EP1011730A2 - Gezielte hinzufügung von wasserlöslichen polymeren zu rekombinanten proteinen - Google Patents

Gezielte hinzufügung von wasserlöslichen polymeren zu rekombinanten proteinen

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Publication number
EP1011730A2
EP1011730A2 EP97945549A EP97945549A EP1011730A2 EP 1011730 A2 EP1011730 A2 EP 1011730A2 EP 97945549 A EP97945549 A EP 97945549A EP 97945549 A EP97945549 A EP 97945549A EP 1011730 A2 EP1011730 A2 EP 1011730A2
Authority
EP
European Patent Office
Prior art keywords
protein
soluble polymer
proteins
cheland
soluble polymers
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97945549A
Other languages
English (en)
French (fr)
Inventor
Brian Edge Biosystems Inc. SEED
Wen Edge Biosystems Inc. SHAO
John Edge Biosysems Inc. SEED
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Edge Biosystems Inc
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
Publication of EP1011730A2 publication Critical patent/EP1011730A2/de
Withdrawn legal-status Critical Current

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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)
EP97945549A 1996-10-07 1997-10-07 Gezielte hinzufügung von wasserlöslichen polymeren zu rekombinanten proteinen Withdrawn EP1011730A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2809196P 1996-10-07 1996-10-07
US28091P 1996-10-07
PCT/US1997/018104 WO1998015293A2 (en) 1996-10-07 1997-10-07 Targeted addition of soluble polymers to recombinant proteins

Publications (1)

Publication Number Publication Date
EP1011730A2 true EP1011730A2 (de) 2000-06-28

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EP (1) EP1011730A2 (de)
AU (1) AU4672297A (de)
WO (1) WO1998015293A2 (de)

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* 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
US7045283B2 (en) 2000-10-18 2006-05-16 The Regents Of The University Of California Methods of high-throughput screening for internalizing antibodies
CA2975432A1 (en) 2015-02-13 2016-08-18 Transgene Sa Immunotherapeutic vaccine and antibody combination therapy
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 Priority date Publication date Assignee Title
CA2123234A1 (en) * 1991-11-11 1993-05-27 Lanfranco Callegaro Synthesis and purification of truncated and mutein forms of human ciliary neuronotrophic factor
AU688778B2 (en) * 1993-07-09 1998-03-19 Avant Immunotherapeutics, Inc. Protein purification
JP3365635B2 (ja) * 1994-09-23 2003-01-14 ゾナジェン，インコーポレイテッド キトサン誘導性免疫強化

Non-Patent Citations (1)

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Title
See references of WO9815293A2 *

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AU4672297A (en) 1998-05-05
WO1998015293A3 (en) 1998-06-18

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