WO1996040792A1 - Modification de polypeptides - Google Patents

Modification de polypeptides Download PDF

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Publication number
WO1996040792A1
WO1996040792A1 PCT/DK1996/000250 DK9600250W WO9640792A1 WO 1996040792 A1 WO1996040792 A1 WO 1996040792A1 DK 9600250 W DK9600250 W DK 9600250W WO 9640792 A1 WO9640792 A1 WO 9640792A1
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Prior art keywords
polymer
polypeptide
poly
process according
kda
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PCT/DK1996/000250
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English (en)
Inventor
Arne Agerlin Olsen
Thomas Christian Beck
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Novo Nordisk A/S
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Priority to AU58937/96A priority Critical patent/AU5893796A/en
Publication of WO1996040792A1 publication Critical patent/WO1996040792A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/18—Introducing halogen atoms or halogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds

Definitions

  • the present invention relates to a process for producing po ⁇ lymer-polypeptide conjugates with reduced immunogenicity, a polymer-polypeptide conjugate with reduced immunogenicity, an activated polymer for conjugating polypeptides, the use of said process for reducing the immunogenicity of polypeptides, and the use of said polymer-polypeptide conjugate for a number of purposes.
  • polypeptides such as especially proteins and enzymes
  • This exposure may inflict problems for people having an enhanced inclination for eliciting an immune response toward polypeptides or to people frequently in direct contact with relatively large amounts of polypeptides.
  • the above mentioned groups of people include people regularly using pharmaceuticals comprising polypeptides. This group makes up a great many people, as people having a chronical disease, such as diabetes, need to take pharmaceutical products up to several times a day.
  • polypeptides being intentionally introduced into the circulatory system (i.e. the blood stream) of humans and animals may inflict the risk of causing an immune re ⁇ sponse as a result of antibody (or immunoglobulin) formation, such as the formation of mainly IgG, but also IgM.
  • antibody or immunoglobulin
  • the way of challenge is believed to have an important impact on the immune system's response.
  • the po ⁇ lypeptides e. g. being inhaled through the respiratory passage the normal response will be formation of the antibody IgE leading to an allergic response.
  • This type of immune response may also be caused by "industrial polypeptides", such as e. g. enzymes comprised in detergents, personal care products, including cosmetics and the like, which are not intended to enter the circulatory system of the body.
  • Industrial polypeptides such as e. g. enzymes comprised in detergents, personal care products, including cosmetics and the like, which are not intended to enter the circulatory system of the body.
  • PAO polyalkylene oxides
  • PAG polyalkylene glycol
  • Naturally occurring polymers such as the polysaccharides dextran and pullulan are known to be used for conjugating polypeptides.
  • polypeptides having increased stabil ⁇ ity, increased resistance to proteolytic inactivation, reduced immunogenicity, antigenicity and/or allergenicity, prolonged lifetime in the bloodstream, and a low toxicity.
  • PAO polymer polyalkylene oxide
  • PEG polyethylene glycol
  • mPEG methoxypolyethylene glycol
  • PAG activation methods are known.
  • One of the first com ⁇ monly used methods involved activating the hydroxyl end-group of PAG using cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578).
  • this method is not suitable for modifying polypeptide for foods and pharmaceuticals as cyanuric chloride (2,4, 6-trichloro-s-triazine) are toxic, and the activated PAG is non-specific, as it reacts with polypeptides having functional groups other that amines, such as free essential cysteine or tyrosine residues.
  • Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divenylsulfone, 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 chemis ⁇ try.
  • Poly(ethylene glycol) succinoyl-N-hydroxysuccinimide ester (SS- PEG) (Abuchowski et al., (1984), Cancer Biochem. Biophys., 7, 175-186) is an example of such an activated PAO releasing only non-toxic residues, which are readily separable from the PAO- polypeptide conjugate.
  • SS- PEG Poly(ethylene glycol) succinoyl-N-hydroxysuccinimide ester
  • Prior art patents US patent no. 4,179,337 discloses a process for conjugation of polyalkylene glycol (PEG) to polypeptides for therapeutic use, such as insulin and certain enzymes, all having a physiological activity.
  • PEG polyalkylene glycol
  • the preparations of conjugated polypeptides have re ⁇ cuted immunogenicity, antigenicity, and have further a pro- longed lifetime in the bloodstream as compared to the parent polypeptides.
