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.

Questo protocollo si basa su un disegno sperimentale in cui 10 topi vengono trattati con 10 7 CAR cellule T ciascuno. Ciò significa che saranno necessari 10 8 cellule CAR T; il rendimento dovrebbe essere sovrastimata da 5 x 10 7 -1 x 10 8 per conto per la perdita di redditività. Il protocollo che segue viene scalato per generare circa 200 x 10 6 cellule. Le cellule sono poi somministrati per via endovenosa al femminile C57BL / 6 topi con 9 giorni stabiliti singenici tumori intracranici EGFRvIII-positivo, sviluppato dalle linee esistenti di cellule di melanoma KR158B astrocitoma o B16. In concomitanza con il CAR generazione di cellule T, i topi di tumore vengono somministrati dosi clinicamente rilevanti di lymphodepletive TMZ (60 mg / kg) e WBI (16.5 Gy). I topi sono stati mantenuti e allevati in condizioni esenti da organismi patogeni della Duke University Medical Center (DUMC). Tutti gli esperimenti sugli animali sono stati eseguiti secondo protocolli approvati dalla Duke University Istituzioneal Animal Care and Use Committee (IACUC). 1. La somministrazione di Temozolomide per Mice tumore-cuscinetto Giorni 0 a 4: <ol><li> Calcolare il numero totale di topi da iniettare e moltiplicare tale da 0,5 a determinare il volume di soluzione TMZ necessario (ad esempio, 30 topi x 0.5 ml = 15 ml TMZ) e aggiungere 1,5 ml di volume per tenere conto di ricaduta (16.5 ml TMZ ). </li><li> Moltiplicate questo volume totale da 3 mg / ml per determinare il peso di liofilizzato TMZ necessario (ad esempio, 16,5 x 3 = 49.5 mg TMZ). Pesare questo in una conica da 50 ml. ATTENZIONE: TMZ è tossico per inalazione, ingestione e contatto con gli occhi, la pelle e le membrane mucose. Consultare la sicurezza dell&#39;istituzione del lavoro e / o di salute ambientale e il reparto wellness per le raccomandazioni in materia di riduzione del rischio di esposizione. </li><li> Moltiplicare il volume totale di 0,15 per determinare il volume di dimetilsolfossido (DMSO) necessaria (ad esempio, 16,5 x0,15 = 2.475 ml DMSO). Filtro sterilizzare questo volume di DMSO (più altri 2 ml per spiegare ricaduta) attraverso un filtro da 0,2 micron in una provetta sterile. Aggiungere 2,475 ml di DMSO sterile per la polvere TMZ. </li><li> Posizionare la soluzione TMZ / DMSO in un becher di acqua su una piastra calda a 65 ° C per 10-15 min. Una volta TMZ è completamente sciolto, la soluzione dovrebbe diventare colore giallo paglierino; se la soluzione diventa rosa, allora il TMZ è degradata, e una nuova soluzione dovrebbe essere preparata con un nuovo sacco di TMZ. </li><li> Calcolare il volume appropriato di soluzione salina sterile necessario moltiplicando 0,85 per il volume totale (0,85 x 16,5 ml = 14,025 ml di soluzione salina). Aggiungere 15 ml di soluzione salina sterile per un conica da 50 ml. Mentre TMZ sta dissolvendo in DMSO, saline di calore a 65 ° C. </li><li> Vortex la soluzione / DMSO TMZ per garantire che la polvere TMZ è completamente sciolto e aggiungere lentamente salina riscaldata alla soluzione / DMSO TMZ. </li><li> Immediatamente dopo la preparazione, caricare completamente 15 x 1 ml tubercolina syringes con la soluzione TMZ per l&#39;amministrazione di quattro giorni stabiliti tumorali cuscinetto topi. </li><li> Ripetere questa procedura nei giorni da 1 a 4 per un totale di cinque amministrazioni TMZ. </li></ol> 2. Whole Brain irradiazione dei topi tumore-cuscinetto Giorni 2 a 4: <ol><li> Accendere il irradiatore X-ray e lasciarlo riscaldare-fino a piena tensione. Assicurarsi che il filtro appropriato è a posto e ingresso la tensione corretta e le impostazioni correnti (Figura 2a). NOTA: La dosimetria del irradiatore andrà stabilita anticipatamente per stabilire i provvedimenti impostazioni di tensione e layout a griglia (figura 2A, B). </li><li> Calcolare la lunghezza di tempo che è necessario per provocare 5,5 Gy X-ray irradiazione. Ad esempio, se i raggi X vengono forniti ad una velocità di 2 Gy / min, poi 2,75 min è necessario fornire un totale di 5,5 Gy. Inserire la durata di tempo adeguato. NOTA: La tabella 2 fornisce il mouse WBI dosa unnd loro equivalenti clinico nell&#39;uomo. Nel contesto di gliomi ad alto grado, 60 Gy frazionata WBI (2 frazioni Gy, 5 giorni / settimana per 6 settimane) è lo standard clinico di cura, e l&#39;equivalente murino