JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
암 연구학

Paramyxovirus for Tumor-målrettet 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 protokol beskriver en detaljeret arbejdsgang for generation og ex vivo karakterisering af oncolytic virus for udtryk for hæmmende, ved hjælp af mæslinger virus kodning bispecific T-celle ager som eksempel. Ansøgning og tilpasning til andre vektor platforme og transgener vil fremskynde udviklingen af nye immunovirotherapeutics for klinisk oversættelse.

Abstract

Vellykket cancer immunterapi har potentiale til at opnå langsigtet tumor kontrol. Trods nylige kliniske succeser stadig der et presserende behov for sikre og effektive behandlinger skræddersyet til individuelle tumor immun profiler. Oncolytic virus aktiverer induktion af anti-tumor immunrespons samt tumor-begrænset genekspression. Denne protokol beskriver generation og ex vivo analysen af immunmodulerende oncolytic vektorer. Med fokus på mæslinger vaccine virus kodning bispecific T-celle ager som eksempel, kan den generelle metode tilpasses til andre virus arter og transgener. Arbejdsprocessen præsenteres omfatter design, kloning, redning og formering af rekombinant vira. Assays til at analysere replikation kinetik og lytisk aktivitet af vektoren samt funktionalitet af den isolerede Immunmodulator ex vivo er inkluderet, således at lette generation af roman agenter for yderligere udvikling i prækliniske modeller og i sidste ende kliniske oversættelse.

Introduction

Oncolytic virus (OVs) er ved at blive udviklet som anti-cancer terapi, der specifikt replikere inden for og dræbe tumorceller mens forlader raske væv intakt. Det er nu blevet fælles forståelse at oncolytic virotherapy (OVT), i de fleste tilfælde, ikke stole udelukkende på komplet tumor lysering af effektiv replikering og spredning af virus, men kræver yderligere virkningsmekanismer for behandling succes, herunder vaskulære og stromale målretning og vigtigere, immunstimulering1,2,3,4. Mens mange snarlig OV undersøgelser anvendes uændret vira, Aktuel forskning har nydt godt af en bedre biologisk forståelse, virus biobanker, der potentielt indeholder roman OVs, og genteknologi muligheder for at skabe avancerede OV platforme5,6,7.

I betragtning af den seneste succes for immunterapi, er immunmodulerende transgener af særlig interesse med hensyn til genteknologi af OVs. Målrettet udtryk for sådanne genprodukter af OV-inficerede tumorceller reducerer toksicitet i forhold til systemisk administration. Målretning opnås enten ved hjælp af vira med iboende oncoselectivity eller ved at ændre virale tropisme8. Lokale immunomodulation forbedrer de mangesidede anti-tumor mekanismer af OVT. Denne strategi er desuden medvirkende til spørgekriterierne samspillet mellem vira, tumorceller og vært immunsystemet. Med henblik herpå giver denne protokol en gældende og justerbar workflow til at designe, klone, redde, udbrede og validere oncolytic paramyxovirus (specielt mæslingevirus) vektorer kodning sådan transgener.

Graduering af immunresponset kan opnås ved en bred vifte af transgene produkter rettet mod forskellige trin af kræft-immunitet cyklus9, herunder styrkelse af tumor antigen anerkendelse [fx tumor-associerede antigener (TAAs) eller induktorer af store histocompatibility complex (MHC) klasse i molekyler] over støtte dendritiske celle modning for effektiv antigen præsentation (cytokiner); rekruttering og aktivering ønskede immunceller som cytotoksiske og hjælper T-celler [kemokiner, bispecific T-celle ager (BTEs)]; målretning smittespredningshæmmende celler som regulerende T-celler, myeloid-afledte suppressor celler, tumor-associerede makrofager og kræft-associerede fibroblaster (antistoffer, BTEs, cytokiner); og forebyggelse af effektor celler hæmning og udmattelse (checkpoint hæmmere). Således, en overflod af biologiske agenser er tilgængelige. Evaluering af sådanne virus-kodet hæmmende vedrørende terapeutiske virkning og mulige synergier samt forståelse af respektive mekanismer er nødvendigt at forbedre kræftbehandling.