  • the surplus amounts of polymers necessary to conjugate the polypeptide makes the method expensive.
  • WO 90/13540 (Enzon Inc.) concerns activated PEG derivatives, namely, poly(ethylene glycol) succinimide carbonates (SC-PEG) , the bifunctional derivative of PEG, namely, poly(ethylene glycol-bis-succinimide carbonate (BSC-PEG) and heterobifunctional derivatives of PEG, which in one end have the succinimide carbonate group and in the other end have a group readily reacting with amino groups of proteins to afford PEG attachment through stable urethane linkages.
  • SC-PEG poly(ethylene glycol) succinimide carbonates
  • BSC-PEG poly(ethylene glycol-bis-succinimide carbonate
  • heterobifunctional derivatives of PEG which in one end have the succinimide carbonate group and in the other end have a group readily reacting with amino groups of proteins to afford PEG attachment through stable urethane linkages.
  • GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.
  • US patent no. 5,133,968 (Kanebo, LTD.) describes a modified protease linked to a polysaccharide via a triazine ring leading to a suppressing effect on antigenicity and dermal hypersensitivity.
  • the employed polysaccharide has an average molecular weight not less than 10 kDa.
  • the modification rate for surface amino acid groups in the modified protease is not less that 30%.
  • EP 632 082 (Heyleclina) describes the preparation of activated (methoxy)poly(alkylene glycol) carbamate (C-(m)PEG) from (m) PEG via (m) PEG-chloroformiate.
  • the described activated (m) PEG car ⁇ bamate has a high degree of selectivity, as it reacts only with amine groups.
  • (m) PEG chloroformiate readily reacts with amines, alcohols, phenols, and carboxylic acids.
  • PAG polyalkylene glycols
  • PEG polyethylene glycols
  • PEG polysaccharides
  • the present inventors have found an advantageous process for producing polypeptides with reduced immunogenicity using less process steps than expected on the basis of prior art.
  • the invention relates to a process for pro ⁇ ducing polymer-polypeptide conjugates with reduced immunogeni- city, using a polymer as the starting material, comprising the steps of
  • the invention is directed towards an activated polymer capable of linking to attachment groups on a polypeptide by acylation.
  • the activated polymer has the general structure:
  • Y is a halogen or nitrile m is an integer from 1 to 25, and
  • Poly defines a synthetic or a naturally occurring polymer.
  • Poly is a synthetic polymer, such as a polyalkylene oxide (PAO) .
  • PAO polyalkylene oxide
  • the activated polymer has the general structure
  • RI is hydrogen, methyl, hydroxyl or methoxy
  • R2 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated,
  • R3 is an alkyl group, which may be straight, .branched, disubstituted, or unsaturated,
  • R4 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated.
  • a is an integer between 1 and 1000
  • b is an integer between 0 and 1000
  • c is an integer between 0 and 1000
  • Y is a halogen or a nitrile.
  • Poly is a naturally occurring polymer, such as the polysaccharides dextran and pullulan.
  • the invention relates to polymer-polypeptide conjugates, wherein the polymer can be a synthetic or naturally occurring polymer having the structure:
  • n is an integer between 1 and 25,
  • Poly can be a synthetic polymer or a naturally occurring polymer, z is an integer between 1 and 25, Pep can be any polypeptide, and
  • X is a coupling group between the polymer (Poly) and a polypeptide (Pep) which has been formed by reaction with a polymer halogen formiate.
  • polymer moiety (poly) of the conjugate is a synthetic polymer, such as a polyalkylene oxide (PAO) having the general formula
  • RI is hydrogen, methyl, hydroxyl or methoxy
  • R2 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated
  • R3 is an alkyl group, which may be straight, -branched, disubstituted, or unsaturated
  • R4 is an alkyl group, which may be straight, branched. disubstituted, or unsaturated, a is an integer between 1 and 1000, b is an integer between 0 and 1000, c is an integer between 0 and 1000, n is an integer between 1 and 100, and
  • X is a coupling group between the polymer (Poly) and a polypeptide (Pep) which has been formed by reaction with a polyalkylene oxide halogen formiate.
  • Poly moiety of the conju ⁇ gate is an naturally occurring polysaccharide, such as dextran or pullulan.
  • the invention relates to the use of the above process for reducing the immunogenicity of polypeptides, which include polypeptides for use in pharmaceutical and industrial products.
