HRP20211159T1 - Postupci identifikacije bakterija koje sadrže vezujuće polipeptide - Google Patents

Postupci identifikacije bakterija koje sadrže vezujuće polipeptide Download PDF

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HRP20211159T1
HRP20211159T1 HRP20211159TT HRP20211159T HRP20211159T1 HR P20211159 T1 HRP20211159 T1 HR P20211159T1 HR P20211159T T HRP20211159T T HR P20211159TT HR P20211159 T HRP20211159 T HR P20211159T HR P20211159 T1 HRP20211159 T1 HR P20211159T1
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antibody
bacterium
binding polypeptide
target molecule
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Twyla Noelle Lombana
Rebekah McKENNA
Christoph Spiess
Michael Dillon
Karthik VEERAVALLI
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F. Hoffmann - La Roche Ag
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Claims (9)

1. Postupak za identifikaciju bakterije (i) koja sadrži vezujući polipeptid koji se specifično veže na ciljnu molekulu ili (ii) s poboljšanom ekspresijom, u odnosu na referentnu razinu ekspresije, vezujućeg polipeptida koji se specifično veže na ciljnu molekulu, naznačen time, da obuhvaća sljedeće korake: (a) pribavljanje bakterije koja obuhvaća mutaciju koja omogućuje difuziju molekula nepropusnih za membranu preko vanjske membrane u periplazmu, pri čemu je mutacija u genu koji kodira glavni lipoprotein Lpp (Lpp) vanjske membrane, pri čemu bakterija eksprimira nukleinsku kiselinu koja kodira vezujući polipeptid kandidat, pri čemu je vezujući polipeptid kandidat prisutan u periplazmi bakterije; (b) kontaktiranje bakterije s prepoznatljivo označenom ciljnom molekulom; i (c) identificiranje bakterije kao bakterije (i) koja sadrži vezujući polipeptid koji se specifično veže na ciljnu molekulu ili (ii) koja ima poboljšanu ekspresiju, u odnosu na referentnu razinu ekspresije, vezujućeg polipeptida putem prisutnosti označene ciljne molekule unutar periplazme, pri čemu bakterija ostaje vijabilna nakon koraka (c).
2. Postupak prema patentnom zahtjevu 1, naznačen time, da nadalje obuhvaća podvrgavanje bakterije uvjetima koji čine vanjsku membranu bakterije permeabilnom prije koraka (b), nadalje opcionalno obuhvaća ponovno zatvaranje vanjske membrane bakterije nakon kontakta bakterije s prepoznatljivo označenom ciljnom molekulom; pri čemu nadalje, opcionalno, ponovno zatvaranje vanjske membrane bakterije obuhvaća kontakt bakterije sa soli kationa odabranog iz skupine koju čine Mg2+, Mn2+, Ca2+, Zn2+, Fe2+, Na+ ili K+; pri čemu nadalje, opcionalno, kation je Mg2+ ili sol Mg2+ je MgCl2.
3. Postupak prema patentnom zahtjevu 2, naznačen time, da podvrgavanje bakterije uvjetima koji čine vanjsku membranu bakterije permeabilnom obuhvaća tretiranje bakterije permeabilizacijskim sredstvom, opcionalno gdje se permeabilizacijsko sredstvo opcionalnobira iz skupine koja sadrži dvovalentni kationski kelat, NaCl, saharozu, antibiotik, detergent, lizozim, Tris, Tris-EDTA, askorbat, polilizin, benzalkonijev klorid, protamin, baktericidni protein/protein koji povećava permeabilnost (BPI), serum, komplement i Ca2+; pri čemu nadalje, opcionalno, dvovalentni kationski kelator je EDTA ili se antibiotik bira iz skupine koju čine aminoglikozid, polimiksin B, deacilirani polimiksin, oktapeptin i benzil penicilin.
4. Postupak prema bilo kojem od patentnih zahtjeva od 1 do 3, naznačen time, da: (i) korak kontaktiranja nadalje uključuje kontakt bakterije s bojom za nukleinske kiseline, pri čemu se, opcionalno, boja za nukleinske kiseline bira iz skupine koju čine SYTO® 9, SYTO® 41, SYTO® 43, SYTO® 42, SYTO® 44, SYTO® 40 i SYTO® 45; pri čemu je nadalje, opcionalno, boja nukleinske kiseline SYTO® 9 ili SYTO® 41; (ii) prepoznatljivo označena ciljna molekula sadrži fluorescentnu oznaku, pri čemu opcionalno fluorescentna oznaka sadrži fluorescentnu boju ili fluorescentni polipeptid; pri čemu se nadalje, opcionalno, fluorescentna boja bira iz skupine koju čine: boja ALEXA® ili boja DYLIGHT®, pri čemu nadalje opcionalno boja ALEXA® je ALEXA FLUOR® 488 ili ALEXA FLUOR® 647 ili boja DYLIGHT® je DYLIGHT® 649; (iii) postupak nadalje obuhvaća najmanje jedan korak ispiranja koji obuhvaća resuspendiranje bakterije u puferu za ispiranje nakon kontakta bakterije s prepoznatljivo označenom ciljnom molekulom; (iv) korak identificiranja obuhvaća protočnu citometriju, pri čemu, opcionalno, protočna citometrija obuhvaća sortiranje pojedinačnih stanica i sortiranje na temelju signala prepoznatljivo označene ciljne molekule; pri čemu nadalje, opcionalno, obuhvaća sortiranje na temelju signala boje za nukleinske kiseline; pri čemu nadalje, opcionalno, protočna citometrija uključuje sekvencijalno sortiranje na temelju signala boje za nukleinske kiseline, sortiranje za pojedinačne stanice i sortiranje na temelju signala prepoznatljivo označene ciljne molekule; (v) bakterija je gram negativna bakterija, pri čemu, opcionalno, gram negativna bakterija je bakterija E. coli; (vi) vezujući polipeptid se izražava u topljivom obliku u periplazmi bakterije; (vii) postupak nadalje obuhvaća izolaciju nukleinske kiseline nakon koraka identifikacije; i/ili (viii) referentna razina ekspresije je razina ekspresije vezujućeg polipeptida divljeg tipa ili razina ekspresije vezujućeg polipeptida u bakteriji divljeg tipa.