Negativ forstand enkeltstrenget RNA-vira af familien Paramyxoviridae er kendetegnet ved flere funktioner bidrager til deres anvendelse som oncolytic vektorer. Disse omfatter en naturlig oncotropism, stor genomisk kapacitet for transgener (mere end 5 kb)10,11, effektiv spredning, herunder syncytia dannelse og høj immunogenicitet12. Derfor, OV platforme baseret på canine distemper virus13, fåresyge virus14, Newcastle disease virus15, Sendai virus16,17, simian virus 518og Tupaia paramyxovirus19 er blevet udviklet. De mest fremtrædende, live svækkede mæslinger virus vaccine stammer (MV) er kommet i prækliniske og kliniske udvikling20,21. Disse virusstammer har været anvendt i årtier for rutinemæssig immunisering med en fremragende sikkerhed post22. Derudover er der ingen risiko for pattedyrsceller mutagenese på grund af den strengt cytosole replikering af paramyxovirus. En alsidig omvendt genetik system baseret på anti-genomisk cDNA, der giver mulighed for indsættelse af transgener i yderligere transskription enheder (ATUs) er tilgængelige11,23,24. MV vektorer kodning natrium-Iodid symporter (MV-NIS) til billedbehandling og strålebehandling eller opløselige carcioembryonisk antigen (MV-CEA) som en surrogat markør for viral genekspression evalueres i øjeblikket i kliniske forsøg (NCT02962167, NCT02068794, NCT02192775, NCT01846091, NCT02364713, NCT00450814, NCT02700230, NCT03456908, NCT00408590 og NCT00408590). Sikker administration er blevet bekræftet og har indberettet tilfælde af anti-tumor effekt i tidligere undersøgelser25,26,27,28,29, 30 (gennemgået af Msaouel et al.31), baner vejen for yderligere oncolytic mæslinger vira, der er blevet udviklet og testet preclinically. MV kodning immunmodulerende molekyler rettet mod forskellige trin af kræft-immunitet cyklus har vist sig at forsinke tumorvækst og/eller forlænge overlevelsen i mus, med dokumentation for immun-medieret effekt og langsigtede beskyttende immun hukommelse i syngeneic musemodeller. Vektor-kodet transgener omfatter granulocyt-makrofag koloni stimulerende faktor (GM-CSF)32,33, H. pylori aktivering af neutrofile protein34, immun checkpoint hæmmere35, interleukin-12 (IL-12)36, TAAs37og BTEs38, som krydse-link en tumor overflade antigen med CD3 og dermed fremkalde anti-tumor aktivitet af polyklonale T celler, uanset T-celle receptoren specificitet og co stimulation ( Figur 1). De lovende prækliniske resultater opnået for disse konstruktioner efterspørgslen yderligere translationel indsats.

Talimogene laherparepvec (T-VEC), en type jeg herpes simplex virus kodning GM-CSF, er den eneste oncolytic terapeutiske godkendt af de Forenede Stater mad og Drug Administration (FDA) og det Europæiske Lægemiddelagentur (EMA). Fase III undersøgelse fører til godkendelser i slutningen 2015 har ikke kun vist effekt i stedet for samhandel tumorsygdomme injektion, men også abscopal effekter (dvs. remissioner af ikke-indsprøjtning læsioner) i avanceret melanom39. T-VEC har siden indgået yderligere forsøg for anvendelse i andre tumor enheder (f.eks., ikke-melanom hudkræft, NCT03458117, kræft i bugspytkirtlen, NCT03086642) og for evaluering af Kombinationsbehandlinger, især med immun checkpoint hæmmere (NCT02978625, NCT03256344, NCT02509507, NCT02263508, NCT02965716, NCT02626000, NCT03069378, NCT01740297 og Ribas et al.40).

Det viser ikke kun potentialet i oncolytic immunterapi, men også behovet for yderligere forskning for at identificere superior kombinationer af OVT og immunomodulation. Rationelt design af yderligere vektorer og deres udvikling til præklinisk afprøvning er nøglen til denne virksomhed. Dette vil også fremme forståelse af de underliggende mekanismer og har betydning for progression mod mere personlig kræftbehandling. Med henblik herpå præsenterer denne publikation metoden til ændring og udvikling af paramyxovirus for målrettede cancer immunterapi og mere specifikt af oncolytic mæslinger virus kodning T-celle-engagerende antistoffer (figur 2).

Protocol

Bemærk: [O], [P], og [M] angive underafsnit gælder: OVs i general, (de fleste) paramyxovirus eller MV kun, henholdsvis. [B] angiver sektioner specifikke for BTE transgener. 1 kloning af Immunmodulator-kodning transgener til mæslinger Virus vektorer [O] design indsætte sekvens. [O] træffe beslutning om en Immunmodulator af interesse baseret på litteratur forskning eller sonderende data såsom genetiske skærme41</sup…

Representative Results

Figur 1 illustrerer virkningsmekanisme af oncolytic mæslinger virus kodning bispecific T-celle ager. Et flowchart viser arbejdsprocesser af denne protokol er præsenteret i figur 2. Figur 3 viser et eksempel på en modificeret oncolytic mæslinger virus genomet. Dette giver en visuel repræsentation af de specifikke ændringer anvendes på mæslinger virus anti-genom og særlige fu…

Discussion

Oncolytic immunterapi (dvs.., OVT i kombination med immunomodulation) holder meget lovende til behandling af cancer, kræver yderligere udvikling og optimering af oncolytic virus kodning immunmodulerende proteiner. Denne protokol beskriver metoder til at generere og validere sådanne vektorer til efterfølgende test i relevante prækliniske modeller og potentielle fremtidige kliniske oversættelse til nye anti-cancer terapi.

Mange forskellige oncolytic virus platforme med forskellige …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Disse metoder blev etableret i gruppen Virotherapy, ledet af Prof. Dr. Dr. Guy Ungerechts på det nationale Center for Tumor sygdomme i Heidelberg. Vi står i gæld til ham og alle medlemmer af teamet laboratorium især Dr. Tobias Speck, Dr. Rūta Veinalde, Judith Förster, Birgit Hoyler og Jessica Albert. Dette arbejde blev støttet af andet Kröner-Fresenius-Stiftung (Grant 2015_A78 til C.E. Engeland) og tysk National Science Foundation (DFG, give C.E. Engeland da 1119/2-1). J.P.W. Heidbuechel modtager et stipendium af Helmholtz International Graduate School for kræftforskning.

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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