DK2560683T3 - Fremstilling af heteromultimeriske proteiner - Google Patents

Fremstilling af heteromultimeriske proteiner Download PDF

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DK2560683T3
DK2560683T3 DK11718831.8T DK11718831T DK2560683T3 DK 2560683 T3 DK2560683 T3 DK 2560683T3 DK 11718831 T DK11718831 T DK 11718831T DK 2560683 T3 DK2560683 T3 DK 2560683T3
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Justin Scheer
Christoph Spiess
Daniel G Yansura
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Hoffmann La Roche
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Claims (18)

1. Fremgangsmåde til fremstilling af et heteromultimert protein omfattende et første hængsels-indeholdende polypeptid med et første heterodimerisationsdomæne og et andet hængsels-indeholdende polypeptid med et andet heterodimerisationsdomæne, hvor det andet heterodimerisationsdomæne interagerer med det første heterodimerisationsdomæne, og hvor de første og anden hængsels-indeholdende polypeptider er bundet ved mindst én interkædedisulfidbinding, og hver af de første og anden hængsels-indeholdende polypeptider er et antistof tung kæde og er parret med et antistof let kæde for at danne et første par og et andet par, og hvor de lette kæder af det første par og det andet par omfatter forskellige variable domænesekvenser, hvilken fremgangsmåde omfatter trinnene at: (a) dyrke en første værtscelle omfattende en nukleinsyre der koder for det første hængsels-indeholdende polypeptid under betingelser hvor det hængselsindeholdende polypeptid udtrykkes og er parret med et antistof let kæde for at danne det første par; (b) dyrke en anden værtscelle omfattende en nukleinsyre der koder for det andet hængsels-indeholdende polypeptid under betingelser hvor det hængselsindeholdende polypeptid udtrykkes og er parret med et antistof let kæde for at danne det andet par; (c) ødelægge celleemembranerne af de første og anden værtsceller for at frigive de første og anden hængsels-indeholdende polypeptider fra værtscellerne for at danne et heteromultimert protein, hvor de første og anden værtsceller kombineres sammen i en enkelt suspension; og (d) genvinde det heteromultimeriske protein, hvor fremgangsmåden ikke kræver tilsætningen af et reduktionsmiddel.
2. Fremgangsmåde til fremstilling af et heteromultimert protein omfattende et første hængsels-indeholdende polypeptid med et første heterodimerisationsdomæne og et andet hængsels-indeholdende polypeptid med et andet heterodimerisationsdomæne, hvor det andet heterodimerisationsdomæne interagerer med det første heterodimerisationsdomæne, og hvor de første og anden hængsels-indeholdende polypeptider er bundet at mindst én interkædedisulfidbinding, og hver af de første og anden hængsels-indeholdende polypeptider er et antistof tung kæde og er parret med et antistof let kæde for at danne et første par og et andet par, og hvor de lette kæder af det første par og det andet par omfatter forskellige variable domænesekvenser, hvilken fremgangsmåde omfatter trinnene at: (a) dyrke en første værtscelle omfattende en nukleinsyre der koder for det første hængsels-indeholdende polypeptid under betingelser hvor det hængselsindeholdende polypeptid udtrykkes, parret med et antistof let kæde for at danne det første par og secerneret i dyrkningsmediet; (b) dyrke en anden værtscelle omfattende en nukleinsyre der koder for det andet hængsels-indeholdende polypeptid under betingelser hvor det hængselsindeholdende polypeptid udtrykkes, parret med et antistof let kæde for at danne det andet par og secerneret i dyrkningsmediet; og (c) genvinde det heteromultimeriske protein fra dyrkningsmediet, hvor dyrkningsmediet af den første og anden værtscelle kombineres sammen til at danne det heteromultimeriske protein, hvor fremgangsmåden ikke kræver tilsætningen af et reduktionsmiddel.
3. Fremgangsmåden ifølge krav 1 eller 2, hvor det første heterodimerisationsdomæne og det andet heterodimerisationsdomæne mødes ved en forbindelse, og forbindelsesdelen af det andet heterodimerisationsdomæne omfatter en protuberans (en knop) der er positionerbar i et hulrum (et hul) i hængselsdelen af det første heterodimerisationsdomæne, hvor protuberansen omfatter en aminosyresidekæde der projektere ud fra hængselsdelen af det andet heterodimerisationsdomæne, og hulrummet omfatter en aminosyresidekæde der er forsænket fra hængselsdelen af det første heterodimerisationsdomæne.
4. Fremgangsmåden ifølge krav 1 eller 2, hvor interkædedisulfidbindingen er mellem hængselsregionerne.
5. Fremgangsmåden ifølge krav 1 eller 2, hvor værtscellen er en prokaryot-celle, især en E. coli-celle, mere specielt en Ipp deficient E. coli-celle.
6. Fremgangsmåden ifølge krav 1 eller 2, hvor værtscellen er en pattedyrcelle, især en CHO-celle.
7. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 6, hvor cellerne fra trinnene (a) og (b) er i separate cellekulturer.
8. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 6, hvor cellerne fra trinnene (a) og (b) er i en blandet kultur omfattende begge værtsceller.
9. Fremgangsmåden ifølge krav 7, hvor cellerne fra trinnene (a) og (b) (i) kombineres, (ii) centrifugeres og (iii) resuspenderes i buffer før ødelæggelsen af cellemembranen, eller (i) centrifugeres, (ii) resuspenderes i buffer og (iii) kombineres før ødelæggelse af cellemembranen.
10. Fremgangsmåden ifølge krav 1 eller 2, hvor genvindelsestrinnet yderligere omfatter mindst et oprensningstrin.
11. Fremgangsmåden ifølge krav 1 eller 2, hvor det heteromultimere protein er et bispecifikt antistof eller et multispecifikt antistof.
12. Fremgangsmåden ifølge krav 11, hvor antistoffet er humaniseret eller humant.
13. Fremgangsmåden ifølge krav 11, hvor antistoffet et er fuldlængdeantistof.
14. Fremgangsmåden ifølge krav 11, hvor antistoffet er valgt fra gruppen bestående af IgG, IgA og IgD, især er antistoffet IgG, mere specielt IgGl eller IgG2.
15. Fremgangsmåden ifølge krav 1 eller 2, hvor mindst 45% af polypeptiderne er i et kompleks omfattende det første hængsels-indeholdende polypeptid og let kæde par og det andet hængsels-indeholdende polypeptid og let kæde par.
16. Fremgangsmåden ifølge krav 10, hvor ikke over end 10% af de isolerede polypeptider er til stede som monomerer eller homodimerer før oprensningstrinnet af det heteromultimeriske protein.
17. Fremgangsmåde til at generere et kombinatorisk heteromultimert proteinbibliotek omfattende et første hængsels-indeholdende polypeptid med et første heterodimerisations-domæne og et andet hængsels-indeholdende polypeptid med et andet heterodimeri-sationsdomæne, hvor det andet heterodimerisations-domæne interagerer med det første heterodimerisationsdomæne, og hvor de første og anden hængsels-indeholdende polypeptider er bundet at mindst én interkædedisulfidbinding, og hver af de første og anden hængsels-indeholdende polypeptider er et antistof tung kæde og er parret med et antistof let kæde for at danne et første par og et andet par, og hvor de lette kæder af det første par og det andet par omfatter forskellige variable domænesekvenser, hvilken fremgangsmåde omfatter trinnene at: (a) dyrke en første værtscelle og mindst to yderligere værtsceller, hvor i. den første værtscelle omfatter en første nukleinsyre der koder for et første hængsels-indeholdende polypeptid; og ii. de yderligere værtsceller omfatter en nukleinsyre der koder for et andet hængsels-indeholdende polypeptid; under betingelser hvor de hængsels-indeholdende polypeptider udtrykkes og parres med et antistof let kæde for at danne det første og det andet par; (b) ødelægge cellemembranerne af den første værtscelle og de mindst to yderligere værtsceller så at de første og anden hængsels-indeholdende polypeptider frigives i det ekstracellulære miljø, hvor den første værtscelle og de mindst to yderligere værtsceller kombineres sammen i en enkelt suspension; og (c) genvinde de heteromultimeriske proteinkomplekser, hvor fremgangsmåden ikke kræver tilsætningen af et reduktionsmiddel.
18. Fremgangsmåde til at generere et kombinatorisk heteromultimert protein-bibliotek omfattende et første hængsels-indeholdende polypeptid med et første heterodimerisationsdomæne og et andet hængsels-indeholdende polypeptid med et andet heterodimerisationsdomæne, hvor det andet heterodimerisationsdomæne interagerer med det første heterodimerisationsdomæne, og hvor de første og anden hængselsindeholdende polypeptider er bundet at mindst én interkædedisulfidbinding, og hver af de første og anden hængsels-indeholdende polypeptider er et antistof tung kæde og er parret med et antistof let kæde for at danne et første par og et andet par, og hvor de lette kæder af det første par og det andet par omfatter forskellige variable domænesekvenser, hvilken fremgangsmåde omfatter trinnene at: (a) dyrke en første værtscelle og mindst to yderligere værtsceller, hvor i. den første værtscelle omfatter en første nukleinsyre der koder for et første hængsels-indeholdende polypeptid; og ii. de yderligere værtsceller omfatter en nukleinsyre der koder for et andet hængsels-indeholdende polypeptid; under betingelser hvor det hængsels-indeholdende polypeptider udtrykkes, parres med et antistof let kæde for at danne det første og det andet par og secerneret i dyrkningsmediet; og (b) genvinde de heteromultimeriske proteinkomplekser fra dyrknings-mediet, hvor dyrkningsmediet af den første værtscelle og de mindst to yderligere værtsceller kombineres sammen for at danne det heteromultimeriske protein, hvor fremgangsmåden ikke kræver tilsætningen af et reduktionsmiddel.
DK11718831.8T 2010-04-23 2011-04-22 Fremstilling af heteromultimeriske proteiner DK2560683T4 (da)

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