  • polypeptides includes proteins, peptides and/or enzymes for pharmaceutical or industrial uses.
  • Is is especially contemplated to introduce polypeptides into the circulatory system of the body of humans and/or animals, which may inflict an immunogenic response.
  • polypeptides used as ingredients in industrial products such as detergents and personal care products, including cos- metics, are contemplated.
  • industrial polypeptides come into direct contact with the circulatory system of the body of humans or animals, as such polypeptide (or products comprising such polypeptides, are not injected (or the like) into the bloodstream.
  • Such industrial polypeptides have a much greater risk of inflicting an allergic response, as a consequence of inhalation through the respiratory passage.
  • 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 0 2 , nutritious matter, hormones, and other substances of importance for the cell regulation into the tissue. Further the blood removes C0 2 from 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 defence mechanisms of the body, which include the immune system.
  • pharmaceutical polypeptides is defined as polypep ⁇ tides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body. Examples of “pharmaceutical polypeptides” will be mentioned below.
  • “industrial polypep ⁇ tides” are defined as polypeptides, including peptides, proteins and/or enzymes, which are not to be introduced into the circulatory system of the body of humans and animals.
  • polypeptides which are specifically contem ⁇ plated, are polypeptides used in products such as detergents, household article products, agrochemicals, personal care prod ⁇ ucts, such as cosmetics, toiletries, oral and dermal pharmaceuticals, composition use for processing textiles, compositions for hard surface cleaning, and compositions used for manufacturing food and feed etc.
  • polymer-polypeptide conjugates such as polyalkylene oxide-polypeptide conjugates and polysaccharide-polypeptide conjugates by acylating the polymer with at least one polypeptide.
  • HOSu N- hydroxysuccinimide
  • TAA triethylamine
  • the present invention relates to a process for producing polymer-polypeptide conjugates using a polymer (Poly) as the starting material.
  • the conjugation process com ⁇ prises the steps of
  • polymer in ⁇ cludes synthetic polymers and naturally occurring polymers.
  • suitable synthetic polymers are polymers which can be selected from the group comprising polyalkylene oxide (PAO) , including polyalkylene glycols (PAG) , such as polyethylene gly ⁇ cols (PEG) or methoxypolyethylene glycols (mPEG) , polypropylene glycols (PPG) , and poly-vinyl alcohol (PVA) , poly-carboxylates, poly- (vinylpyrolidone) and poly-D,L-amino acids.
  • PAO polyalkylene oxide
  • PAG polyalkylene glycols
  • PEG polyethylene gly ⁇ cols
  • mPEG methoxypolyethylene glycols
  • PPG polypropylene glycols
  • PVA poly-vinyl alcohol
  • the polymer is a polyalkylene oxide (PAO) , such as an polyalkylene glycol (PAG) or ethoxypolyalkylene glycol (mPAG) .
  • PAO polyalkylene oxide
  • PAG polyalkylene glycol
  • mPAG ethoxypolyalkylene glycol
  • the PAG may advantageously be an polyethylene glycol (PEG) or a methoxypolyethylene glycol (mPEG) .
  • Suitable naturally occurring polymers include poly ⁇ saccharide, such as agarose, guar gum, inulin, starch, dextran, pullulan, xanthan gum, carrageenin, pectin, alginic acid hy- drolysates of chitosan etc., and derivatives thereof including hydroxypropylcellulose, methylcellulose. ethylcellulose, car- boxymethylcellulose.
  • Especially contemplated according to the invention are dextran and pullulan.
  • Polymers having a molecular weight (M r ) between 1 and 60 kDa may be used according to the invention.
  • Polypeptides with reduced immunogenicity can be produced in a less cumbersome way and also faster by the use of a process of the invention in comparison to prior art processes. Consequently, the process costs are less in comparison to the cost of equivalent prior art processes.
  • EP 0 632 082-A1 see page 19, line 41
  • mPEG chloroformiate readily reacts with a number of chemical groups.
  • chloroformiates will mainly react with (and attach to) a ine groups on the polypeptide. Reaction with (attachment to) other groups will be much less pronounced.
  • the reaction solvent is mainly aqueous. If a less specific attachment to the polypeptide is needed, the composition of the reaction solvent is less important.
  • the process of the invention provides the possi ⁇ bility of obtaining a "designed" conjugated polymer-polypeptide product, as the conjugation product is dependant on the solvent in which the conjugation reaction is carried out.
