese Patent No. 3,723,250. E. coli MC1000 (MJ Casadaban and SN Cohen (1980): J. Mol. Biol. 138:
179-207) was converted to r-, m + by conventional methods and is also described in U.S. Patent No. 039,298.

Vector: pPD498: E containing the wild-type gene encoding PD498 protease (SEQ ID NO: 2)
Coli-B. Subtilis shuttle vector (described in US Pat. No. 5,621,089 under section 6.2.1.6). The same vector is used for mutagenesis in E. coli and expression in B. subtilis. Materials, Chemicals and Solutions: Anti-rat-Ig labeled with horseradish peroxidase (Dako
, DK, P162, # 031; dilution 1: 1000). Mouse anti-rat IgE (Serotec MCA193; dilution 1: 200). Rat anti-mouse IgE (Serotec MCA419; dilution 1: 100). Mouse anti-rat IgG1 monoclonal antibody labeled with biotin (Zyme
d03-9140; dilution 1: 1000). A rat anti-mouse IgG1 monoclonal antibody labeled with biotin (Sero
tec MCA 336 B; dilution 1: 1000).

Streptavidin-Horse Radish Peroxidase (Kirkegard & Perry
14-30-00, dilution 1: 1000). CovaLink NH ₂ plate (Nunc. Cat. No. 459439); cyanuric chloride (Aldri
ch); acetone (Merck); rat anti-mouse IgG1, biotin (Serotec, catalog number MCA336B); streptavidin, peroxidase (KPL); ortho-phenylene-diamine (OPD) (Kem-en-Tec, catalog number 4260); H ₂ O ₂ , 30% (M
erck); Tween 20 (Merck) ; skimmed milk powder _{(Difco); H 2 SO 4} (Merck).

[0102] Buffers and solutions: carbonate buffer (0.1M, pH10 (1L)) : Na 2 CO 3 10.60g PBS (pH7.2 (1L)): NaCl 8.00g KCl 0.20g K 2 HPO 4 1.04g KH ₂ PO ₄ 0.32g

Wash Buffer PBS, 0.05% (v / v) Tween 20; Inhibition Buffer PBS, 2% (wt / v) skim milk powder; Dilution Buffer PBS, 0.05% (v / v) Tween 20, 0.5 % (Wt / v) skim milk powder; citrate buffer (0.1 M, pH 5.0-5.2 (1 L)): sodium citrate 20.60 g citric acid 6.30 g

Sodium borate, borax (Sigma); 3,3-dimethylglutaric acid (Sigma); CaCl ₂ (Sigma); Trecis chloride (2,2,2-trifluoroethanesulfonyl chloride) (Fluka); 1- Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (F
luka);

N-hydroxysuccinimide (Fluka art. 56480); phosgene (Fluka art. 79280); lactose (Merck 7656); PMSF (phenylmethylsulfonyl fluoride) from Sigma; succinyl-alanine-alanine-proline-phenylalanine- Para-nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g / mol.

Activation of Covalink plates: a fresh stock solution of 10 mg of cyanuric chloride per ml of acetone. -Immediately before use, while stirring, dilute the cyanuric chloride stock solution in PBS, 1 mg / m
Bring to a final concentration of l. · CovaLink NH ₂ plates adding the diluted solution 100ml to each well and incubated at room temperature for 5 minutes. -Wash 3 times with PBS. Dry the freshly prepared activated plate at 50 ° C. for 30 minutes. Immediately seal the individual plates with sealing tape. -Store preactivated plates for 3 weeks at room temperature if kept in plastic bags.

Test animals: Female Balb / C mice (about 20 g) were purchased from Bomholdtgaard, Ry, Denmark. Female Brown-Norway rats weigh on average 180 g.

Apparatus: XCELII (Novex); ELISA reader (Uvmax, Molecular Devices); HPLC (Waters); PFLC (Pharmacia); Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW; SLT: SLT LabInstrument Size exclusion chromatography (Sepherogel TSK-G2000 SW); Size exclusion chromatography (Superdex 200, Pharmacia, SW); Amicon Cell.