utilizzato è 16,5 Gy (5,5 Gy x 3). </li><li> Preparare una soluzione fresca di ketamina / xylazina per anestesia sistemica aggiungendo 2 ml di ketamina e 1 ml a 17 ml xilazina soluzione salina in modo che la soluzione finale è di 10 mg / ml di ketamina e 1 mg / ml xylazina. </li><li> Pesare topi e amministrare 10 ml di ketamina / xilazina intraperitoneale per grammo di peso corporeo tale che gli animali ricevono una dose di ketamina a 100 mg / kg e 10 mg / kg xilazina. I topi dovrebbe essere pienamente sedato e visibilmente la respirazione entro 2 minuti di somministrazione / xylazina ketamina. </li><li> Per mantenere l&#39;umidità oftalmica negli animali sedati, strofinare delicatamente una piccola quantità di lacrime artificiali unguento su ciascun occhio. </li><li> Inserire topi sedati sulla griglia in modo tale che le teste sono posizionati nella zona che riceve il massimo raggi X intensity (Figura 2B, C). Corpi Shield dal collo in giù, con opportunamente dimensionate tubi di piombo per bloccare la consegna a raggi X sistemica (Figura 2D). </li><li> Inserire topi posizionati sotto il fascio di raggi X in modo che il laser indica il punto focale del fascio di raggi X è al di coordinate (0,0) sul layout della griglia. Inizia consegna X-ray. </li><li> Quando la consegna a raggi X, rimuovere gli animali dal irradiatore e posto su una piastra elettrica caldo. Non accogliere animali sedati con animali coscienti fino animali hanno ripreso conoscenza sufficiente per mantenere prona sternale. Una volta che gli animali possono mantenere prona sternale, posizionare animali coscienti nel loro gabbie appropriate. </li><li> Ripetere questa procedura nei giorni 3 e 4 per un totale di tre dosi frazionate di radiazioni. </li></ol> 3. Trasfezione -1 ° giorno: <ol><li> Preparare supporti D10 aggiungendo 50 ml di siero bovino fetale (FBS) a 500 ml di Dulbecco Modified (DMEM). </li><li> Preparare supporti di cellule T (TCM) con l&#39;aggiunta di 5,5 ml di L-glutammina, 5,5 ml di piruvato di sodio, 5,5 ml aminoacidi non essenziali, e 5.5 ml Pencillin / di streptomicina (pen / strep) a 500 supporti ml RPMI-1640. Avanti, aggiungere 550 ml di 2-mercaptoetanolo e 550 ml gentamicina. Infine, aggiungere 50 ml di FBS. </li><li> Harvest vitro cellule in coltura HEK293T e portare a una concentrazione di 7,5 x 10 6 cellule / ml nei media D10. </li><li> La tavola 10 ml supporti D10 e aggiungere 1 ml di sospensione cellulare HEK293T in ciascuno di 16 x 10 centimetri poli-D-lisina (PDL) piastre rivestite. </li><li> Incubare per una notte a 37 ° C con 5% di CO 2. </li></ol> Giorno 0: <ol start="6"><li> Sostituire i media con 10 ml D10 fresco almeno 30 minuti prima di trasfezione le cellule. </li><li> Determinare la quantità di vettore plasmidico, PCL-Eco vettoriale, e liposomiale trasfezione reagente necessario per trasfezione moltiplicando il numero totale di piatti + 1 (16 + 1 = 17) 14.1 mgplasmide vettore (14.1 x 17 = 239,7 mg), 9.9 mg PCL-Eco vettore (9,9 x 17 = 168,3 mg), e 60 ml di reagente liposomiale trasfezione (60 x 17 = 1.020 ml). Tutti i reagenti devono essere a temperatura ambiente prima di preparare soluzioni. </li><li> Etichettare due provette A e B. In tubo A, aggiungere 239,7 mcg vettore plasmidico e 168,3 mg PCL-Eco vettore a (1,5 x 17) ML-siero ridotto modificato mezzo di Eagle (RS-MEM). Nel tubo B, aggiungere 1020 ml liposomiale trasfezione reagente (1,5 x 17) ml RS-MEM. </li><li> Incubare A e B separatamente per 5 min a temperatura ambiente. </li><li> Mescolare A e B insieme delicatamente (vortex per 1-2 sec capovolgere varie volte) e incubare per 20 min a temperatura ambiente per formare lipidi complessi / DNA. </li><li> Aggiungere lipidi / DNA complesso goccia a goccia per le cellule HEK293T a 10 centimetri piatto. </li><li> Incubare a 37 ° C per 6-8 ore o durante la notte (non superare 24 ore). </li><li> Dopo 6-8 ore di incubazione, sostituire medie con 12 ml di TCM fresco per la produzione virale. Questo placcato supportisaranno utilizzati al Day 2 come surnatante virale per la trasduzione di cellule T. </li></ol> 4. La splenectomia e preparazione delle cellule T Giorno 0: <ol><li> Versare 10 ml di TCM in una conica da 50 ml e posto in ghiaccio per la raccolta della milza. </li><li> Sacrifica il numero appropriato di animali da CO 2 asfissia e decapitazione secondario: Posizionare gli