Name: generation of car t cells for adoptive therapy in the context of glioblastoma standard of care
Uploaded: 2015-02-16T14:00:00
Description: Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

Denne protokol er baseret på en eksperimentel design, hvor 10 mus behandles med 10 7 CAR T-celler hver. Det betyder, at der bliver brug 10 8 BIL T-celler; udbyttet skal være overvurderet med 5 x 10 7 -1 x 10 8 til grund for tab i levedygtighed. Følgende protokol er skaleret til at generere ca. 200 x 10 6 celler. Cellerne bliver derefter administreret intravenøst ​​til C57BL / 6-mus med ni dage etablerede syngene EGFRvlll-positive intrakranielle tumorer udvik...

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Behandlingen tidslinje beskrives her var designet til at modellere klinisk standard for pleje og udnytte dens virkninger for CAR adoptiv terapi. CAR T-celle doser TMZ regimer og strålebehandling indgivelse kan modificeres til at forøge in vivo T-celle-aktivitet, lymphodepletion og tumordrab. TMZ regimer kan øges til opnåelse vært myeloablation og øget udvidelse af adoptivt overførte celler 30. Endvidere kan lymphodepletive virkninger af TMZ blive sammenfattet af lav-dosis (4 - 6 Gy) single-fra...

Glioblastoma (GBM) er den mest almindelige primære maligne hjernetumor og er uvægerligt dødelig. Kirurgisk resektion kombineret med ikke-specifik standard pleje kemoterapi og strålebehandling ikke helt at eliminere maligne celler, hvilket resulterer i en trist prognose på mindre end 15 måneder hos patienter med denne sygdom 1. I modsætning hertil immunterapi tilbyder en præcis fremgangsmåde til specifikt at målrette tumorcellerne og således har potentiale til at tjene som en yderst effektiv behandling platform med reduceret risiko for sikkerhed toksicitet 2-4. T-celler manipuleret ex vivo til at udtrykke kimære antigen-receptorer (biler) tilbyder en alsidig strategi for tumor immunterapi. Biler er genereret ved sammensmeltning af det ekstracellulære variable region af et antistof med en eller flere intracellulære T-celle signalering molekyle (r), i stedet for en fuld-længde større histokompatibilitetskompleks (MHC) -begrænset T-cellereceptor 5. Denne tilstand af antistof-lignende antigen recognitipå tillader reaktive antigenspecifikke T-celler til at genkende og reagere på tumorantigener i fravær af MHC, og kan tilpasses til en praktisk talt uendelig antigen repertoire. CAR T-celler manipuleret mod forskellige tumorantigener har vist præklinisk effektivitet og enestående løfte i klinikken 6-9. Specifikt i forbindelse med GBM, en målretning CAR T-celle platform epidermal vækstfaktor receptor variant III (EGFRvlll), en tumor-specifik mutation udtrykt på celleoverfladen 10, blev vist at forlænge overlevelsen i gliom-bærende mus 11. På trods af deres alsidighed, men den kliniske fordel af CAR adoptiv behandling er ikke blevet realiseret fuldt ud, dels på grund af tumor-associerede immunosuppression og immun unddragelse 12-16 samt udfordringer i at etablere og opretholde antigen-specifikke T-celler in vivo. Udnyttelse standardbehandling (SOC) med immunterapi potentielt kan overvinde flere af disse lefterligninger, hvilket resulterer i øget effektivitet i både den prækliniske og kliniske omgivelser. SOC for post-resektion GBM består af højdosis temozolomid (TMZ), et DNA-alkyleringsmiddel 17, og hele hjernen bestråling (WBI) 1. Disse behandlinger formodes at virke synergistisk med tumorvacciner via opregulering af tumor MHC-ekspression 18-20 samt afgivelse af antigener ved døde tumorceller 17,19,21,22. Faktisk tilsætning af TMZ 20,23 eller WBI 18,24 fører til øget antitumorvirkning af immun-baserede behandlinger i prækliniske indstilling. Desuden, ligesom mange uspecifikke cytotoksiske kemoterapeutika, TMZ kendt for at forårsage systemisk lymfopeni 25,26, som kan udnyttes som et middel til host-conditioning til adoptiv terapi platforme 27-29. TMZ-medieret lymphodepletion har vist sig at forbedre frekvensen og funktion af antigen-specifikke T-celler, hvilket fører til forøget effektivitet af en vedtative terapi platform mod intrakranielle tumorer 30. I forbindelse med CAR terapi, lymphodepletion tjener som et middel til host-konditionering ved både at reducere antallet af endogene suppressor-T-celler 31 og inducere homeostatiske proliferation 32 via reduceret konkurrence om cytokiner 33 og dermed øge antitumoraktivitet 11,34. På grund af den synergistiske forhold mellem GBM SOC og immunterapi platforme, evaluere nye adoptivforældre behandlingsformer og vaccine-platforme i forbindelse med SOC er kritisk for udarbejdelsen meningsfulde konklusioner om effekten. I denne protokol, vi skitsere en metode til generering og intravenøs administration af murine EGFRvlll-specifikke CAR T-celler sammen TMZ og WBI i mus med EGFRvIII-positive intrakranielle tumorer (se figur 1 for behandling tidslinje). Kort fortalt CAR T-celler fremstillet ex vivo ved retroviral transduktion. Humane embryoniske nyre (HEK) 293T celler transficeres under anvendelse af en DNA / lipid-komplekset (indeholdende CAR vektor og PCL-Eco plasmider) for at fremstille virus, der derefter anvendes til at transducere aktiverede murine splenocytter, der er høstet og dyrket i parallel. I løbet af CAR generation, er murine værter bærer EGFRvlll-positive intrakranielle tumorer indgives fraktioneret hel-hjerne røntgenbestråling og systemisk TMZ behandling ved doser svarende til klinisk SOC. CAR T-celler leveres derefter intravenøst ​​til lymphodepleted værter. Følgende procedure er beskrevet i syv separate faser: (1) Indgivelse af temozolomid til tumorbærende mus, (2) hele hjernen Bestråling af tumorbærende mus, (3) transfektion (4) splenektomi og T-celle Preparation (5 ) transduktion, (6) CAR T-celle kultur og Harvest, og (7) CAR T-celle administration til tumorbærende mus. Disse faser består af flere trin, der strækker sig 6-7 dage og udføres samtidig.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Name of Material</td>
			<td style="width:172px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:449px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCL-Eco Retrovirus Packaging Vector</td>
			<td>Imgenex</td>
			<td>10045P</td>
			<td>Helper vector for generating CAR retrovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Concanavalin A</td>
			<td>Sigma Aldrich</td>
			<td>C2010</td>
			<td>Non-specific mitogen to induce T cell proliferation and viral transduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retronectin</td>
			<td>ClonTech/Takara</td>
			<td>T100B</td>
			<td>Facilitates retroviral transduction of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>HEK293 culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Life technologies</td>
			<td>11058-021</td>
			<td>Transfection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM L-Glutamine&#160;</td>
			<td>Life technologies</td>
			<td>25030-081</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Sodium Pyruvate</td>
			<td>Life technologies</td>
			<td>11360-070</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Life technologies</td>
			<td>11140-050</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">55 mM 2-Mercaptoethanol&#160;</td>
			<td>Life technologies</td>
			<td>21985-023</td>
			<td>Reducing agent to remove free radicals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin (50 mg/mL)&#160;</td>
			<td>Life technologies</td>
			<td>15750-060</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GemCell U.S. Origin Fetal Bovine Serum</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Provides growth factors and nutrients for in vitro cell growth&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA), Fraction V&#8212;Standard&#160;Grade&#160;</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pharm Lyse (10X concentrate)&#160;</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm BioCoat Culture Dishes with Poly-D-Lysine&#160;</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temozolomide</td>
			<td>Best Pharmatech</td>
			<td>N/A</td>
			<td>Lyophilized powder prepared on the day of administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL&#160;</td>
		</tr>
	</tbody>
</table>