Enzymes for DNA manipulation: Unless otherwise stated, all enzymes for DNA manipulation, such as endonucleases, rivases, etc., are obtained from New England Biolabs. Inc. Medium: BPX: tissue (1L per) Potato starch 100g milled barley 50g Soybean flour _{20g Na 2 HPO 4 · 12H 2} O 9g Pluronic 0.1g starch in sodium caseinate 10g medium is liquefied by α- amylase and the medium But 120
Sterile by heating at 45 ° C for 45 minutes. After sterilization, the pH of the medium is adjusted to 9 by adding sodium bicarbonate to a concentration of 0.1M.

Methods: General molecular biology methods: Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory ma
nual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM, etc. (eds.) “Current protocols in Molecular Biology”. John Wiley and So
ns, 1995; Harwood, CR, and Cutting, s.M. (eds.) “Molecular Biologic
al Methods for Bacillus ". John Wiley and Sons, 1990. Enzymes for DNA manipulation were used according to the supplier's regulations.

Fermentation of PD498 mutants : Fermentation of PD498 mutants in B. subtilis was performed on a rotary shake table (300 rpm) in a 500 ml baffled Erlenmeyer flask containing 100 ml of BPX medium.
Perform for 5 days at ° C. For example, 20 Erlenmeyer flasks are fermented simultaneously to produce 2 L of broth.

Purification of PD498 mutants : Approximately 1.6 L of PD498 mutant fermentation broth was added to a 1 L beaker at 5000 rpm for 35 minutes.
Centrifuge for minutes. The supernatant was adjusted to pH 10 using 10% acetic acid and
Filter on itz Supra S100 filter plate. The filtrate is concentrated to about 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate was centrifuged and filtered on a Bacitracin affinity column at pH 7 at room temperature before absorption. PD498 mutants were prepared using a 25% 2-propanol and 1 M sodium chloride solution in a buffer solution adjusted to pH 7 and containing 0.01 M dimethyl-glutaric acid, 0.1 M boric acid and 0.002 M calcium chloride. Elute from the bacitracin column at room temperature.

Bacitracin The fractions with protease activity from the purification step are combined and equilibrated with a buffer adjusted to pH 6.0, containing 0.01 M dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride. 750 ml Sephadex G25 column (5c
m diameter).

The proteolytically active fractions of the Sephadex G25 column are combined and the pH 6.
Apply to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethyl glutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to 0. Protease was added from 0 to 0 in 1 L of the same buffer.
Elute with a linear gradient of 0.5M sodium chloride. The protease containing fractions from the CM Sephasose column are combined and filtered through a 2μ filter.

Determination of molecular weight: Electrophoretic separation of proteins was carried out by standard methods on SDS polyacrylamide gels (Novex) with a 4-20% gradient. Protein was detected by silver staining. The molecular weight was measured in relation to the mobility of the Mark-12 ™ broad molecular weight standard from Novex.

Protease activity: Analysis by Suc-Ala-Ala-Pro-Phe-pNa: Protease breaks down the bond between the peptide and p-nitroaniline, resulting in 405 nm
To give a visible yellow color that is absorbed by. Buffer: eg Britton and Robinson buffer (pH 8.3). Substrate: 100 mg of suc-AAPF-pNa is dissolved in 1 ml of dimethylsulfoxide (DMSO) and 100 ml of this is diluted in 10 ml of Britton and Robinson buffer. The substrate and the protease solution are mixed and the absorbance is measured at 405 n as a function of time.
Monitor at m and ABS ₄₀₅ nm / min. The temperature should be adjusted (20-50 ° C., depending on the protease). This is a measure of the protease activity in the sample.

Proteolytic activity: In the present invention, the proteolytic activity is determined by the Kilo NOVO protease unit (KN
PU). The activity is determined relative to the enzyme standard (SAVINASE), and the determination is made under standard conditions, ie 50 ° C., pH 8.3, reaction time of 9 minutes, measurement time of 3 minutes, dimethyl by protease Based on digestion of casein (DMC) solution. Folder AF220 / 1 is available on request from Novo Nordisk A / S, Denmark.