animali in gabbia ricevere CO 2 ad una portata di 10-30% gabbia volume / minuto, secondo le linee guida American Veterinary Medical Association (non più di 5 animali possono essere sacrificato contemporaneamente) fino termina respirazione e per due minuti successivi. Rimuovere gli animali dalla camera di CO 2 e di decapitarlo. NOTA: Uno spleen di un 6-12 settimane vecchia femmina C57BL / 6 del mouse produrrà circa 4,5-5 x 10 7 splenociti. Qui, 4 milze saranno raccolte per circa 200 x 10 6 cellule. </li><li> Posare il mouse in modo che il suo lato destro rivolto verso l&#39;alto, espruzzare con il 70% di etanolo. Con le pinze, afferrare un sottile lembo di pelle sotto la gabbia toracica sinistra e tagliare una leggera incisione con le forbici. Rimuovere la pelle, afferrare con cura un sottile piega del peritoneo con una pinza, e tagliare una piccola cavità. </li><li> La milza è un piccolo, allungato, organo rosso scuro che ricorda un fagiolo appiattita; delicatamente afferrare la milza con le pinze e le accise tagliando via il tessuto connettivo circostante. Posizionare le milze asportati nel conica contenente 10 ml di TCM su ghiaccio. </li><li> Versare milze oltre un filtro 70 micron cella maglie e disaggregare da schiacciare con l&#39;estremità smussata della parte interna di una siringa da 5 ml per generare una cella singola sospensione. Un massimo di due milze dovrebbe essere disaggregate per filtro a rete. </li><li> Utilizzare un piccolo volume di TCM di lavare accuratamente il filtro seguente disaggregazione per raccogliere eventuali splenociti rimanenti. Piscina tutto milze disaggregati in una cella singola sospensione. Portare il volume finale di 50 ml con TCM per un lavaggio did girare a 300 xg per 10 min. </li><li> Preparare una soluzione di tampone di lisi 1x aggiungendo 5 ml 10x tampone di lisi a 45 ml di acqua sterile. Per eliminare i globuli rossi, risospendere pellet in 5 ml di 1x tampone di lisi per milza in una conica da 50 ml, mescolando bene pipettando gentilmente su e giù. Se superiore a 5 milze, utilizzare un tubo da centrifuga da 250 ml. Inserire conica o tubo da centrifuga in un bagno d&#39;acqua 37 ° per 5 minuti. </li><li> Rimuovere la reazione lisi dal bagno di acqua e aggiungere TCM in un rapporto 1: 1 con tampone di lisi per neutralizzare la reazione. Lavare facendo girare a 300 xg per 10 min. </li><li> Aspirare il surnatante e pellet completamente risospendere in MTC (2 ml / milza, 8 ml totale per 4 milza) pipettando su e giù. </li><li> Contare le cellule con l&#39;aggiunta di 10 ml di sospensione cellulare a 190 ml blu trypan (1:20 diluizione). Moltiplicare il numero ottenuto in uno dei quattro quadrati reticolati da 20 x 10 x 4 volume totale (8 ml) per ottenere il numero totale di cellule (ad esempio, 125 cellule x 20 x 10 4 </sup&gt; x 8 ml = 200 x 10 6 splenociti). </li><li> Diluire le cellule ad una concentrazione di 2 x 10 6 cellule / ml in TCM, supplementato con 2 ug / ml concanavalina A (ConA) e 50 UI / ml ricombinante umano interleuchina-2 (rhIL-2). Così, per 200 x 10 6 splenociti, aggiungere 92 ml di media per 8 ml cellule, 200 mcg Cona e 5.000 UI rhIL-2. </li><li> Aggiungere 2 mL cellule in ciascun pozzetto 24 pozzetti tessuto-cultura piastre trattati, tale che 4 x 10 6 cellule sono in ogni pozzetto (es, 100 ml di cellule richiederanno circa 4 piastre). </li><li> Incubare per una notte a 37 ° C con 5% di CO 2. </li></ol> 5. Trasduzione 1 ° giorno: <ol><li> Calcolare il numero della cultura non-tessuto trattato piastre da 24 pozzetti necessari per la trasduzione moltiplicando il numero di milza raccolte da 2 (8 piastre sono necessari per 4 milza). </li><li> Successivamente, calcolare il volume richiesto di soluzione ricombinante frammento fibronectina umana (RHFF)necessario per lastre cappotto moltiplicando (numero totale di pozzi + 3) x 0,5 ml (8 piastre x 24 pozzi = 192 + 3 = 195) x 0,5 ml = 97,5 ml. </li><li> Preparare 97,5 ml di soluzione salina tamponata con fosfato (PBS) contenente RHFF ad una concentrazione di 25 ug / ml moltiplicando il volume totale da 25 mg (97,5 x 25 = 2437,5 mg). Aggiungi questa quantità di RHFF a 97,5 ml di PBS. </li><li> Coat coltura non-tessuto trattato piastre da 24 pozzetti aggiungendo 0,5 ml di soluzione PBS / RHFF per pozzetto. </li><li> Incubare una notte a 4 ° C. </li></ol> 2 ° giorno: <ol start="6"><li> Dump