DK2732034T3 - Hbv-polymerasemutanter - Google Patents

Hbv-polymerasemutanter Download PDF

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DK2732034T3
DK2732034T3 DK12733748.3T DK12733748T DK2732034T3 DK 2732034 T3 DK2732034 T3 DK 2732034T3 DK 12733748 T DK12733748 T DK 12733748T DK 2732034 T3 DK2732034 T3 DK 2732034T3
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Perrine Martin
Nathalie Silvestre
Jean-Baptiste Marchand
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Transgene Sa
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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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Claims (23)

1. Mutant-polypeptid, som omfatter et muteret HBV-polymerase-domæne med en intern deletion, der funktionelt forstyrrer polymerase-aktiviteten, hvor den interne deletion er højst 30 aminosyrerester, hvilket muteret polymerase-domæne omfatter aminosyresekvensen vist i SEQ ID NO: 1, men mangler mindst Tyr-resten i position 203, Met-resten i position 204, Asp-resten i position 205, Asp-resten i position 206, Val-resten i position 207, Val-resten i position 208 og Leu-resten i position 209, hvor mutant-polypeptidet yderligere omfatter et muteret RNaseH-domæne omfattende en deletion af mindst 8 aminosyrer og højst 60 aminosyrer, herunder mindst den del af SEQ ID NO: 3, der strækker sig fra Glu-resten (E) i position 39 til Ala (A)-resten i position 46.
2. Mutant-polymerase-polypeptid ifølge krav 1, hvor det muterede RNaseH-domæne omfatter aminosyresekvensen vist i SEQ ID NO: 3, men (a) som mangler den del af 33 aminosyrerester, der strækker sig fra rest Xaa i position 31 til ca. Leu (L)-resten i position 63, og omfatter (b) substitutionen af Asp (D)-resten i position 10 med en His (H)-rest (D689H); (c) substitutionen af Val (V)-resten i position 90 med en Tyr (Y)-rest (V769Y); (d) substitutionen af resten i position 97 med en Tyr (Y)-rest (T/A776Y) og (e) substitutionen af Asp (D)-resten i position 98 med en His (H)-rest (D777H).
3. Mutant-polymerase-polypeptid ifølge krav 1 eller 2, som omfatter aminosyresekvensen vist i SEQ ID NO: 5.
4. Fusionsprotein omfattende mutant-polypeptidet ifølge et hvilket som helst af kravene 1 til 3 og et HBV-kerne-polypeptid, hvilket HBV-kerne-polypeptid fortrinsvis er C-terminalt trunkeret, mere fortrinsvis C-terminalt trunkeret ved rest 148 eller 149.
5. Fusionsprotein ifølge krav 4, hvor HBV-kerne-polypeptidet er fusioneret i ramme til N-terminussen af mutant-polymerase-polypeptidet.
6. Fusionsprotein ifølge krav 4 eller 5, hvor fusionsproteinet omfatter amino-syresekvensen vist i SEQ ID NO: 6.
7. Fusionsprotein omfattende mutant-polymerase-polypeptidet ifølge et hvilket som helst af kravene 1 til 3 og et eller flere immunogene HBsAg-domæne(r) eller fusionsproteinet ifølge et hvilket som helst af kravene 5 og 6, yderligere omfattende et eller flere immunogene HBsAg-domæne(r).
8. Fusionsprotein ifølge et hvilket som helst af kravene 4 til 7 omfattende ved dets N-terminus et kerne-polypeptid, der er fusioneret på mutant-polymerase-polypeptidet, og et eller to immunogene HbsAg-domæner, der er fusioneret i stedet for den interne deletion i det muterede polymerase-domæne og/eller i stedet for deletionen i det muterede RNaseH-domæne.
9. Fusionsprotein ifølge krav 8, omfattende aminosyresekvensen vist i SEQ ID NO: 8.
10. Nukleinsyremolekyle, der koder for mutant-polypeptidet ifølge et hvilket som helst af kravene 1 til 3 eller fusionsproteinet ifølge et hvilket som helst af kravene 4 til 9.
11. Vektor omfattende et nukleinsyremolekyle ifølge krav 10.
12. Vektor ifølge krav 11, hvor vektoren er en replikationsdefekt adenovirus-vektor, der stammer fra en human eller fra en chimpanse-adenovirus, i hvilken nukleinsyremolekylet fortrinsvis er insereret i adenovirus-E1-regionen og placeret under styring af en CMV-promoter.