GU has a proteolytic enzyme activity that produces, under standard conditions, with N-acetylcasein as a substrate during a 15-minute incubation at 40 ° C., an amount of NH ₂ -groups equal to 1 mmol of glycine. Defined. Enzyme activity is also measured by the Journal of American Oil Chemists Society, Rothgeb, TM
, Goodlander, BD, Garrison, PH, and Smith, LA, (1988) using a PNA assay according to the reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol. Can also be measured.

ELISA IgE test system (for Brown Norway rats): The relative concentration of a particular antibody is determined using a three-layer sandwich ELISA. The immunized molecules were used as 10 mg and 50 ml of coated antigen per well in neutral phosphate buffer and incubated overnight at 4 ° C. All remaining binding spots on the well surface are blocked in 200 ml of 2% skim milk per well in phosphate buffer for at least 30 minutes at room temperature. All sera tested by this antigen are added to the plate at 50 ml / well using an 8-channel pipette in a dilution series from a 10-fold dilution, followed by a 3-fold dilution series.

Dilutions are made in phosphate buffer with 0.5% skim milk and 0.05% Tween 20 and incubated for 2 hours at RT on a stirring platform. The “trace” molecule is 50 ml of biotinylated mouse anti-rat IgE per well, diluted 2000-fold in phosphate buffer with 0.5% delipidation and 0.05% Tween 20 and RT for 2 hours on a stirring platform Incubate. The control (blank)
Identical order but without rat serum.

50 ml of Streptavidin horseradish peroxidase, diluted 2000-fold, was incubated for 1 hour on a stirring platform. Coloring substrate at 50 ml per well was determined using OPD (6 ml) per 10 ml of pH 5.2 citrate buffer.
mg) is and H ₂ O ₂ (30% solution 4 ml). The reaction is stopped with 2N H ₂ SO ₄ in 100ml per well. All readings were taken at 486 nm and 620 nm (as controls)
Done on T. Data is calculated and presented in Lotus.

An ELISA method for determining the relative concentration of IgE antibodies in Balb / C mice: The relative concentration of specific IgE serum antibodies is determined using a three-layer sandwich ELISA. 1) 10 mg of rat anti-mouse IgE or mouse anti-rat IgE / ml buffer 1
Cover LISA-plate. 50 ml / well. Incubate overnight at 4 ° C. 2) Empty plate and block with blocking buffer for at least 30 minutes at room temperature. 200 ml / well. Shake gently. Wash plate 3 times with wash buffer.

3) Incubate with mouse / rat serum, starting undiluted and continuing with 2-fold dilution. Some wells are kept free of buffer 4 only (blank). 50 ml / well. Incubate for 30 minutes at room temperature. Shake gently. The plate is washed three times with wash buffer. 4) Dilute enzyme to appropriate protein concentration in dilution buffer. 50 ml / well. Incubate for 30 minutes at room temperature. Shake gently. Wash plate 3 times with wash buffer. 5) Dilute specific polyclonal anti-enzyme antiserum (pIg) to detect bound antibody in dilution buffer. Incubate for 30 minutes at room temperature. Shake gently. Wash plate three times with wash buffer.

6) Horseradish peroxidase-conjugated anti-pIg- in dilution buffer
Dilute the antibody. 50 ml / well. Incubate for 30 minutes at room temperature. Shake gently. Wash plate three times with wash buffer. 7) Mix 0.6 mg ODP / ml + 0.4 μl H ₂ O ₂ / ml in substrate buffer. Just before use, bring into solution. Incubate for 10 minutes. 50 μl / well. 8) Add stop solution to stop the reaction. 50 μl / well. 9) Read plate at 492 nm and at 620 nm as control. Data is calculated and presented in Lotus.

[0125] Example Example 1. Distance and orientation criteria are applied to identify the subtilisin BPN ' residues to be modified. As disclosed above, residues with their Cα-atoms closer than 15 ° to the ligand are targets for modification. Preferably, having those Cβ-atoms closer to the bound ligand than the Cα-atoms (and thereby allowing the orientation of potential side chains in the direction of the ligand) is Target for

Suitable distances can be easily measured, for example, using a molecular graphics program like Insight II from Molecular Simulations Inc. In particular, when applying the DSSP program to the appropriate protein part of the structure, the ACC
Surface exposed residues, defined as having> 0, are targets for modification. The DSSP program is based on W. Kabsch and C. Sander, BIOPOLYMERS 22 (198
3) It is disclosed in P.2577-2637. Thomas E. Creighton, PROTEINS; Structure and Molecular Principles, WH
Freeman and Company, NY, ISBN: 0-7167-1566-X (1984) discloses a table listing the accessible surface area of individual amino acid residues. In the table below,
15% and 20% accessibility has been determined.