  • the present inventors have developed the process of the invention especially for large scale processing of polypeptides. However, the advantages of the process will also be present when producing smaller amounts of polymer polypeptide conjugates.
  • Another advantage of the invention is that the prepared poly- peptide-polymer conjugate has an improved stability in compari ⁇ son to the parent polypeptide.
  • the process of the invention has all of the above mentioned advantages. Further, only a minimum of process steps are used to obtain polypeptides with reduced immunogenicity, in comparison to the corresponding prior art processes. Assessment of Immunogenicity
  • 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.
  • immunoglobulin may be defined as a substance which, when intro ⁇ quizzed into circulatory system of animals and humans, is capable of stimulating an immunologic response resulting in formation of immunoglobulin.
  • antigen refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule.
  • 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) .
  • This model seek to identify the immunogenic response in the form of the IgG response in BALB/C mice being injected subcutaneously with modified and unmodified polypeptides.
  • Example 6 it is shown that by using the process of the invention the immunogenicity of polypeptides modified according to the process of the invention is reduced.
  • 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.
  • the polypeptide to be conjugated according to the invention may be of plant, animal or microbial origin, although the polypep- tides preferably is of microbial origin, such as of bacterial or fungal origin.
  • polypeptide is a protein or peptide having a biological activity, such as an anti-microbial activity.
  • the polypeptide can be a "pharmaceutical polypeptide such as 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, thro bopoietin (TPO) and prolactin.
  • pharmaceutical polypeptide such as 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, thro bopoietin (TPO) and prolactin.
  • polypeptide is an enzyme used in pharmaceuticals or industrial products including an enzyme selected from the group of proteases, upases. transferases, carbohydrases, oxidoreductases, and phytases.
  • Polypeptides with a molecular weight in the range between about 10 kDa and 100 kDa are especially contemplated.
  • a “substantially” maintained activity is, in the context of the present invention, defined as an activity which is 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 parent polypeptide.
  • polymer molecules preferably from 1 to 10 polymer molecules, are conjugated to each polypeptide molecule. This is significantly less than corresponding prior art tech ⁇ niques. Consequently the expense to polymer is reduced. To some extent it entails that the activity of the enzyme is substan ⁇ tially retained, as it is to be anticipated that the activity to a certain extent varies inversely with the number and the size of polymer conjugated to the polypeptide.
  • the activated polymer such as an polyalkylene oxide halogen formiate generated in step a) is preferably a polyalkylene oxide chloroformiate, such as methoxypolyalkylene glycol chloroformiate, and may be generated by using phosgene.
  • Example 1 the preparation of methoxypolyethylene glycol chloroformiate 5,000 ( PEG 5,000) and 15,000 (mPEG 15,000), respectively, is described.
  • the halogen may be any other halogen, such as Br or F.
  • the attachment groups on the polypeptide can be any group selected from the group including amines, hydroxyls, alcohols, phenols, or carboxylic acids.
  • polymer halogen formiate may, if desired, attach to the side chain of e.g. serine, threonine, tyrosine, lysine, arginine, aspartate, glutamate in the polypeptide chain.
  • the invention is directed towards an activated polymer capable of linking to at least one attachment group on a polypeptide by an acylation reaction.
  • the activated polymer has the general structure:
  • Y is a halogen or nitrile m is an integer between 1 and 25, and Poly defines a synthetic polymer or a naturally occurring polymer.
  • the activated polymer is an polyalkylene oxide (PAO) or polysaccharide capable of linking to attachment groups on a polypeptide.
  • PAO polyalkylene oxide
  • the polymer moiety (Poly) of the activated polymer is a synthetic polymer, such as polyalkylene oxide (PAO) .
  • PAO polyalkylene oxide
  • RI is hydrogen, methyl, hydroxyl or methoxy
  • R2 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated.
  • R3 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated,
  • R4 is an alkyl group, which may be straight. branched. disubstituted, or unsaturated, a is an integer between 1 and 1000, b is an integer between 0 and 1000, c is an integer between 0 and 1000, and
  • Y is a halogen or nitrile
  • the halogen Y may be either of Cl, Br, or F, although Cl is preferred.
  • polymer moiety is one of the above mentioned naturally occurring polysaccharides.
  • the activated polymer of the invention may preferably have a molecular weight in the range from about 1 kDa to 60 kDa, preferably from about 2 kDa to 35 kDa, especially from about 2 kDa to 25 kDa.