generation of car t cells for adoptive therapy in the context of glioblastoma standard of care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

CAR T-celler dannes ved transduktion med EGFRvlll CAR retrovirusvektor 11. Denne vektor, MSGV1, blev udviklet fra SFGtcLuc_ITE4 vektor 35, som indeholder den murine stamcelle virus (MSCV) lange terminale gentagelser, den udvidede gag regionen og kuvert splejsningssted (splejsningsdonor, SD, og splejsningsacceptor, SA), og viral emballage signal (ψ). Den EGFRvlll CAR indeholdende det humane anti-EGFRvlll variable fragment enkelt kæde (scFv) 139, i takt med murine CD8TM, CD28, 4-1BB og CD3ζ intrac...

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Generation af CAR T-celler til adoptiv terapi i forbindelse med glioblastom standard for pleje

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

Generering af inducerede regulatoriske T-celler fra primære humane naive og memory T-celler

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Ændring af primære humane T-celler

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Accelerated Type 1 Diabetes Induktion i mus ved adoptiv overførsel af diabetogene CD4 + T-celler

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Vurdere udviklingen i murine Plasmacytoid dendritiske celler i Peyer&#39;ske Patches Brug adoptiv overførsel af Hematopoietic Progenitorer

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Generation af human alloantigen-specifikke T-celler fra perifert blod

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

Automatiseret Cell Berigelse af cytomegalovirus-specifikke T-celler til kliniske applikationer ved hjælp af Cytokin-capture system

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...