[0127]

[Table 1]

The accessible surface area (ACC) found for individual amino acids of the protein
If is divided by the accessible surface area for that individual amino acid (found in the Creighton table), accessibility in% is obtained. To find the residues to be modified, the above method was applied to the X-ray structure of subtilisin BPN 'in a complex with inhibitor CI-2 (Brookhaven Protein
Data Bank entry 2 SNI).

Only the subtilisin BPN ′ and the two metal ions in the above structure were used for analysis. Both ions are calcium ions. They are found at site 1 and site 2. The results of the analysis are found in the table below. The columns indicate the distance in C from the metal ion to Cα and Cβ, and the accessibility as determined by DSSP for the individual residues to be modified.

[0130]

[Table 2]

[0131]

[Table 3]

[0132]

[Table 4]

[0133] The following formulas, BPN ^- shows a preferred functional substitution at sites 1 and 2. For Gly80, the substitutions G to S / T, G to N / Q and G to K / D are glycine at position 80, preferably serine / threonine or asparagine / glutamine or lysine /
It means that it can be replaced by aspartic acid.

[0134]

[Table 5]

[0135]

[Table 6]

Example 2 Determined by X-ray crystallography in PD498 Brookhaven Protein Data Bank (PDB) format
Three-dimensional structure of PD498 as described : The sequence used to elucidate the three-dimensional structure that forms the basis for the present invention is Bacillus sp. PD498, NCIMB No., as disclosed in SEQ ID NO: 2. Consists of 280 amino acids from .40484.

The structure of PD498 is described in “X-Ray Structure Determination”, Stout, GK and Jen.
sen, LH, John Wiley & Sons, Inc. NY, 1989, and “Protein Crystallograp
hy ”by Blundell, TL and Johnson, LN, Academic Press, London, 1990, elucidated according to the principles of X-ray crystallography methods. Using the isomorphous replacement method, PS498 was determined with a 2.2Å determination. Appendix 1 shows the structural coordinates of the crystal structure.
In the standard PDB format (Brookhaven Protein Database). It should be understood that Appendix 1 forms part of the present application.

In Appendix 1, the amino acid residues of the enzyme are represented by the three letter amino acid code (uppercase).
Is identified by PD498 has three types of bound metal ions. Site 1 is subtilisin BPN
Equivalent to site 1 in 'and contains calcium ions. Site 2 has no equivalent in subtilisin BPN 'and contains calcium ions. Site 3 is located in the same region as the second site in subtilisin BPN 'and contains a sodium ion and a monopropylene glycol ligand. Application of the above method disclosed in Example 1 results in:

[0139]

[Table 7]

[0140]

[Table 8]

[0141]

[Table 9]

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

[Table 10]

[0143]

[Table 11]

[0144]

[Table 12]

Example 3 Savinase For this example, the X-ray structure entry 1 SVN in the Brookhaven Protein Data Bank was used. Site 1 contains calcium ions and is located 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 table, sequential numbering applies and is not the numbering system used for structure files.

[0146]

[Table 13]

[0147]

[Table 14]

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

[Table 15]

[0149]

[Table 16]

Example 4 Amylase (AA560) For this example, the structure of AA560 was found by homology modeling using the BAN / Termamyl α-amylase structure disclosed in WO 96/23874, incorporated herein by reference. This structure contains two metal ions. Both parts 1 and 2
Contains calcium ions. This example is used to identify residues at the ligand binding site, how the three-dimensional structure determined by the model constructed using coordinates from the homologous structure can be modified to reduce the immune response. Indicate what you get. Application of the above disclosed method results in:

[0151]

[Table 17]

[0152]

[Table 18]

[0153]

[Table 19]

[0154]

[Table 20]

The table below shows preferred functional substitutions at sites 1 and 2 of amylase AA560. With respect to ASN126, the substitution "N to D / E" means that the asparagine in the substitution 126 can preferably be replaced by aspartic or glutamic acid, lysine or arginine, or alanine or cysteine.