soluzione PBS / RHFF dalla cultura non-tessuto trattato piastre da 24 pozzetti. </li><li> Aggiungere 1 ml / pozzetto di albumina sierica bovina 2% (BSA) in PBS e incubare a temperatura ambiente per 30 min. </li><li> Rimuovere BSA saldamente ribaltare piatto. Lavare con l&#39;aggiunta di 2 ml di PBS. </li><li> Raccogliere surnatante virale trasferendo file multimediali da ogni HEK293T PDL piastra in una provetta da 250 ml centrifuga e spin 10 min a 500 x g. </li><li> Trasferire accuratamente su viralepernatant in un fresco 250 ml tubo da centrifuga, assicurandosi di non disturbare il pellet che possono essersi formate. </li><li> Aggiungere TCM fresco al surnatante virale tale che il volume finale è 3 ml più che la quantità necessaria per pozzi RHFF rivestite. Ad esempio, per 192 pozzetti RHFF rivestite, portare il volume del surnatante virale a 192 + 3 ml = 195 ml. </li><li> Rimuovere splenociti coltivate dal incubatore e risospendere pipettando gentilmente su e giù 2 - 3 volte in ogni pozzetto. Trasferire in un conica 250 ml. Se si utilizza una pipetta multicanale, con un serbatoio sterile prima del trasferimento contribuirà ad accelerare questo passaggio. Conta come descritto in precedenza e far girare a 300 xg per 10 min. </li><li> Aggiungere rhIL-2 per surnatante virale ad una concentrazione di 50 UI / ml. Ad esempio, aggiungere 50 x 195 = 9.750 UI a 195 ml di surnatante virale-2 rhIL. </li><li> Risospendere splenociti di surnatante virale a 1 x 10 6 cellule / ml </li><li> Aggiungere 1 ml / pozzetto di sospensione splenocyte a piastre da 24 pozzetti RHFF rivestite. </li><li>Spin per 90 min in base alle seguenti impostazioni: 770 xg, accelerazione = 4, freno / decelerazione = 0, 32 ° C. </li><li> Preparare una soluzione TCM con 50 UI / ml rhIL-2. Ad esempio, preparare una soluzione 200 TCM aggiungendo 10.000 UI rhIL-2. Aggiungere 1 ml di rhIL-2 / TCM a ciascun pozzetto dopo centrifugazione. </li><li> Culture notte a 37 ° C in 5% CO 2. </li></ol> 6. CAR T cellule Cultura e Harvest Giorni 3 e 4: <ol><li> Se le cellule T raggiungono&gt; 80% di confluenza, le cellule possono essere divise (questo di solito avviene il giorno 3 o 4 giorni). Per dividere le celle, delicatamente pipettare su e giù in ciascun pozzetto 2-3 volte, e passare 1 ml da ogni pozzetto in nuovi pozzetti di una 24-pozzetti trattati tessuto-coltura fresco. Quindi, aggiungere 1 ml di TCM fresca con 50 UI / ml di IL-2 in ciascun pozzetto tale che il volume finale di 2 ml in tutti i pozzetti. </li><li> Se le cellule non raggiungono&gt; 80% di confluenza, effettuare un cambiamento mezza mezzi lentamente pipettando off 1 ml del supporto dalla parte superiore di ciascun pozzetto. Evitare disturbing cellule insediate sul fondo durante la rimozione dei media. Aggiungere 1 ml di TCM fresco contenente 50 UI / ml rhIL-2. </li></ol> 5 ° giorno: <ol start="3"><li> Risospendere le cellule T CAR pipettando gentilmente su e giù per 3 volte in ogni pozzetto e il trasferimento ad un tubo da centrifuga da 250 ml. Spin cellule a 300 xg per 10 min. </li><li> Surnatante completamente aspirate senza disturbare pellet. Risciacquare una volta con PBS, contare le cellule come precedentemente descritto, e lavare con PBS una seconda volta. Una resa di cellule CAR T tipico è di circa 1 x 10 6 cellule per bene (8 piastre cellule x 24 pozzi = 192 x 10 6 CAR T). </li><li> Risospendere le cellule in PBS ad una concentrazione di 5 x 10 7 / ml per una iniezione di 1 x 10 7 in un volume di 200 mg (per esempio, per 192 x 10 6 cellule T CAR, risospendere pellet lavate in PBS 3,84 ml). </li></ol> 7. CAR T cellule Administration per Mice tumore fruttiferi 5 ° giorno: <ol><li> Transpocellule rt sul ghiaccio per stabulario per iniezione endovenosa. Load 500-1000 ml in siringa da insulina con 27-31 G ago, assicurando che tutte le bolle vengono espulsi. </li><li> Prendete un mouse alla base della coda e mettere in fermo tubi. </li><li> Estrarre la coda tesa con la vena rivolto verso l&#39;alto, e scivolare l&#39;ago di circa 1-2 mm sotto la pelle nella vena inserendolo parallelo alla vena della coda. Lentamente espellere 200 microlitri nella vena. Se l&#39;ago è correttamente inserito nella vena, il volume sarà facile flusso in; altrimenti ci sarà la resistenza e l&#39;ago dovranno essere rimossi dalla coda e ri-inserito correttamente. </li></ol>

