WO1998015293A2 - Apport cible de polymeres solubles a des proteines de recombinaison - Google Patents

Apport cible de polymeres solubles a des proteines de recombinaison Download PDF

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Publication number
WO1998015293A2
WO1998015293A2 PCT/US1997/018104 US9718104W WO9815293A2 WO 1998015293 A2 WO1998015293 A2 WO 1998015293A2 US 9718104 W US9718104 W US 9718104W WO 9815293 A2 WO9815293 A2 WO 9815293A2
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WO
WIPO (PCT)
Prior art keywords
protein
soluble polymer
proteins
cheland
soluble polymers
Prior art date
Application number
PCT/US1997/018104
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English (en)
Other versions
WO1998015293A3 (fr
Inventor
Brian Seed
Wen Shao
John Seed
Original Assignee
Edge Biosystems, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Edge Biosystems, Inc. filed Critical Edge Biosystems, Inc.
Priority to EP97945549A priority Critical patent/EP1011730A2/fr
Priority to AU46722/97A priority patent/AU4672297A/en
Publication of WO1998015293A2 publication Critical patent/WO1998015293A2/fr
Publication of WO1998015293A3 publication Critical patent/WO1998015293A3/fr

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Classifications

    • 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
    • A61K47/61—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 the organic macromolecular compound being a polysaccharide or a derivative 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