  • n is an integer between 1 and 25,
  • Poly can be a synthetic polymer or a naturally occurring polymer, z is an integer between 1 and 25, polypeptide can be any polypeptide, and X is a coupling group between the polymer (Poly) and a polypeptide (Pep) which has been formed by reaction with a polymer halogen formiate.
  • the polymer moiety (Poly) of the conjugate is a synthetic polymer, such as a polyalkylene oxide (PAO) having the general formula
  • RI, R2, R3, R4, a, b, c, and n are defined as above.
  • X is a coupling group between the polymer (Poly) and a polypeptide (Pep) .
  • polymer moiety (Poly) of the conjugate is a naturally occurring polysaccharide, such as dextran or pullulan.
  • the coupling group has been formed by reacting the activated polymer (polymer halogen formiate) of the invention with an attachment group on the polypeptide (Pep) .
  • Said attachment group is preferably an amine, but can be any group selected from the group of amines, hydroxyls, alcohols, phenols, and/or carboxylic acids on the polypeptide (Pep) .
  • the attachment group may be the side chain of serine, threonine, tyrosine, lysine, arginine, aspartate, glutamate in the polypeptide chain.
  • polypeptide conjugate of the invention is produced by the process of the invention.
  • R2 is CH 2 -CH 2 , CH 2 -CH-CH3, or CH2-CH2-CH2-CH2.
  • Activated polymers of the invention have a molecular weight (M r ) between 1 and 60 kDa may be used according to the inven ⁇ tion. Polymers are preferred having a molecular weight (M r ) of between 2 kDa and 35 kDa, especially between 2 kDa and 25 kDa.
  • the polypeptide to be conjugated is preferably a protein or peptide having a biological activity or an enzyme as mentioned above.
  • a polypeptide conjugate of the invention has a total molecular weight in the range of 50 kDa to 250 kDa, preferably between 80 and 200 kDa.
  • a conjugate of the invention can be stored by freezing e.g. at about 18°C.
  • the invention also relates to pharmaceutical or industrial compositions comprising at least one polymer-polypeptide conjugate of the invention.
  • composition may further comprise other polypeptides/pro- teins/enzymes/peptides and/or ingredients normally used in e.g. pharmaceuticals, detergents, agrochemicals, personal care prod ⁇ ucts, composition use for treating textiles, compositions used of hard surface cleaning, compositions used for manufacturing food, e.g. baking, and feed etc.
  • ingredients normally used in e.g. pharmaceuticals, detergents, agrochemicals, personal care prod ⁇ ucts composition use for treating textiles, compositions used of hard surface cleaning, compositions used for manufacturing food, e.g. baking, and feed etc.
  • polypeptides examples include enzymes/polypeptides from the group including proteases, lipases, oxidoreductases, carbohydrases, transferases, such as transglutaminases, anti ⁇ microbial polypeptides, and phytases.
  • the polypeptide/peptide is 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.
  • the invention also relates to the use of a conjugate of the invention or a composition of the invention in pharmaceuticals or indistrial products defined above.
  • the invention relates to a method for reducing the immunogenicity of polypeptides by using the process of the invention.
  • the polypeptide may be any of the above mentioned.
  • ELISA reagents Horseradish Peroxidase-conjugated anti-IgG and anti IgE (Serotex Ldt, oxford, England)
  • Tresyl chloride (2,2, 2-triflouroethansulfonyl chloride) (Fluka)
  • Tween 20 Poly oxyethylene sorbitan mono laurate (Merck cat no.
  • OPD o-phenylene-diamine
  • mice about 20 grams (purchased from Bomholdtgaard, Ry, Denmark) )
  • Proteases cleave the bond between the peptide and p- nitroaniline to give a visible yellow colour absorbing at 405 nm.
  • Buffer e.g. Britton and Robinson buffer pH 8.3
  • Substrate 100 mg suc-AAPF-pNA is dissolved into 1 ml dimethyl sulfoxide (DMSO) . 100 ⁇ l of this is diluted into 10 ml with Britton and Robinson buffer.
  • DMSO dimethyl sulfoxide
  • the substrate and protease solution is mixed, and the absorbance is monitored at 405 nm as a function of time and
  • ABS 4 0 5 - un /min The temperature should be controlled (20-50°C depending on protease) . This is a measure of the protease activity in the sample.