<ol>
	<li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N Engl J Med. 352 (10), 987-996 (2005).</li><li>Kantoff, P., Higano, C., Shore, N., Berger, E., Small, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sipuleucel-T+immunotherapy+for+castration-resistant+prostate+cancer.">Sipuleucel-T immunotherapy for castration-resistant prostate cancer.</a> N Engl J Med. (363), 411-422 (2010).</li><li>Hodi, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+survival+with+ipilimumab+in+patients+with+metastatic+melanoma.">Improved survival with ipilimumab in patients with metastatic melanoma.</a> N Engl J Med. 363 (8), 711-723 (2010).</li><li>Schwartzentruber, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gp100+peptide+vaccine+and+interleukin-2+in+patients+with+advanced+melanoma.">gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma.</a> N Engl J Med. 364 (22), 2119-2127 (2011).</li><li>Gross, G., Gorochov, G., Waks, T., Eshhar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+effector+T+cells+expressing+chimeric+T+cell+receptor+with+antibody+type-specificity.">Generation of effector T cells expressing chimeric T cell receptor with antibody type-specificity.</a> Transplant Proc. 21 (1 Pt 1), 127-130 (1989).</li><li>Pule, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+T+cells+engineered+to+coexpress+tumor-specific+receptors:+persistence+and+antitumor+activity+in+individuals+with+neuroblastoma.">Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma.</a> Nat Med. 14 (11), 1264-1270 (2008).</li><li>Kochenderfer, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+depletion+and+remissions+of+malignancy+along+with+cytokine-associated+toxicity+in+a+clinical+trial+of+anti-CD19+chimeric-antigen-receptor-transduced+T+cells.">B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.</a> Blood. 119 (12), 2709-2720 (2012).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+in+chronic+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.</a> N Engl J Med. 365 (8), 725-733 (2011).</li><li>Brentjens, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+persistence+of+adoptively+transferred+autologous+CD19-targeted+T+cells+in+patients+with+relapsed+or+chemotherapy+refractory+B-cell+leukemias.">Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.</a> Blood. 118 (18), 4817-4828 (2011).</li><li>Wikstrand, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+against+EGFRvIII+are+tumor+specific+and+react+with+breast+and+lung+carcinomas+and+malignant+gliomas.">Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.</a> Cancer Res. 55 (14), 3140-3148 (1995).</li><li>Sampson, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFRvIII+mCAR-modified+T-cell+therapy+cures+mice+with+established+intracerebral+glioma+and+generates+host+immunity+against+tumor-antigen+loss.">EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss.</a> Clin Cancer Res. 20 (4), 972-984 (2014).</li><li>Kuppner, M. C., Hamou, M. F., Sawamura, Y., Bodmer, S., de Tribolet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+lymphocyte+function+by+glioblastoma-derived+transforming+growth+factor+beta+2.">Inhibition of lymphocyte function by glioblastoma-derived transforming growth factor beta 2.</a> J Neurosurg. 71 (2), 211-217 (1989).</li><li>Wintterle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+B7-related+molecule+B7-H1+by+glioma+cells:+a+potential+mechanism+of+immune+paralysis.">Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis.</a> Cancer Res. 63 (21), 7462-7467 (2003).</li><li>Fecci, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+regulatory+T-cell+fraction+amidst+a+diminished+CD4+compartment+explains+cellular+immune+defects+in+patients+with+malignant+glioma.">Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains cellular immune defects in patients with malignant glioma.</a> Cancer Res. 66 (6), 3294-3302 (2006).</li><li>Wilmotte, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-homolog+1+expression+by+human+glioma:+a+new+mechanism+of+immune+evasion.">B7-homolog 1 expression by human glioma: a new mechanism of immune evasion.</a> Neuroreport. 16 (10), 1081-1085 (2005).</li><li>Yang, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediation+of+enhanced+transcription+of+the+IL-10+gene+in+T+cells,+upon+contact+with+human+glioma+cells,+by+fas+signaling+through+a+protein+kinase+A-independent+pathway.">Mediation of enhanced transcription of the IL-10 gene in T cells, upon contact with human glioma cells, by fas signaling through a protein kinase A-independent pathway.</a> Journal of Immunology. 171 (8), 3947-3954 (2003).</li><li>Reilly, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temozolomide:+a+new+oral+cytotoxic+chemotherapeutic+agent+with+promising+activity+against+primary+brain+tumours.">Temozolomide: a new oral cytotoxic chemotherapeutic agent with promising activity against primary brain tumours.</a> Eur J Cancer. 29A (7), 940-942 (1993).</li><li>Newcomb, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Combination+of+Ionizing+Radiation+and+Peripheral+Vaccination+Produces+Long-term+Survival+of+Mice+Bearing+Established+Invasive+GL261Gliomas.">The Combination of Ionizing Radiation and Peripheral Vaccination Produces Long-term Survival of Mice Bearing Established Invasive GL261Gliomas.</a> Clin Cancer Res. 12, 4730-4737 (2006).</li><li>Park, B., Yee, C., Lee, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+radiation+on+the+immune+response+to+cancers.">The effect of radiation on the immune response to cancers.