13. Vektor ifølge krav 12, som er en defekt adenovirusvektor, der fortrinsvis stammer fra en Ad5-adenovirus, omfattende, insereret i stedet for E1-region, et nukleinsyremolekyle, der er placeret under styring af en CMV-promoter, og som koder for et fusionsprotein omfattende en aminosyresekvens som vist i SEQ ID NO: 8.
14. Vektor ifølge krav 12, som er en replikationsdefekt adenovirusvektor, der fortrinsvis stammer fra en Ad5-adenovirus, omfattende, insereret i stedet for E1-region, et nukleinsyremolekyle, der er placeret under styring af en CMV-promoter, og som omfatter nukleotidsekvensen vist i SEQ ID NO: 15.
15. Vektor ifølge krav 11, hvor vektoren er en poxvirusvektor, der stammer fra en kanariefuglepox-, en fjerkræpox- eller en vaccinia-virus, fortrinsvis en vaccinia-virus af Copenhagen-stammen, Wyeth-stammen eller den modificerede Ankara (MVA)-stamme.
16. Vektor ifølge et hvilket som helst af kravene 12 til 15, hvor vektoren er i form af infektiøse viruspartikler.
17. Proces til fremstilling af vektoren ifølge krav 16, omfattende trinnene at indføre virusvektoren i en egnet cellelinje, at dyrke cellelinjen under egnede betingelser til muliggørelse af fremstilling af den infektiøse viruspartikel, at genindvinde den fremstillede infektiøse viruspartikel fra kulturen af cellelinjen og eventuelt at oprense viruspartiklen.
18. Værtscelle omfattende nukleinsyremolekylet ifølge krav 10 eller vektoren ifølge et hvilket som helst af kravene 11 til 16.
19. Fremgangsmåde til rekombinant fremstilling af mutant-polypeptidet ifølge krav 1 til 3 eller fusionsproteinet ifølge krav 4 til 9, omfattende trinnene at indføre vektoren ifølge et hvilket som helst af kravene 11 til 16 i en egnet værtscelle til fremstilling af en transficeret eller inficeret værtscelle, at dyrke den transficerede eller inficerede værtscelle in vitro under betingelser, der er egnede til vækst af værtscellen, at genindvinde cellekulturen og eventuelt at oprense det fremstillede mutant-polypeptid eller fusionsprotein.
20. Sammensætning omfattende mindst mutant-polypeptidet ifølge et hvilket som helst af kravene 1 til 3, fusionsproteinet ifølge et hvilket som helst af kravene 4 til 9, nukleinsyremolekylet ifølge krav 10, vektoren ifølge et hvilket som helst af kravene 11 til 16, værtscellen ifølge krav 18 eller en hvilken som helst kombination deraf.
21. Mutant-polypeptid ifølge et hvilket som helst af kravene 1 til 3, fusionsprotein ifølge et hvilket som helst af kravene 4 til 9, nukleinsyremolekyle ifølge krav 10, vektor ifølge et hvilket som helst af kravene 11 til 16, værtscelle ifølge krav 18 eller sammensætning ifølge krav 20 til anvendelse ved behandling eller forebyggelse af en HBV-infektion eller HBV-associerede sygdomme og patologiske tilstande.
22. Mutant-polypeptid ifølge et hvilket som helst af kravene 1 til 3, fusionsprotein ifølge et hvilket som helst af kravene 4 til 9, nukleinsyremolekyle ifølge krav 10, vektor ifølge et hvilket som helst af kravene 11 til 16, værtscelle ifølge krav 18 eller sammensætning ifølge krav 20 til anvendelse til fremkaldelse eller stimulering af en immunreaktion i den behandlede organisme.
23. Kit af dele til anvendelse ved behandling af en HBV-infektion eller til fremkaldelse af en immunreaktion hos et individ, hvor kittet omfatter en flerhed af aktive midler udvalgt fra gruppen bestående af mutant-polypeptidet ifølge et hvilket som helst af kravene 1 til 3, fusionsproteinet ifølge et hvilket som helst af kravene 4 to 9, nukleinsyremolekylet ifølge krav 10, vektoren ifølge et hvilket som helst af kravene 11 til 16, værtscellen ifølge krav 18 eller sammensætningen ifølge krav 20.
DK12733748.3T 2011-07-12 2012-07-12 Hbv-polymerasemutanter DK2732034T3 (da)

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