[0156]

[Table 21] Example 5 Conjugation of Savinase Variant R241K with Activated Bis-PEG-1000 Sodium (p
H9.5). The reaction was brought to pH by adding 0.5 M NaOH,
Performed at ambient temperature using magnetic stirring while maintaining 9.0-9.5. The reaction time is
2 hours.

The reaction was stopped by adding 1 M HCl to a final pH of 6.0. Excess reagent is removed by ultrafiltration using a Filtron-Ultrasette, and the final product is treated with 50 nM sodium borate, 150 mM NaCl, 1 mM CaCl ₂ , 50% monopropylene glycol solution (pH 6.0). ) And stored at −20 ° C. When compared to the parent enzyme, the residual activity is determined by the peptide substrate (succinyl-Ala-Ala
-Pro-Phe-p-nitro-anilide).

Example 6 Conjugation of Savinase Variant R24K with Activated Bis-PEG-2000 353 mg of Savinase variant was combined with 1621 mg of N-succinimidyl carbonate-activated Bis-PEG2000 in 50 mM reaction volume in approximately 35 ml Sodium (p
H9.5). The reaction was brought to pH by adding 0.5 M NaOH,
Performed at ambient temperature using magnetic stirring while maintaining 9.0-9.5. The reaction time is
2 hours. The reaction was stopped by adding 1 M HCl to a final pH of 6.0. Excess reagent is removed by ultrafiltration using a Filtron-Ultrasette and the final product is treated with 50 nM sodium borate, 150 mM NaCl, 1 mM CaCl ₂ , 50 mM
The solution was stored at −20 ° C. in a solution of 0.1% monopropylene glycol (pH 6.0). When compared to the parent enzyme, the residual activity is determined by the peptide substrate (succinyl-Ala-Ala
-Pro-Phe-p-nitro-anilide).

Example 7 Methods for determining R241KbPEG1000 and R241KbPEG2000 IgE levels in rats : Sample management: Dilute individual samples to 0.075 mg protein / ml and
Aliquoted into 1.5 ml. The fractions were sent to storage for storage at -20 C until use. In addition, 100 μl of each fraction was stored in a laboratory refrigerator at −20 ° C. for immunochemical analysis at the beginning, middle and end of the study. Fresh fractions were collected for individual immunizations and individual analyses.

Immunization: 20 intratracheal immunizations were performed with 100 μl of 0.9% (w / v) NaCl (control group)
, Or weekly with 100 μl of the diluted protein solution. Group 5 receives the unmodified R241k variant of Savinase and Group 6 receives R241K-bis-S-P
EG1000 and Group 7 received R241K-bis-S-PEG2000. Each group contained 10 rats. Blood samples (2 ml) were taken from the eyes one week after any second immunization. Serum was obtained by blood coagulation and centrifugation.

ELISA: Specific IgE levels were determined using an ELISA specific for rat IgE. Serum was titrated at 1/2 dilution starting from undiluted. Optical density, 492/6
Measured at 20 nm. The results are shown in FIG. As can be found there, the IgE levels of the conjugated savinase mutant R241K are reduced compared to the savinase mutant R241K.

Example 8 IgE levels in mice of savinase mutants R241Q, R241E, R241H and R241K
Bell Determination Nine week old female Balb / c mice were challenged with wild-type savinase and mutants with a single mutation at position R241 (R241Q, R241E, R241H, R241K) for 20 consecutive weeks. Subcutaneous immunization was performed. Every other week, IgG1 and IgE serum levels were determined by ELISA.

Sample management: Dilute individual samples to 0.010 mg protein / ml and
Aliquoted into 1.5 ml. The fractions were sent to storage for storage at -20 C until use. In addition, 100 μl of each fraction was stored in a laboratory refrigerator at −20 ° C. for immunochemical analysis at the beginning, middle and end of the study. Fresh fractions were collected for individual immunizations and individual analyses.

