JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
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Pesquisa do Câncer

Paramyxoviruses for svulst målrettede Immunomodulation: Design og evaluering Ex Vivo

Published: January 07, 2019
doi:
10.3791/58651
,
1Department of Translational Oncology,German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), 2Faculty of Biosciences,Heidelberg University, 3Department of Medical Oncology,NCT and Heidelberg University Hospital

Summary

Denne protokollen beskriver en detaljert arbeidsflyt for generering og ex vivo karakterisering av kan virus for uttrykk for immunomodulators, ved hjelp av meslinger virus koding bispecific T celle engagers som et eksempel. Programmet og tilpasning til andre vektor plattformer og effekter av transgener vil akselerere utviklingen av romanen immunovirotherapeutics for klinisk oversettelse.

Abstract

Vellykket kreft immunterapi har potensial til å oppnå langsiktig svulst kontroll. Til tross for siste klinisk suksess, er det fortsatt et presserende behov for sikker og effektiv terapi skreddersydd til individuelle svulst immun profiler. Kan virus aktiverer induksjon av anti-tumor immunreaksjoner samt svulst-begrenset genuttrykk. Denne protokollen beskriver generasjon og ex vivo analyse av immunmodulerende kan vektorer. Fokusere på meslinger vaksine virus koding bispecific T celle engagers som et eksempel, kan generelle metodikken tilpasses til andre virus arter og effekter av transgener. Presentert arbeidsflyten inkluderer design, kloning, redning og overføring av rekombinant virus. Analyser analysere replikasjon kinetikk og lytisk aktivitet av vektoren og funksjonaliteten til den isolerte immunomodulator ex vivo er inkludert, dermed tilrettelegge generering av romanen agenter for videre utvikling i preklinisk modeller og til slutt klinisk oversettelse.

Introduction

Kan virus (OVs) utvikles som anti-kreft legemiddelselskap som spesielt replikere innen og drepe kreftceller mens røres sunt vev. Det har nå blitt vanlig forstå at det kan virotherapy (overtid), i de fleste tilfeller, ikke stole utelukkende på komplett svulst lysis av effektiv replikering og spredning av viruset, men krever flere virkningsmekanismer for suksess, inkludert vaskulær og stromal målretting og, viktigst, immune stimulering1,2,3,4. Mens mange OV studier brukt uforandret virus, har nåværende forskning profittert fra en forbedret biologiske forståelse, virus biobanker som inneholde romanen OVs og mulighetene som tilbys av genteknologi for å lage avanserte OV plattformer5,6,7.

Gitt den nylige suksessen immunterapi, er immunmodulerende effekter av transgener av spesiell interesse om genteknologi av OVs. Målrettet uttrykk for slike genet produkter av OV-infiserte kreftceller reduserer toksisitet sammenlignet med systemisk administrasjon. Målretting oppnås ved å bruke virus med iboende oncoselectivity eller ved å endre viral tropism8. Lokale immunomodulation forbedrer mangesidig anti-tumor mekanismer for overtid. Videre, denne strategien er instrumental i avhør samspillet mellom virus, tumorceller og immun vertssystemet. Dette gir denne protokollen en anvendelig og justerbar arbeidsflyt for å utforme, klone, redde, overføre og validere kan paramyxovirus (spesielt meslinger viruset) vektorer koding slike effekter av transgener.

Modulering av immun respons oppnås ved en rekke forskjellige trinn av kreft-immunitet syklus9, inkludert styrke svulst antigen anerkjennelse [f.eks svulst-assosiert antigener (TAAs) eller indusere av transgene spesialprodukter store histocompatibility kompleks (MHC) klasse I molekyler] over støtter dendrittiske celle modning for effektiv antigen presentasjon (cytokiner); rekruttering og aktivere ønsket immunceller som cytotoksiske og hjelper T celler [chemokines, bispecific T celle engagers (BTEs)]; målretting undertrykkende celler som regulatoriske T-celler, myelogen-avledet suppressor cellene, tumor-assosiert makrofager og kreft-assosiert fibroblaster (antistoffer, BTEs, cytokiner); og hindre effektor celle hemming og utmattelse (checkpoint hemmere). Således, en overflod av biologiske midler er tilgjengelige. Evaluering av slike virus-kodede immunomodulators terapeutiske effekten og synergimuligheter samt forståelse av respektive mekanismer er nødvendig å forbedre kreft terapi.

