DK2150617T3 - Regioner med forøget ekspression og stabilitet - Google Patents

Regioner med forøget ekspression og stabilitet Download PDF

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Publication number
DK2150617T3
DK2150617T3 DK08756678.2T DK08756678T DK2150617T3 DK 2150617 T3 DK2150617 T3 DK 2150617T3 DK 08756678 T DK08756678 T DK 08756678T DK 2150617 T3 DK2150617 T3 DK 2150617T3
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goi
expression
seq
cell
recombinase recognition
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Gang Chen
James P Fandl
Robert Babb
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Regeneron Pharma
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
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  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (16)

1. Isoleret celle, som omfatter en ekspressionsforøgende sekvens, der er valgt blandt SEQ ID NO: 1,3 og 5, og mindst ét rekombinase-genkendelsessted, som er placeret i en nukleotidsekvens ifølge SEQ ID NO: 1,3 eller 5.
2. Isoleret celle ifølge krav 1, hvor det mindst ene rekombinase-genkendelsessted er valgt fra gruppen bestående af et loxp-sted, et lox 511-sted, et lox 2272-sted og et frt-sted.
3. Isoleret celle ifølge krav 1, hvilken celle endvidere omfatter et første gen af interesse (GOI, gene of interest), som koder for et protein, hvor det første GOI er integreret på det mindst ene rekombinase-genkendelsessted; og hvor GOl'et eventuelt er operativt forbundet med en promotor.
4. Isoleret celle ifølge krav 3, hvilken celle endvidere omfatter et andet GOI, som koder for et protein, hvor der forefindes et rekombinase-genkendelsessted mellem det første GOI og det andet GOI; og hvor cellen eventuelt endvidere omfatter et rekombinase-genkendelsessted 5' i forhold til det første GOI og et rekombinase-genkendelsessted 3' i forhold til det andet GOI.
5. Isoleret celle ifølge krav 1, hvor cellen er en CHO-celle, og hvor den ekspressionsforøgende sekvens er SEQ ID NO: 5.
6. Isoleret celle ifølge krav 5, hvor rekombinase-genkendelsesstedet er placeret i en stilling mellem nukleotiderne 5.000 og 7.400 ifølge SEQ ID NO: 5; nukleotiderne 5.000 og 6.500 ifølge SEQ ID NO: 5; nukleotiderne 6.400 og 7.400 ifølge SEQ ID NO: 5 eller nukleotiderne 6.400 og 6.500 ifølge SEQ ID NO: 5.
7. CHO-celle, som omfatter en ekspressionsforøgende sekvens, der er nukleotiderne 6.400 til 6.500 ifølge SEQ ID NO: 5, hvilken CHO-celle endvidere omfatter mindst ét rekombinase-genkendelsessted, der er flankeret af et humant gen af interesse (GOI) 5' eller 3', hvor det humane GOI eventuelt er flankeret 5' af det første rekombinase-genkendelsessted, og hvilken CHO-celle endvidere omfatter et andet rekombinase-genkendelsessted, der flankerer det humane GOI 3'.
8. CHO-celle ifølge krav 7, hvilken CHO-celle endvidere omfatter et andet humant GOI, som flankerer det andet rekombinase-genkendelsessted 3'; og hvilken CHO-celle eventuelt endvidere omfatter et tredje rekombinase-genkendelsessted, som flankerer det andet humane GOI 3'.
9. CHO-celle ifølge krav 8, hvilken CHO-celle endvidere omfatter mindst ét markørgen mellem det andet rekombinase-genkendelsessted og det andet gen af interesse.
10. CHO-celle ifølge krav 9, hvilken CHO-celle endvidere omfatter en promotor, som er operativt forbundet med det første humane GOI, og en promotor, som er operativt forbundet med det andet GOI; hvor promotorerne eventuelt er eukaryote promotorer, og de eukaryote promotorer er operativt forbundet med en prokaryot operator.
11. CHO-celle ifølge krav 10, hvor det andet og det tredje rekombinase-genkendelsessted er i modsat orientering i forhold til det første rekombinase-genkendelsessted.
12. Isoleret celle, som omfatter en ekspressionsforøgende sekvens, der er valgt blandt SEQ ID NO: 1,3 og 5, og mindst ét eksogent tilføjet GOI i den ekspressionsforøgende sekvens.
13. Isoleret celle ifølge krav 12, hvor cellen er en CHO-celle, den ekspressionsforøgende sekvens omfatter SEQ ID NO: 5, det mindst ene eksogent tilføjede GOI er et humant gen, og det humane gen er operativt forbundet med en eksogent tilføjet promotor.
14. Fremgangsmåde til fremstilling af et protein af interesse, hvilken fremgangsmåde omfatter: (a) tilvejebringelse af en værtscelle, som omfatter en ekspressionsforøgende sekvens, der er valgt blandt SEQ ID NO: 1, 3 og 5, eller nukleotiderne 6.400 til 6.500 ifølge SEQ ID NO: 5, (b) indføring i værtscellen, i den ekspressionsforøgende sekvens, af et gen af interesse (GOI), som er operativt forbundet med en promotor; (c) bibeholdelse af værtscellen fra (a) under betingelser, der gør det muligt for GOl'et at udtrykke et protein af interesse; og (d) indvinding af proteinet af interesse.
15. Isoleret celle, CHO-celle eller fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor det første og/eller andet GOI koder for et protein valgt blandt en let kæde fra et antistof eller et antigenspecifikt fragment deraf, en tung kæde fra et antistof eller et antigenspecifikt fragment deraf, en ligand og en receptor eller et ligandspecifikt fragment deraf.
16. Anvendelse af den isolerede celle ifølge krav 1 eller CHO-cellen ifølge krav 7 til fremstilling af et antistof eller et fragment deraf.
DK08756678.2T 2007-06-04 2008-06-04 Regioner med forøget ekspression og stabilitet DK2150617T3 (da)

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US93321307P 2007-06-04 2007-06-04
PCT/US2008/065733 WO2008151219A1 (en) 2007-06-04 2008-06-04 Enhanced expression and stability regions

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DK2150617T3 true DK2150617T3 (da) 2015-01-12

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US (6) US7771997B2 (da)
EP (1) EP2150617B1 (da)
DK (1) DK2150617T3 (da)
ES (1) ES2522615T3 (da)
PL (1) PL2150617T3 (da)
WO (1) WO2008151219A1 (da)

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US20130130372A1 (en) 2013-05-23
US20100291626A1 (en) 2010-11-18
ES2522615T3 (es) 2014-11-17
US10415055B2 (en) 2019-09-17
WO2008151219A1 (en) 2008-12-11
PL2150617T3 (pl) 2015-04-30
US20160097059A1 (en) 2016-04-07
EP2150617B1 (en) 2014-10-22
EP2150617A1 (en) 2010-02-10
US9562238B2 (en) 2017-02-07
US9222106B2 (en) 2015-12-29
US8389239B2 (en) 2013-03-05
US20170096681A1 (en) 2017-04-06
US9932605B2 (en) 2018-04-03
US20180327777A1 (en) 2018-11-15
US20090124005A1 (en) 2009-05-14
US7771997B2 (en) 2010-08-10

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