Immunization: 20 subcutaneous immunizations were performed with 100 μl of 0.9% (w / v) NaCl (control group),
Alternatively, it was performed weekly with 100 μl of the protein dilution. Group 1 received wild-type sabinase, group 2 received (R241Q), group 3 received (R241H), group 4 received (R241E), and group 5 received (R241K). Each group contained 10 mice. A blood sample (100 μl) can be
One week after the second immunization, they were collected from the eyes. Serum was obtained by blood coagulation and centrifugation.

ELISA: Specific IgG1 levels were determined using an ELISA specific for mouse IgG1. Serum was titrated at 1/2 dilution starting at 1: 160. Specific IgE levels were determined using an ELISA specific for mouse IgE. Serum was titrated at 1/2 dilution starting from undiluted. Optical density was measured at 492/620 nm. Statistical analysis: Differences between data sets were determined by a nonparametric method, ie, Kruskal-Wallis
Analyzed by using Test and Dunn's Multiple Comparison Test. The results are shown in FIG. As can be found there, the IgE levels of the savinase variants are significantly reduced.

[0166]

[Table 22]

[0167]

[Table 23]

[0168]

[Table 24]

[0169]

[Table 25]

[0170]

[Table 26]

[0171]

[Table 27]

[0172]

[Table 28]

[0173]

[Table 29]

[0174]

[Table 30]

[0175]

[Table 31]

[0176]

[Table 32]

[0177]

[Table 33]

[0178]

[Table 34]

[0179]

[Table 35]

[0180]

[Table 36]

[0181]

[Table 37]

[0182]

[Table 38]

[0183]

[Table 39]

[0184]

[Table 40]

[0185]

[Table 41]

[0186]

[Table 42]

[0187]

[Table 43]

[0188]

[Table 44]

[0189]

[Table 45]

[0190]

[Table 46]

[0191]

[Table 47]

[0192]

[Table 48]

[0193]

[Table 49]

[0194]

[Table 50]

[0195]

[Table 51]

[0196]

[Table 52]

[0197]

[Table 53]

[0198]

[Table 54]

[0199]

[Table 55]

[0200]

[Table 56]

[0201]

[Table 57]

[0202]

[Table 58]

[0203]

[Table 59]

[0204]

[Table 60]

[0205]

[Table 61]

[0206]

[Table 62]

[0207]

[Table 63]

[0208]

[Table 64]

[0209]

[Table 65]

[0210]

[Table 66]

[0211]

[Table 67]

[0212]

[Table 68]

[0213]

[Table 69]

[0214]

[Table 70]

[0215]

[Table 71]

[0216]

[Table 72]

[0219]

[Table 73]

[0218]

[Table 74]

[0219]

[Table 75]

[0220]

[Table 76]

[0221]

[Table 77]

[0222]

[Table 78]

[0223]

[Table 79]

[0224]

[Table 80]

[0225]

[Table 81]

[0226]

[Table 82]

[0227]

[Table 83]

[0228]

[Table 84]

[0229]

[Table 85]

[0230]

[Table 86]

[0231]

[Table 87]

[0232]

[Table 88]

[0233]

[Table 89]

[0234]

[Table 90]

[0235]

[Table 91]

[0236]

[Table 92]

[0237]

[Table 93]

[0238]

[Table 94]

[0239]

[Table 95]

[0240]

[Table 96]

[0241]

[Table 97]

[0242]

[Table 98]

[0243]

[Table 99]

[0244]

[Table 100]

[0245]

[Table 101]

[0246]

[Table 102]

[0247]

[Table 103]

[0248]

[Table 104]

[0249]

[Table 105]

[0250]

[Table 106]

[0251]

[Table 107]

[0252]

[Table 108]

[0253]

[Table 109]

[0254]

[Table 110]

[0255]

[Table 111]

[0256]

[Table 112]

[0257]

[Table 113]

[0258]

[Table 114]

[0259]

[Table 115]

[0260]

[Table 116]

[0261]

[Table 117]

[0262]

[Table 118]

[0263]

[Table 119]

[0264]

[Table 120]

[0265]

[Table 121]

[0266]

[Table 122]

[0267]

[Table 123]

[0268]

[Table 124]

[0269]

[Table 125]

[0270]

[Table 126]

[0271]

[Table 127]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows integrated IgE antibody levels in rats.