</a> Int J Mol Sci. 15 (1), 927-943 (2014).</li><li>Fritzell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+temozolomide+synergizes+with+immunotherapy+in+a+T+cell-dependent+fashion.">Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion.</a> Cancer Immunol Immunother. 62 (9), 1463-1474 (2013).</li><li>Park, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+by+temozolomide+enhances+antitumor+immunity+of+dendritic+cell+vaccination+in+murine+brain+tumor+model.">Cross-priming by temozolomide enhances antitumor immunity of dendritic cell vaccination in murine brain tumor model.</a> Vaccine. 25 (17), 3485-3491 (2007).</li><li>Emens, L. A., Jaffee, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leveraging+the+activity+of+tumor+vaccines+with+cytotoxic+chemotherapy.">Leveraging the activity of tumor vaccines with cytotoxic chemotherapy.</a> Cancer Res. 65 (18), 8059-8064 (2005).</li><li>Murphy, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+immunotherapy+screen+of+costimulatory+molecules+identifies+Fc-OX40L+as+a+potent+reagent+for+the+treatment+of+established+murine+gliomas.">An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas.</a> Clin Cancer Res. 18 (17), 4657-4668 (2012).</li><li>Newcomb, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+enhances+antitumor+effect+of+anti-CD137+therapy+in+a+mouse+Glioma+model.">Radiotherapy enhances antitumor effect of anti-CD137 therapy in a mouse Glioma model.</a> Radiat Res. 173 (4), 426-432 (2010).</li><li>Su, Y. B., Krown, S. E., Livingston, P. O., Wolchok, J. D., Chapman, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lymphotoxic+is+dose-intensified+temozolomide?+The+glioblastoma+experience.">How lymphotoxic is dose-intensified temozolomide? The glioblastoma experience.</a> J Clin Oncol. 23 (18), 4235-4236 (2005).</li><li>Neyns, B., Tosoni, A., Hwu, W. J., Reardon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dense+temozolomide+regimens:+antitumor+activity,+toxicity,+and+immunomodulatory+effects.">Dose-dense temozolomide regimens: antitumor activity, toxicity, and immunomodulatory effects.</a> Cancer. 116 (12), 2868-2877 (2010).</li><li>Muranski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+intensity+lymphodepletion+and+adoptive+immunotherapy-how+far+can+we+go.">Increased intensity lymphodepletion and adoptive immunotherapy-how far can we go.</a> Nat Clin Pract Oncol. 3 (12), 668-681 (2007).</li><li>Wrzesinski, C., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Less+is+more:+lymphodepletion+followed+by+hematopoietic+stem+cell+transplant+augments+adoptive+T-cell-based+anti-tumor+immunotherapy.">Less is more: lymphodepletion followed by hematopoietic stem cell transplant augments adoptive T-cell-based anti-tumor immunotherapy.</a> Current Opinion in Immunology. 17 (2), 195-201 (2005).</li><li>Dudley, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+therapy+for+patients+with+metastatic+melanoma:+evaluation+of+intensive+myeloablative+chemoradiation+preparative+regimens.">Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens.</a> J Clin Oncol. 26 (32), 5233-5239 (2008).</li><li>Sanchez-Perez, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloablative+Temozolomide+Enhances+CD8(+)+T-Cell+Responses+to+Vaccine+and+Is+Required+for+Efficacy+against+Brain+Tumors+in+Mice.">Myeloablative Temozolomide Enhances CD8(+) T-Cell Responses to Vaccine and Is Required for Efficacy against Brain Tumors in Mice.</a> Plos One. 8 (3), (2013).</li><li>Su, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+CD4++lymphopenia+in+melanoma+patients+treated+with+temozolomide:+a+toxicity+with+therapeutic+implications.">Selective CD4+ lymphopenia in melanoma patients treated with temozolomide: a toxicity with therapeutic implications.</a> J Clin Oncol. 22 (4), 610-616 (1200).</li><li>Dummer, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+homeostatic+proliferation+elicits+effective+antitumor+autoimmunity.">T cell homeostatic proliferation elicits effective antitumor autoimmunity.</a> J Clin Invest. 110 (2), 185-192 (2002).</li><li>Gattinoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+homeostatic+cytokine+sinks+by+lymphodepletion+enhances+the+efficacy+of+adoptively+transferred+tumor-specific+CD8++T+cells.">Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells.</a> J Exp Med. 202 (7), 907-912 (2005).</li><li>Wrzesinski, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+intensity+lymphodepletion+enhances+tumor+treatment+efficacy+of+adoptively+transferred+tumor-specific+T+cells.">Increased intensity lymphodepletion enhances tumor treatment efficacy of adoptively transferred tumor-specific T cells.</a> J Immunother. 33 (1), 1-7 (2010).</li><li>Hughes, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+a+TCR+gene+derived+from+a+patient+with+a+marked+antitumor+response+conveys+highly+active+T-cell+effector+functions.">Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.</a> Hum Gene Ther. 16 (4), 457-472 (2005).</li><li>Morgan, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+glioma+stem+cells+by+genetically+modified+T+cells+targeting+EGFRvIII+and+development+of+adoptive+cell+therapy+for+glioma.">Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.</a> Hum Gene Ther. 23 (10), 1043-1053 (2012).</li><li>Kerkar, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+murine+CD8++and+CD4++T+cells+for+preclinical+adoptive+immunotherapy+studies.">Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.</a> J Immunother. 34 (4), 343-352 (2011).</li></ol>