  • Enzymatic activity was measured as release of blue dye from azurine-crosslinked HE-cellulose (Cellazyme-C®) .
  • the reaction was carried out at 40°C in 20 mM Na-phosphate pH 7 for 10 minutes. Release of dye was monitored by reading the absorbance at 595 nm in a UVmax® Elisa-reader.
  • cellulytic activity was measured as described in "EAL-SM-0373.01/01" (available from Novo Nordisk on request) .
  • Antigen is incubated with dilutions of sera in Dilution Buffer. Make those solutions just before adding them to the wells.
  • BALB/C mice (20 grams) are immunised 10 times (intervals of 14 days) by subcutaneous injection of the modified or unmodified polypeptide in question, respectively by standard proceedures known in art.
  • Electrophoretic separation of proteins was performed by stan- dard methods using 4-20% gradient SDS poly acrylamide gels (Novex) . Proteins were detected by silver staining. The mole ⁇ cular weight was measured relative to the mobility of Mark-12® wide range molecular weight standards from Novex.
  • the resulting Subtilisin Novo-mPEG-15, 000 conjugate was purified by size-exclusion chromatography using a Superdex-75 column in an HPLC-system.
  • the residual activity of the conjugate was assessed by using suc-AAPF-pNP and casein as substrates:
  • reaction was performed at ambient temperature using magnetic stirring. At time 120 minutes the reaction was terminated by addition of 0.5 ml of 2 M Glycine.
  • the resulting Subtilisin Novo-mPEG-5,000 conjugate was purified by size-exclusion chromatography using a Superdex-75 column in an HPLC-system.
  • the residual activity of the conjugate is assessed by using suc-AAPF-pNP as substrate.
  • the conjugated retained activity towards the peptide substrate.
  • the catalytic core-domain of Carezyme ⁇ was prepared according to Boisset, C. et al. (1995), FEBS Lett. 376, p. 49-52.
  • reaction was performed at ambient temperature using magnetic stirring. At time 30 minutes the reaction was terminated by addition of 1 ml of 2 M Glycine.
  • the resulting Cellulase-core-domain-mPEG-5, 000 conjugate was purified by desalting in 0.05 M NaHC0 3 and concentrated to the original volume using an Amicon Cell.
  • the obtained conjugate was PEGylated a second time using 1.0 g of methoxypolyethylene glycol 5, 000 chloroformiate at otherwise identical conditions.
  • Sera from immunized mice were tested in the ELISA assay (described above) to elucidate whether the molecules had activated the immune response system giving rise to a IgG response.

Abstract

Cette invention concerne un procédé de production de conjugués de polymère-polypeptide présentant une immunogénicité réduite; ce procédé consistant à: a) générer un formiate d'halogène polymère; et b) conjuguer au moins un formiate d'halogène polymère avec au moins un groupe de fixation situé sur le polypeptide (Pep). Cette invention concerne un conjugué de polymère-polypeptide présentant une immunogénicité réduite, un polymère activé utile pour conjuguer les polypeptides (Pep), ainsi que l'utilisation dudit procédé pour réduire l'immunogénicité des polypeptides utilisés dans des applications pharmaceutiques et/ou industrielles. L'objet de cette invention est d'utiliser ledit conjugué de polymère-polypeptide dans un grand nombre d'applications thérapeutiques et industrielles, par exemple dans des produits pharmaceutiques, des produits d'hygiène et de beauté et dans des compositions détergentes.