Definitions

  • This invention is related to modification of recombinant proteins to increase their favorable characteristics for use as therapeutic agents.
  • PEG Polyethylene glycol
  • PEG modification (often referred to as PEGylation) reduces immunogenicity has not been advanced, and it may be that PEGylation also has an effect on the fate of proteins internalized by surface antibody on B cells, thereby compromising processing of the antigen for MHC class II presentation and preventing subsequent recognition by T cells.
  • Qualitatively different behaviors have been shown to attend very high degrees of PEG modification, and highly modified proteins appear to be able to induce tolerance (Wie et al. , 1981; Holford et al. , 1982; Wilkinson et al., 1987; Wilkinson et al., 1987; Maiti et al., 1988; Chen et al., 1992; Bitoh et al., 1995).
  • Targeting of the mPEG can be performed to a limited extent by increasing the density of reactive groups in the protein of interest, for example by providing a short oligo-lysyl tag element engineered into the protein, or by the addition of cysteine residues which can react with relatively cysteine-specific reactive groups on derivatized mPEG, such as maleimide or haloacetyl groups (Goodson and Katre, 1990; Benhar et al., 1994).
  • cysteine residues which can react with relatively cysteine-specific reactive groups on derivatized mPEG, such as maleimide or haloacetyl groups (Goodson and Katre, 1990; Benhar et al., 1994).
  • cysteines can affect protein global structure through the formation of unwanted or unintended disulfide linkages.
  • Natural sites for N- or O-linked glycan addition also afford the potential for targeting the PEG modification to nonpeptide constituents of the protein.
  • not all secreted proteins bear these substituents and the reaction conditions for scission of carbohydrates to generate reactive aldehydes are relatively oxidizing.
  • soluble polymers have been identified which also confer immunologic privilege and enhanced serum half life on proteins. These include, dextran, polyvinyl alcohol, polyvinylpyrrolidone, Ficoll ® and albumin. Protein modification with these soluble polymers frequendy requires the same or similar approaches as were described for PEGylation. Thus, the problems of broad and heterogeneous coupling, protein oxidation and unintended changes to protein global structure apply here as well.
  • a method of modifying a recombinant protein with a soluble polymer comprises the step of: mixing in an aqueous solution to form a complex comprising the protein and a soluble polymer:
  • a molecular complex in another embodiment of the invention, comprises a recombinant protein and a soluble polymer, wherein the recombinant protein comprises an oligohistidine tag and the soluble polymer is conjugated to a metal chelate, wherein the molecular complex is formed by interaction of the metal chelate with the oligohistidine tag.
  • recombinant proteins can be modified with soluble polymers to alter their physical and immunological properties using oligohistidine tags and metal chelates.
  • the modification employs gentle conditions which do not harm the protein's biochemical properties.
  • the method is specifically targeted to one portion of the protein, so that unwanted modifications do not occur, and so that the product is relatively homogeneous.
  • the method provides a high yield of the reaction products.
  • the method of the present invention exploits precise targeting to predetermined residues through very high affinity noncovalent bonds.
  • the targeting allows the soluble polymers to be added to the protein under very mild conditions and the reaction allows convenient purification of the modified protein.
  • a derivatized methoxypolyethyleneglycol (mPEG) which bears a metal ion in a tetradentate cheland is complexed with a recombinant protein engineered to comprise an oligohistidine tag.
  • mPEG methoxypolyethyleneglycol
  • a recombinant protein engineered to comprise an oligohistidine tag The very high affinity of cheland-His tag complexes (estimated K ⁇ about 10 ⁇ 13 M) allows the complex to persist over the biological lifetime of the protein.
  • Oligohistidine tags are known in the art and can be introduced into a protein by introducing an oligonucleotide which encodes oligohistidine into a protein-encoding gene. This will preferably be done at either the amino terminal or carboxy terminal or both so as to minimize disruption of the protein's structure.
  • the tag will be between 6 and 20 amino acids in length. More preferably the tag will be between 6 and 12 amino acids in length.
  • Any metal ion can be used to form the chelate. These include but are not limited to transistion metals such as nickel, copper, zinc, and iron, lanthanides such as lanthanum, terbium and ytterbium, and actinides such as uranium.
  • the coordination number of the metal ion is greater than the number of coordination sites of the cheland. The extra coordination sites are used for binding to the histidines in the oligohistidine tag.
  • Any soluble polymer can be used to modify the physical, immunological, or other biological properties of the recombinant protein. Particularly preferred polymers are methoxypolyethyleneglycol, and dextran. Others as are known in the art and which impart desirable properties to the modified protein can be used.
  • Any metal cheland as is known in the art can be used in the practice of the present invention. They may be tetradentate, octadentate, hexadentate, etc.