FIG. 2 shows integrated specific IgE levels in mice.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/06 A61P 37/08 37/08 A61K 37/48 (81) Designated countries EP (AT, BE, CH) , CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN) , GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, (KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, C , DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Andersen, Kim Billbowl, Denmark, Daco-2100 Copenhagen A, 4 Tahoe, Tesingegarde 31 ( 72) Inventor Ernst, Steffen, Denmark, DECO-2880 Bagsbaerto, Novo Ale, Novo Nordisk Actiselskab (72) Inventor Loggen, Elwind, Denmark, DEー −2880 Bagsbaerto, Novo Are, Novo Nordisk Actiesel Scab F-term (Reference) 4B018 MD20 ME07 ME14 4B035 LC09 LG15 LG51 LP59 4B050 CC02 CC07 GG03 LL01 LL02 LL04 LL10 4C076 CC41 EE01 EE06 EE08 EE31 EE37 AA02 AA07 DC01 DC02 DC23 DC24 DC25 DC26 DC27 DC28 MA02 NA06 ZB08 ZB13 ZC80

Claims (34)

[Claims] 1. A polypeptide having a reduced immune response, wherein one or more amino acid residues are modified, wherein the Cα-atom of said amino acid residue is 15 ° from the ligand attached to said polypeptide. A polypeptide characterized by being located at a shorter position. 2. The polypeptide according to claim 1, wherein said polypeptide has reduced allergenicity. 3. The polypeptide according to claim 1, wherein the Cβ-atom of the amino acid residue is located closer to the ligand than the Cα-atom. 4. The Cα-atom of the amino acid residue is located less than 10 ° from the ligand and the amino acid residue has at least 15% accessibility (acces).
The polypeptide according to any one of claims 1 to 3, which has sibility). 5. The polypeptide according to claim 1, wherein the ligand is a metal or a metal ion. 6. The polypeptide according to claim 1, wherein the polypeptide is modified by amino acid residue substitution. 7. The polypeptide according to any one of claims 1 to 6, wherein said modified polypeptide is selected from various libraries of variants. 8. The amino acid to be substituted comprises an amino group in the form of a lysine residue, a carboxyl group in the form of an aspartic acid or glutamic acid residue, or an SH-group in the form of a cysteine residue. 7. The polypeptide according to 6. 9. The modification is prepared by a conservative substitution of an amino acid residue, such as an arginine: lysine substitution, or an asparagine: aspartic acid / glutamic acid or glutamine: aspartic acid / glutamic acid substitution, or a threonine / serine: cysteine substitution. The polypeptide according to claim 6. 10. The polypeptide of claim 1, wherein the polypeptide is modified by attaching one or more polymer molecules to the polypeptide, thereby providing a polypeptide-polymer conjugate. The polypeptide according to Item. 11. The polypeptide according to claim 10, wherein the parent polypeptide component of the conjugate has a molecular weight of 1 to 1000 kDa, preferably 4 to 100 kDa, more preferably 12 to 60 kDa. 12. The method according to claim 12, wherein the polymer molecule attached to the polypeptide is 0.1 to 100.
kDa, preferably 0.1-60 kDa, more preferably 0.3-5 kDa, most preferably 1-2.
The polypeptide of claim 10 having a molecular weight of kDa. 13. The polypeptide or the parent polypeptide may be an oxidoreductase, such as laccase and superoxide dismutase (SOD); a hydrolase, such as carbohydrase, amylase, protease, especially subtilisin; a transferase, such as transferglutaminase (TGase); 13. A lyase, such as protein disulfide isomerase (PDI); an enzyme selected from the group of pectate lyases.
The polypeptide according to Item. 14. The polypeptide or the parent polypeptide is PD498, Savinas.
e (TM), BPN ^-, amylase, proteinase K, proteinase R, subtilisin DY, Lion Y, Rennilase (R), according to claim 1 which is JA16, Alcalase (TM)
3. The polypeptide according to 3. 15. The polypeptide of the conjugate or the parent polypeptide comprises one of the following:
Or multiple substitutions, i.e. positions 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 PD498 variants in which the amino acid residue is replaced by K, D, E or C, preferably R250k, R250D, R250E, R250C
The polypeptide according to claim 14, which is 16. The polypeptide or the parent polypeptide may comprise one or more of the following substitutions: 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, wherein the amino acid residue at K, D, E or C is replaced by a BPN ^- mutant, preferably R247K, R