PCT/DK1996/000250 1995-06-07 1996-06-07 Modification de polypeptides WO1996040792A1 (fr)

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028336A1 (fr) * 1996-12-20 1998-07-02 Map Medical Technologies Oy Derives de polysaccharide-peptide
WO1998030682A1 (fr) * 1997-01-10 1998-07-16 Novo Nordisk A/S Enzyme couplee a des molecules polymeres pour les soins de la peau
WO1999067370A1 (fr) * 1998-06-23 1999-12-29 Novozymes A/S Conjugue polypeptide-polymere
WO2000004138A1 (fr) * 1998-07-17 2000-01-27 Novozymes A/S Conjugue polypeptide-polymeres a efficacite de lavage amelioree
US6030933A (en) * 1995-12-29 2000-02-29 The Procter & Gamble Company Detergent compositions comprising immobilized enzymes
WO2000022103A1 (fr) * 1998-10-13 2000-04-20 Novozymes A/S Polypeptide modifie a reponse immunitaire reduite
AU725965B2 (en) * 1997-08-14 2000-10-26 Aventisub Llc Method of enhancing bioavailability of fexofenadine and its derivatives
US6416756B1 (en) 1997-01-10 2002-07-09 Novozymes A/S Modified protease having 5 to 13 covalently coupled polymeric molecules for skin care
US6461849B1 (en) 1998-10-13 2002-10-08 Novozymes, A/S Modified polypeptide
WO2002079232A2 (fr) 2001-03-30 2002-10-10 Merck Patent Gmbh Diminution de l'antigenicite de proteines de fusion
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
US6638526B1 (en) 1998-06-23 2003-10-28 Novozymes A/S Polypeptides conjugated to copolymers of ethylene oxide and propylene oxide to reduce allergenicity
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
EP1585817A2 (fr) * 2002-02-20 2005-10-19 The General Hospital Corporation Conjugues contenant un polymere biodegradable, et leurs utilisations
US7714114B2 (en) 2005-02-16 2010-05-11 Nektar Therapeutics Conjugates of an EPO moiety and a polymer
US8420087B2 (en) 2004-01-05 2013-04-16 Antisoma Research Limited Interleukin-12 targeted to oncofoetal fibronectin
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site

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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6030933A (en) * 1995-12-29 2000-02-29 The Procter & Gamble Company Detergent compositions comprising immobilized enzymes
WO1998028336A1 (fr) * 1996-12-20 1998-07-02 Map Medical Technologies Oy Derives de polysaccharide-peptide
US6455025B1 (en) 1996-12-20 2002-09-24 Map Medical Technologies Oy Polysaccharide-peptide derivatives
WO1998030682A1 (fr) * 1997-01-10 1998-07-16 Novo Nordisk A/S Enzyme couplee a des molecules polymeres pour les soins de la peau
US6416756B1 (en) 1997-01-10 2002-07-09 Novozymes A/S Modified protease having 5 to 13 covalently coupled polymeric molecules for skin care
AU725965C (en) * 1997-08-14 2004-04-22 Aventisub Llc Method of enhancing bioavailability of fexofenadine and its derivatives
AU725965B2 (en) * 1997-08-14 2000-10-26 Aventisub Llc Method of enhancing bioavailability of fexofenadine and its derivatives
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
WO1999067370A1 (fr) * 1998-06-23 1999-12-29 Novozymes A/S Conjugue polypeptide-polymere
US6638526B1 (en) 1998-06-23 2003-10-28 Novozymes A/S Polypeptides conjugated to copolymers of ethylene oxide and propylene oxide to reduce allergenicity
WO2000004138A1 (fr) * 1998-07-17 2000-01-27 Novozymes A/S Conjugue polypeptide-polymeres a efficacite de lavage amelioree
US6461849B1 (en) 1998-10-13 2002-10-08 Novozymes, A/S Modified polypeptide
WO2000022103A1 (fr) * 1998-10-13 2000-04-20 Novozymes A/S Polypeptide modifie a reponse immunitaire reduite
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
EP2311882A1 (fr) 2001-03-30 2011-04-20 Merck Patent GmbH Réduction de l'immunogénicité des protéines de fusion
WO2002079232A2 (fr) 2001-03-30 2002-10-10 Merck Patent Gmbh Diminution de l'antigenicite de proteines de fusion
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
EP1585817A2 (fr) * 2002-02-20 2005-10-19 The General Hospital Corporation Conjugues contenant un polymere biodegradable, et leurs utilisations
EP1585817A4 (fr) * 2002-02-20 2005-12-28 Gen Hospital Corp Conjugues contenant un polymere biodegradable, et leurs utilisations
EP2138575A1 (fr) * 2002-02-20 2009-12-30 The General Hospital Corporation Conjugués comportant un polymère biodégradable et utilisations associées
US7785618B2 (en) 2002-02-20 2010-08-31 Elmaleh David R Conjugates comprising a biodegradable polymer and uses therefor
US8420087B2 (en) 2004-01-05 2013-04-16 Antisoma Research Limited Interleukin-12 targeted to oncofoetal fibronectin
US7714114B2 (en) 2005-02-16 2010-05-11 Nektar Therapeutics Conjugates of an EPO moiety and a polymer
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site

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