5. Postupak prema bilo kojem od patentnih zahtjeva od 1 do 4, naznačen time, da je vezujući polipeptid protutijelo, pri čemu, opcionalno, (i) protutijelo je protutijelo pune duljine, pri čemu nadalje, opcionalno, protutijelo pune duljine je IgG protutijelo; (ii) protutijelo je polu-protutijelo; ili (iii) protutijelo je fragment protutijela; pri čemu se, opcionalno, fragment protutijela bira iz skupine koju čine fragmenti Fab, Fab'-SH, Fv, scFv i (Fab')2.
6. Postupak prema bilo kojem od patentnih zahtjeva od 1 do 5, naznačen time, da korak (a) obuhvaća pribavljanje mnoštva bakterija, pri čemu mnoštvo bakterija obuhvaća biblioteku nukleinskih kiselina, svaka od kojih kodira vezujući polipeptid kandidat, pri čemu, opcionalno, biblioteka sadrži mnoštvo nukleinskih kiselina koje kodiraju varijante protutijela na kandidat, pri čemu svaka varijanta protutijela na kandidat sadrži izmjenu aminokiselinskog ostatka u FR VH ili VL, u usporedbi s referentnim protutijelom, pri čemu je izmjena aminokiselinskog ostatka: (a) identificirana ju prirodnom protutijelu koje ima isti podtip kao i referentno protutijelo, i (b) nalazi se u aminokiselinskom ostatku za koji se predviđa da će biti skriven; pri čemu je nadalje, opcionalno, izmjena aminokiselinskog ostatka u prirodnom protutijelu posljedica somatske hipermutacije; pri čemu nadalje, opcionalno, postupak obuhvaća: (i) identificiranje vezujućeg polipeptida kandidata koji ima povećanu razinu ekspresije na temelju količine označene ciljne molekule unutar periplazme; i/ili (ii) identificiranje vezujućeg polipeptida kandidata koji ima povećanu razinu stabilnosti na temelju količine označene ciljne molekule unutar periplazme.
7. Postupak prema bilo kojem od patentnih zahtjeva od 1 do 6, naznačen time, da bakterija sadrži mutaciju koja utječe na gen za regulaciju transkripcije, pri čemu je gen za regulaciju transkripcije RpoD (σ70), pri čemu, opcionalno, korak (a) obuhvaća pribavljanje mnoštva bakterija, pri čemu mnoštvo bakterija obuhvaća biblioteku nukleinskih kiselina, svaka od kojih kodira mutanta gena za regulaciju transkripcije; pri čemu je nadalje, opcionalno, mutacija koja utječe na gen za regulaciju transkripcije prisutna na sintetičkom plazmidu ili u endogenom bakterijskom genomu; pri čemu je nadalje, opcionalno, mutacija koja utječe na gen za regulaciju transkripcije posljedica pogreškama sklone PCR mutageneze, kemijske mutageneze, transpozicijske mutageneze ili ciljane mutageneze; pri čemu nadalje, opcionalno, postupkom identificira bakterija koja ima poboljšanu ekspresiju vezujućeg polipeptida.
8. Bakterija koja sadrži (i) mutaciju koja omogućuje difuziju molekula nepropusnih za membranu preko vanjske membrane u periplazmu, pri čemu je mutacija u genu koji kodira Lpp; (ii) ekspresijski konstrukt koji kodira vezujući polipeptid; i (iii) mutacija koja utječe na gen za regulaciju transkripcije, pri čemu je gen za regulaciju transkripcije RpoD (σ70).
9. Bakterija prema patentnom zahtjevu 8, naznačena time, da: (i) mutacija koja utječe na gen za regulaciju transkripcije prisutna je na sintetskom plazmidu ili je prisutna u endogenom bakterijskom genomu, pri čemu je, opcionalno, mutacija koja utječe na gen za regulaciju transkripcije posljedica pogreškama sklone PCR mutageneze , kemijske mutageneze, transpozicijske mutageneze ili ciljane mutageneze, pri čemu je nadalje, opcionalno, mutacija koja utječe na gen za regulaciju transkripcije posljedica pogreškama sklone PCR mutageneze; i/ili (ii) vezujući polipeptid je protutijelo, pri čemu, opcionalno, (a) protutijelo je protutijelo pune duljine, pri čemu je nadalje, opcionalno, protutijelo pune duljine je IgG protutijelo; (b) protutijelo je polu-protutijelo; ili (c) protutijelo je fragment protutijela; pri čemu se, opcionalno, fragment protutijela bira iz skupine koju čine fragmenti Fab, Fab'-SH, Fv, scFv i (Fab')2.
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