Negativ forstand single-strandet RNA virus av Paramyxoviridae familie er preget av flere funksjoner til deres bruk som kan vektorer. Disse inkluderer en naturlig oncotropism, stor genomisk kapasitet for effekter av transgener (mer enn 5 kb)10,11, effektiv spre inkludert syncytia formasjon og høy immunogenisitet12. Derfor OV plattformer basert på Hjørnetann distemper virus13, kusma virus14, Newcastle sykdom virus15, Sendai virus16,17, simian virus 518og Tupaia paramyxovirus19 har blitt utviklet. Mest fremtredende, live dempes meslinger viruset vaksine stammer (MV) har kommet i preklinisk og klinisk utvikling20,21. Disse viruset stammer har blitt brukt i flere tiår for rutinen immunisering med en utmerket sikkerhet posten22. Dessuten, det er ingen risiko for insertional mutagenese på grunn av strengt cytosolic replikering av paramyxoviruses. Et allsidig omvendt genetikk system basert på anti-genomic cDNA som tillater innsetting av effekter av transgener i flere transkripsjon enheter (ATUs) er tilgjengelig11,23,24. MV vektorer koding natrium-iodide symporter (MV-NIS) for bildebehandling og strålebehandling eller løselig carcinoembryonic antigen (MV-CEA) som et surrogat markør for viral genuttrykk er som blir evaluert i kliniske forsøk (NCT02962167, NCT02068794, NCT02192775, NCT01846091, NCT02364713, NCT00450814, NCT02700230, NCT03456908, NCT00408590 og NCT00408590). Sikker administrasjon har blitt bekreftet og tilfeller av anti-tumor effekten har blitt rapportert i forrige studier25,26,27,28,29, 30 (anmeldt av Msaouel et al.31), banet veien for ytterligere kan meslinger virus som er utviklet og testet preklinisk. MV koding immunmodulerende molekyler målretting ulike trinn av kreft-immunitet syklusen har vist seg å forsinke tumor vekst eller forlenge overlevelse i mus, med bevis for immun-mediert effekt og langsiktig beskyttende immun minne i syngeneic musen modeller. Vektor-kodet effekter av transgener inkluderer granulocytt-macrophage koloni stimulerende faktor (GM-CSF)32,33, H. pylori nøytrofile-aktivere protein34, immun checkpoint hemmere35, interleukin-12 (IL-12)36, TAAs37og BTEs38, som en svulst-link overflateantigen med CD3 og dermed indusere anti-svulst aktivitet av polyklonale T-celler, uavhengig av T celle reseptor spesifisitet og co-stimulering ( Figur 1). Lovende prekliniske resultatene for disse konstruksjoner etterspørsel ytterligere translasjonsforskning innsats.

Talimogene laherparepvec (T-VEC), en type jeg herpes simplex virus koding GM-CSF, er den eneste kan terapeutiske godkjent av USA Food, Drug Administration (FDA) og europeiske Legemidler Agency (EMA). Fase III studien fører til godkjenninger i slutten 2015 har ikke bare vist effekt på stedet av intra-tumoral injeksjon, men også abscopal effekter (dvs. forbedringer av ikke-injisert lesjoner) i avansert melanom39. T-VEC har siden kommet ytterligere forsøk for programmet i andre svulst enheter (f.eks, ikke-melanom hudkreft, NCT03458117, bukspyttkjertelkreft, NCT03086642) og evaluering av kombinasjon terapier, spesielt med immun checkpoint hemmere (NCT02978625, NCT03256344, NCT02509507, NCT02263508, NCT02965716, NCT02626000, NCT03069378, NCT01740297 og Ribas et al.40).