The polypeptide according to claim 14, which is 247D, R247E, or R247C. 17. The method according to claim 17, wherein the polypeptide or parent polypeptide has one or more of the following substitutions: positions 75, 2, 42, 208, 200, 14, 22, 17, 189, 241, 125, 125,
Savinase ™ variants in which the amino acid residues at 141, 245, 249, 237, 254, 157 have been replaced by K, D, E or C, preferably R241K, R241D, R241E, R241C
The polypeptide according to claim 14, which is 18. The method according to claim 18, wherein the polypeptide or parent polypeptide has one or more of the following substitutions: positions 124, 126, 128, 159, 160, 166, 185, 186, 189, 190, 19
3, 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, 4
06, 407, 408, 409, 433, 434, 435, 436, 437, 475, 476, 477, 478 is an amylase variant in which the amino acid residue at K, D, E or C is substituted. 14
The polypeptide of any one of the preceding claims. 19. The method of claim 19, wherein the polymer molecule is a synthetic polymer molecule, for example, a branched polymer molecule.
PEG, polyvinyl alcohol (PVA), polycarboxylic acids, poly- (vinylpyrrolidone) and poly-D, L-amino acids, or naturally occurring polymer molecules such as dextran, such as carboxymethyl dextran, and cellulose, such as methylcellulose, carboxy Methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and chitosan hydrolyzate,
Starches such as hydroxyethyl-starch, hydroxypropyl-starch, glycogen, agarose, guar gum, inulin, pullulan, xantham, carrageenan,
13. A polypeptide according to claim 10 or 12, selected from the group comprising natural or synthetic homo- and heteropolymers selected from the group of pectin and alginic acid. 20. The modified polypeptide is a savinase mutant R241Kb.
The polypeptide according to claim 19, which is PEG1000 or R241KbPEG2000. 21. The modified polypeptide is a savinase variant R241Q,
The polypeptide according to any one of claims 1 to 7, which is R241E, R241H or R241K. 22. A method for preparing a polypeptide having a reduced immune response, comprising: a) identifying amino acids located on the surface of the three-dimensional structure of the parent polypeptide of interest; Selecting a target amino acid residue on the surface of the three-dimensional structure of the parent polypeptide to be c) replacing one or more amino acid residues selected in step b) with another amino acid residue And / or d) coupling a polymer molecule to the amino acid residue in step b) and / or step c). 23. The method of claim 22, wherein the Cα-atom of the amino acid residue is located less than 15 ° from the ligand attached to the polypeptide. 24. The method of claim 22, wherein the Cβ-atom of the amino acid residue is located closer to the ligand than the Cα-atom. 25. The Cα-atom of the amino acid residue is located less than 10 ° from the ligand, and the amino acid residue is at least 15%, preferably at least 20%, more preferably at least 30% closer. Claims 22-
25. The method of any one of 24. 26. The method according to claim 22, wherein the identification of the amino acid residues located on the surface of the polypeptide referred to in the step a) is carried out by a computer program for analyzing the three-dimensional structure of the parent peptide of interest. A method according to any one of the preceding claims. 27. The method of any one of claims 22 to 26, 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) are replaced by lysine residues in step c). 29. The method for reducing the allergenicity of an industrial product according to claim 1.
Use of the modified polypeptide according to any one of the above. 30. Use of a modified polypeptide according to any one of claims 1 to 21 for reducing the immunogenicity of a medicament. 31. A composition comprising the modified polypeptide according to any one of claims 1 to 21 and further components used in industrial products. 32. The composition according to claim 31, wherein the industrial product is a detergent, such as a laundry, dishwashing or hard surface cleaning product, such as a bio-film product or a food or feed product, or a textile product. 33. The composition according to claim 32, further comprising a modified polypeptide according to any one of claims 1 to 21, and further comprising a personal care product, in particular a skin care product. 34. A composition comprising the modified polypeptide of any one of claims 1-21, and further comprising components used in medicine.