  • One particularly preferred cheland is N-(5 -amino- 1-carboxypentyl)- iminodiacetic acid (NT A). Standard methods for conjugating the metal cheland to the soluble polymer can be used. See Example 2, which does not limit the scope of the invention but is merely exemplary.
  • Metal-cheland-directed soluble polymer conjugation represents an improved method for polymer modification of proteins and more complex biological structures, such as viral vectors for gene delivery, and intact cells.
  • the ability to use very mild reaction conditions and to achieve precise molecular targeting represents an opportunity which previous methods for PEGylation could not attain.
  • the immunogenicity of adenoviral vectors is a particularly important problem which has stymied their full development as gene delivery agents. Many workers in the field have focused on the tendency of these vectors to provoke cellular immunity, i.e. , the tendency of the host to mount cytotoxic T cell responses against cells treated with the vectors.
  • N-(5-amino-l-carboxypentyl)iminodiacetic acid was synthesized following a modification of a previously reported procedure (Hochuli et al. , 1987).
  • a prechilled 13.5 ml aliquot of 2M NaOH containing N-e- carbobenzyloxylysine (8.6 mmole, 2.4 g) was added dropwise to 10.8 ml of 2M NaOH containing bromoacetic acid (17.2 mmole, 2.4 g) at 0°C.
  • the resulting solution was stirred for two hours at 0°C, then allowed to warm to room temperature with continuous stirring overnight. The reaction proceeded for another two hours at 55°C.
  • N-(5- benzyloxycarr ⁇ >nyl-ammo-l-carboxypentyl)iminodiacetic acid was 75%. Purity and structure of the compound were confirmed by proton NMR and FT-IR. NMR in perdeuterated DMSO gave the following peaks: ⁇ 2.3 (m, 4H), 2.6(m, 2H), 3.0(m, 2H), 3.3-3.6(m, 5H), 5.0 (s, 1H), 7.2-7.4(m, 5H). In the FT-IR spectrum, peaks at 1265 cm- , 1720 cnrl and broad peaks around 2500-3500 cm"l are associated with carboxy groups.
  • N-(5-benzyloxycarbonylamino-l- carrx)xypentyl)iminodiacetic acid was deprotected by hydrogenolysis.
  • 2.4 g of N-(5-benzyloxycarbonylamino-l-carboxypentyl)iminodiacetic acid dissolved in 15 ml of 1M NaOH was hydrogenated with addition of 0.5 g of 5 % Pd/C.
  • the progress of the reaction was monitored by detection of by-product carbon dioxide with Ba(OH) 2 solution and exposure of the amino group with ninhydrin reagent.
  • the vacuum dried product, N-(5-amino-l-carboxypentyl)iminodiacetic acid weighed 1.9 grams. The complete deprotection was confirmed by proton NMR of a sample dissolved in D 2 O.
  • Methoxypolyethylene glycol carbonyl imidazolide (2, nominal MW 5000 or 20,000; 0.04 mmole) dissolved in chloroform was added dropwise to a 0.1 M Na2CO3 solution of N-(5-amino-l-carboxypentyl)iminodiacetic acid (0.4 mmole) with vigorous stirring.
  • the aqueous phase was removed and chloroform phase was dried with anhydrous sodium sulfate.
  • the dried solution was added dropwise to dry ether.
  • the precipitate which formed was collected by filtration, dissolved in a small amount of chloroform and reprecipitated in ether.
  • the crude product was dried under vacuum.
  • N-(5-amino-l-carboxypentyl)imino diacetic acid terminated methoxypolyethylene glycol (3) was purified by ion exchange chromatography on DEAE sephadex A-25 with a 0.1 - 1.0 M gradient of triethylammonium bicarbonate as an eluent. The conjugation was confirmed by proton NMR, FT-IR and ninhydrin assay. mPEG-NTA was dissolved in 1 % NiSO4 solution and dialyzed against distilled water to remove free nickel ions.
  • any proteins bearing His tags are suitable for using the His- directed PEGylation scheme.
  • GFP green fluorescent protein
  • the fluorophore is formed in a variety of organisms, including E. coli.
  • a completely synthetic gene for green fluorescent protein has been prepared which replaces the naturally occurring codons with those chosen for optimal mammalian expression (Haas et al., 1996).
  • F64L B. Cormack, pers. comm.
  • S65T Heim et al.
  • the synthetic gene was inserted into a prokaryotic expression vector bearing a six histidine amino terminal tag upstream of a clotting factor Xa cleavage site (IEGR) and a consensus protein kinase A (PKA) phosphorylation site (RRAS).
  • IEGR clotting factor Xa cleavage site
  • PKA consensus protein kinase A
  • RRAS consensus protein kinase A
  • the expression vector relies on the regulated induction of transcription by IPTG.
  • the expressed protein is harvested from bacterial cells by EDTA treatment of the harvested cells to remove the outer lipopolysaccharide leaflet of the outer membrane (thereby destabilizing the outer membrane) and then washing to remove EDTA, which would otherwise interfere with the subsequent chromatographic step.
  • a mild nonionic detergent Triton X-100
  • the supernatant from a high speed spin was purified on immobilized nickel nitrilotriacetic acid columns using imidazole step elution (50 mM wash, 250 mM elution). The imidazole was removed by dialysis and the resulting GFP analyzed by SDS polyacrylamide gel electrophoresis.
  • Green fluorescent proteins with one or two histidine tags were mixed with mPEG-NTA-Ni 2 + in aqueous solution. Unreacted mPEG-NTA-Ni 2+ was removed by dialysis using membranes with a nominal molecular weight cutoff of 10 Kd. Gel electrophoresis confirmed the formation of the complex.
  • MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with El-deleted recombinant adenoviruses. Immunity 1, 433-42.
  • Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nature Med. 1, 890-3.