Dette viser ikke bare potensialet i kan immunterapi, men også behov for videre forskning åidentifisere overlegen kombinasjoner av overtid og immunomodulation. Rasjonell utformingen av flere vektorer og deres utvikling for prekliniske tester er nøkkelen til dette prosjektet. Dette vil også fremme forståelse av underliggende mekanismene har implikasjoner for utviklingen mot mer personlig kreftbehandling. Dette presenterer denne publikasjonen metodikken for endring og utvikling av paramyxoviruses for målrettet kreft immunterapi og, mer spesifikt, kan meslinger virus koding T celle engasjerende antistoffer (figur 2).

Protocol

Merk: [O], [P], og [M] angir ledd gjelder: OVs i generelt, (de fleste) paramyxoviruses eller MV, henholdsvis. [B] angir inndelinger bestemt BTE effekter av transgener. 1 kloning av Immunomodulator-koding effekter av transgener i meslinger viruset vektorer [O] design sett inn sekvens. [O] bestemme en immunomodulator rundt basert på forskning i literatur eller utforskende data som genetiske skjermer41 og utlede relev…

Representative Results

Figur 1 illustrerer virkningsmekanismen kan meslinger virus koding bispecific T celle engagers. Et flytskjema som viser arbeidsflyten for denne protokollen vises i figur 2. Figur 3 viser et eksempel på en modifisert kan meslinger viruset genomet. Dette gir en visuell fremstilling av de bestemte endringene gjelder meslinger viruset anti-genom og bestemt funksjonene i innsatte effekt…

Discussion

Kan immunterapi (i.e., overtid i kombinasjon med immunomodulation) har store løftet for kreftbehandling, krevende videre utvikling og optimalisering av kan virus koding immunmodulerende proteiner. Denne protokollen beskriver metoder for å generere og godkjenne slike vektorer for påfølgende testing i relevante prekliniske modeller og potensielle fremtidige klinisk oversettelse til romanen kreft therapeutics.

Mange ulike kan virus plattformer med forskjellige fordeler er tilgjengeli…

Declarações

The authors have nothing to disclose.

Acknowledgements

Disse metodene ble etablert i gruppen Virotherapy ledet av professor Dr. Dr. Guy Ungerechts ved National Center for svulst sykdommer i Heidelberg. Vi står i gjeld til ham og alle medlemmer av laboratoriet team, spesielt Dr. Tobias Speck, Dr. Rūta Veinalde, Judith Förster, Birgit Hoyler og Jessica Albert. Dette arbeidet ble støttet av den andre Kröner-Fresenius-Stiftung (Grant 2015_A78 til CE Engeland) og tysk National Science Foundation (DFG, gi EN 1119/2-1 til CE Engeland). J.P.W. Heidbuechel mottar et stipend av Helmholtz International Graduate School for kreftforskning.