The authors have no conflicts of interests to declare.

La timeline trattamento qui descritto è stato progettato per modello standard clinico di cura e di sfruttare i suoi effetti per l&#39;automobile la terapia adottiva. AUTO dosi T cellulari, regimi di TMZ, e l&#39;amministrazione radioterapia possono essere modificati per aumentare in vivo l&#39;attività delle cellule T, lymphodepletion, e tumore uccisione. Regimi TMZ può essere aumentata per produrre mieloablazione host e una maggiore espansione delle cellule adoptively trasferiti 30. Inoltre, gli ...

Glioblastoma (GBM) è il tumore maligno al cervello primario più comune ed è invariabilmente fatale. La resezione chirurgica accoppiato con standard di non-specifica delle cure di chemioterapia e radioterapia non riesce ad eliminare completamente le cellule maligne, con conseguente prognosi infausta di meno di 15 mesi in pazienti affetti da questa malattia 1. Al contrario, l&#39;immunoterapia offre un metodo preciso per specifico le cellule tumorali, e quindi ha il potenziale per servire come piattaforma trattamento altamente efficace con un ridotto rischio di tossicità garanzie 2-4. Le cellule T progettati ex vivo per esprimere i recettori chimerici antigene (auto) offrono una strategia versatile per tumore immunoterapia. CAR vengono generati fondendo la regione variabile extracellulare di un anticorpo con la molecola di segnalazione uno o più cellule T intracellulare (s), al posto di un integrale complesso maggiore di istocompatibilità (MHC) -restricted recettore delle cellule T 5. Questo modo di anticorpi come att antigeneon permette cellule T antigene-specifiche reattivi per riconoscere e rispondere ad antigeni tumorali, in assenza di MHC e possono essere adattati per un antigene repertorio virtualmente infinita. Cellule T ingegnerizzate CAR contro una varietà di antigeni tumorali hanno dimostrato l&#39;efficacia preclinica e promessa eccezionale nella clinica 6-9. In particolare, nel contesto di GBM, un CAR piattaforma di cellule T di targeting di crescita epidermico recettore del fattore di variante III (EGFRvIII), una mutazione specifica per tumore espresso sulla superficie delle cellule del 10, ha dimostrato di prolungare la sopravvivenza in glioma-cuscinetto topi 11. Nonostante la loro versatilità, tuttavia, il beneficio clinico di CAR terapia adottiva non è stato pienamente realizzato, in parte a causa immunosoppressione associata al tumore e l&#39;evasione immune 12-16 e sfide nel creare e mantenere le cellule T antigene-specifiche in vivo. Sfruttando standard di cura (SOC) con immunoterapia può potenzialmente superare molti di questi limitazioni, con conseguente maggiore efficacia sia in ambito preclinico e clinico. SOC per la post-resezione GBM consiste di temozolomide alte dosi (TMZ), un DNA agente alchilante 17, e tutta l&#39;irradiazione cerebrale (WBI) 1. Questi trattamenti si presume di sinergizzare con i vaccini tumorali attraverso upregulation di tumore MHC espressione 18-20 e lo spargimento di antigeni da parte delle cellule tumorali morte 17,19,21,22. In effetti, l&#39;aggiunta di TMZ 20,23 o 18,24 WBI porta a una maggiore efficacia antitumorale dei trattamenti a base immunitaria nel setting preclinico. Inoltre, come molti chemioterapici citotossici non specifici, TMZ è noto per provocare 25,26 linfopenia sistemica, che può essere sfruttato come mezzo di accoglienza condizionata per le piattaforme di terapia adottivi 27-29. Lymphodepletion TMZ mediata ha dimostrato di aumentare la frequenza e la funzione delle cellule T antigene-specifiche, con conseguente maggiore efficacia di un ADOPPiattaforma terapia tive contro i tumori intracranici 30. Nel contesto della terapia CAR, lymphodepletion serve come mezzo di host-condizionata sia la riduzione del numero di cellule T soppressori endogena 31, e indurre proliferazione omeostatica 32 attraverso concorrenza ridotta per citochine 33, migliorando così l&#39;attività antitumorale 11,34. Dato il rapporto sinergico tra le piattaforme SOC e immunoterapia GBM, valutare nuove terapie adottivi e piattaforme vaccino nel contesto del SOC è fondamentale per trarre conclusioni significative per quanto riguarda l&#39;efficacia. In questo protocollo, delineiamo un metodo per la generazione e la somministrazione endovenosa di cellule murine CAR T specifiche per EGFRvIII accanto TMZ e WBI in topi portatori di tumori intracranici EGFRvIII-positivi (vedi Figura 1 per il trattamento temporale). Brevemente, le cellule T sono realizzati CAR ex vivo da trasduzione retrovirale. Rene embrionale umano (HEK) 293T cellule sono trasfettate con un complesso DNA / lipide (contenente il vettore CAR e plasmidi PCL-Eco) per produrre virus, che viene poi utilizzato per trasdurre splenociti murini attivate che vengono raccolte e coltivate in parallelo. Durante il corso della generazione CAR, ospita murine recanti tumori intracranici EGFRvIII-positivi vengono somministrati frazionata tutto il cervello a raggi X irradiazione e il trattamento sistemico TMZ a dosi paragonabili a SOC clinica. Cellule T auto sono poi consegnati per via endovenosa a host lymphodepleted. La seguente procedura è descritta in sette fasi distinte: (1) La somministrazione di Temozolomide per tumore Mice, (2) irradiazione cerebrale totale di topi tumore-cuscinetto, (3) Transfection, (4) La splenectomia e T cellule Preparazione, (5 ) trasduzione, (6) Cultura celle CAR T e Harvest, e (7) la somministrazione di cellule T di topi CAR tumore-cuscinetto. Queste fasi sono costituite da diversi passaggi che si estendono 6-7 giorni e sono state compiute contemporaneamente.