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  • Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On modifie des protéines avec des polymères solubles afin de modifier leurs propriétés biologiques, de manière à les rendre moins immunogènes et à prolonger leur durée de vie dans l'organisme. On effectue cette modification au moyen d'une complexation forte mais non covalente entre un chélate de métal et un marqueur d'oligohistidine introduit dans la protéine par génie génétique. Ce procédé de modification de protéines permet d'obtenir des préparations homogènes qu'on peut préparer en quantités importantes.
PCT/US1997/018104 1996-10-07 1997-10-07 Apport cible de polymeres solubles a des proteines de recombinaison WO1998015293A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97945549A EP1011730A2 (fr) 1996-10-07 1997-10-07 Apport cible de polymeres solubles a des proteines de recombinaison
AU46722/97A AU4672297A (en) 1996-10-07 1997-10-07 Targeted addition of soluble polymers to recombinant proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2809196P 1996-10-07 1996-10-07
US60/028,091 1996-10-07

Publications (2)

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WO1998015293A2 true WO1998015293A2 (fr) 1998-04-16
WO1998015293A3 WO1998015293A3 (fr) 1998-06-18

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AU (1) AU4672297A (fr)
WO (1) WO1998015293A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6540897B1 (en) * 2000-07-13 2003-04-01 Pierce Chemical Company Direct detection of histidine tagged biomolecules on electrophoretic gel
EP2341346A2 (fr) 2000-10-18 2011-07-06 The Regents of the University of California Procédés de criblage à débit élevé permettant l'internalisation d'anticorps liposomes chelatant des métaux
CN108586291A (zh) * 2018-01-02 2018-09-28 成都傲飞生物化学品有限责任公司 一种n，n-双（羧甲基）-l-赖氨酸的生产工艺

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010233A1 (fr) * 1991-11-11 1993-05-27 Fidia S.P.A. Synthese et purification des formes tronquees et proteiniques mutantes du facteur neurotrophique ciliaire de l'homme
WO1995001797A1 (fr) * 1993-07-09 1995-01-19 Smithkline Beecham Corporation Purification de proteines
WO1996009805A2 (fr) * 1994-09-23 1996-04-04 Zonagen, Inc. Immunostimulation induite par le chitosane

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010233A1 (fr) * 1991-11-11 1993-05-27 Fidia S.P.A. Synthese et purification des formes tronquees et proteiniques mutantes du facteur neurotrophique ciliaire de l'homme
WO1995001797A1 (fr) * 1993-07-09 1995-01-19 Smithkline Beecham Corporation Purification de proteines
WO1996009805A2 (fr) * 1994-09-23 1996-04-04 Zonagen, Inc. Immunostimulation induite par le chitosane

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GERSHON P.D. ET AL: "Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector" JOURNAL OF IMMUNOLOGICAL METHODS, 1995, 183/1 SPEC. ISSUE (65-76), NETHERLANDS, XP004021026 *
HOCHULI E. ET AL.: "New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues" J. CHROMATOGRAPHY, vol. 411, 1987, pages 177-184, XP002059738 *
PORATH J. ET AL: "Metal chelate affinity chromatography, a new approach to protein fractionation" NATURE, vol. 258, 18 December 1975, pages 598-599, XP002059246 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6540897B1 (en) * 2000-07-13 2003-04-01 Pierce Chemical Company Direct detection of histidine tagged biomolecules on electrophoretic gel
EP2341346A2 (fr) 2000-10-18 2011-07-06 The Regents of the University of California Procédés de criblage à débit élevé permettant l'internalisation d'anticorps liposomes chelatant des métaux
CN108586291A (zh) * 2018-01-02 2018-09-28 成都傲飞生物化学品有限责任公司 一种n，n-双（羧甲基）-l-赖氨酸的生产工艺

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Publication number Publication date
EP1011730A2 (fr) 2000-06-28
WO1998015293A3 (fr) 1998-06-18
AU4672297A (en) 1998-05-05

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