Materials

Rapid DNA Dephos & Ligation Kit Roche Life Science, Mannheim, Germany 4898117001
CloneJET PCR Cloning Kit Thermo Fisher Scientific, St. Leon-Rot K1231
Agarose Sigma-Aldrich, Taufkirchen, Germany A9539-500G
QIAquick Gel Extraction Kit QIAGEN, Hilden, Germany 28704
NEB 10-beta Competent E. coli New England Biolabs (NEB), Frankfurt/Main, Germany C3019I
LB medium after Lennox Carl Roth, Karlsruhe, Germany X964.1
Ampicillin Carl Roth, Karlsruhe, Germany HP62.1
QIAquick Miniprep Kit QIAGEN, Hilden, Germany 27104
Restriction enzyme HindIII-HF New England Biolabs (NEB), Frankfurt/Main, Germany R3104S
Dulbecco's Modified Eagle's Medium (DMEM) Invitrogen, Darmstadt, Germany 31966-021
Fetal bovine serum (FBS) Biosera, Boussens, France FB-1280/500
FugeneHD Promega, Mannheim, Germany E2311 may be replaced by transfection reagent of choice
Kanamycin Sigma-Aldrich, Taufkirchen, Germany K0129
Vero cells ATCC, Manassas, VA, USA CCL81
B16-CD46/ B16-CD20-CD46 J. Heidbuechel, DKFZ Heidelberg available upon request
Granta-519 DSMZ, Braunschweig, Germany ACC 342
Opti-MEM (serum-free medium) Gibco Life Technologies, Darmstadt, Germany 31985070
Colorimetric Cell Viability Kit III (XTT) PromoKine, Heidelberg, Germany PK-CA20-300-1000 includes XTT reagent
Dulbecco's Phosphate-Buffered Saline (PBS) Gibco Life Technologies, Darmstadt, Germany 14190-094
QIAquick Ni-NTA Spin Columns QIAGEN, Hilden, Germany 31014
Sodium chloride Carl Roth, Karlsruhe, Germany 3957.3
Imidazole Carl Roth, Karlsruhe, Germany I5513-25G
Amicon Ultra-15, PLGC Ultracel-PL Membran, 10 kDa Merck, Darmstadt, Germany UFC901024
BCA Protein Assay Kit Merck Milipore 71285-3
IgG from human serum Sigma-Aldrich, Taufkirchen, Germany I4506
Anti-HA-PE Miltenyi Biotech, Bergisch Gladbach, Germany 130-092-257 RRID: AB_871939
Mouse IgG1, kappa Isotype Control, Phycoerythrin Conjugated, Clone MOPC-21 antibody BD Biosciences, Heidelberg, Germany 555749 RRID: AB_396091
Anti-HA-biotin antibody, clone 3F10 Sigma-Aldrich, Taufkirchen, Germany 12158167001 RRID: AB_390915
Anti-Biotin MicroBeads Miltenyi Biotech, Bergisch Gladbach, Germany 130-090-485
MS Columns Miltenyi Biotech, Bergisch Gladbach, Germany 130-042-201
MiniMACS Separator Miltenyi Biotech, Bergisch Gladbach, Germany 130-042-102
MACS MultiStand Miltenyi Biotech, Bergisch Gladbach, Germany 130-042-303
RIPA buffer Rockland Immunochemicals, Gilbertsville, PA, USA MB-030-0050
CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega, Mannheim, Germany G1780 includes 10x lysis solution, substrate solution (substrate mix and assay buffer), and stop solution
Cell lifter Corning, Reynosa, Mexico 3008
10 cm dishes Corning, Oneonta, NY, USA 430167
15 cm dishes Greiner Bio-One, Frickenhausen, Germany 639160
96-well plates, U-bottom TPP, Trasadingen, Switzerland 92097
96-well plates, flat bottom Neolab, Heidelberg, Germany 353072
6-well plates Neolab, Heidelberg, Germany 353046
12-well plates Neolab, Heidelberg, Germany 353043
50 mL tubes nerbe plus, Winsen/Luhe, Germany 02-572-3001
T175 cell culture flasks Thermo Fisher Scientific, St. Leon-Rot 159910
0.22 µm filters Merck, Darmstadt, Germany SLGPM33RS
DOWNLOAD MATERIALS LIST

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Tags

ParamyxovirusesTumor-targeted ImmunomodulationOncolytic VectorsReverse Genetic SystemMeasles VirusBispecific T Cell EngagerVirus PropagationSyncytia FormationVirus RescueTransfectionMV Producer CellsReporter Gene ExpressionVirus Harvest

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Heidbuechel, J. P., Engeland, C. E. Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. J. Vis. Exp. (143), e58651, doi:10.3791/58651 (2019).
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