<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.

Cellule T CAR sono generati dalla trasduzione con il EGFRvIII CAR vettore retrovirale 11. Questo vettore, MSGV1, è stato sviluppato dal vettore SFGtcLuc_ITE4 35, che contiene il virus cellule staminali murine (MSCV) lunghe ripetizioni terminali, la estesa regione gag e sito di splicing busta (splice donatore, sd, e splice accettore, sa), e virale Segnale di imballaggio (ψ). Il CAR EGFRvIII contenente l&#39;anti-EGFRvIII catena singola frammento umano variabile (scFv) 139, in tandem con CD8TM muri...

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.

Generazione di cellule CAR T per adottiva terapia nel contesto di Glioblastoma standard di cura

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

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Research

JoVE Journal

Immunology and Infection

21.2K Views.  Duke University. 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. 

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

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 presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

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 ...

Ricerca

Immunologia e infezioni

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 generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generazione di Multivirus-cellule T specifiche per prevenire / curare infezioni virali dopo trapianto allogenico di cellule staminali ematopoietiche

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

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

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

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

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposon modifica del sistema di cellule primarie umane T

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 antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Peptide: MHC Tetramero basata Arricchimento di epitopi specifici per cellule T

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

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

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

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

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

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Arricchimento cellulare automatizzata di Citomegalovirus specifici linfociti T per le applicazioni cliniche che utilizzano il sistema di citochine cattura

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

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Trasferimento adottivo stereotactic di citotossici cellule immunitarie nei modelli murini di xenotrapianti Multiforme di Glioblastoma umano ortotopico

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.