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.

Este protocolo é baseado em um projeto experimental, onde 10 ratos são tratados com 10 células 7 carro T cada. Isto significa que serão necessários 10 8 células CAR T; o rendimento deve ser superestimada por 5 x 10 7 -1 x 10 8 a conta para a perda da viabilidade. O protocolo seguinte é dimensionado para gerar cerca de 200 x 10 6 células. As células são então administrada intravenosamente a fêmea C57BL / 6 com 9 dias estabelecidos os tumores intracranianos EGFRvIII-positivo singeneicos, desenvolvidos a partir das linhas celulares de melanoma B16 ou de astrocitoma KR158B existentes. Concomitante com a geração de células T CAR, os ratinhos portadores de tumor são administradas doses clinicamente relevantes de lymphodepletive TMZ (60 mg / kg) e WBI (16,5 Gy). Os ratos foram mantidos e criados em condições livres de patógenos na Duke University Medical Center (DUMC). Todos os experimentos em animais foram realizados de acordo com protocolos aprovados pela Duke University Institutional Animal Care e Use Committee (IACUC). 1. A administração de temozolomida para portadores de tumor Ratos Dias de 0 a 4: <ol><li> Calcula-se o número total de ratinhos para serem injectados e multiplicar este por 0,5 para determinar o volume de solução necessário TMZ (por exemplo, 30 ratos x 0,5 ml = 15 ml TMZ) e adicionar 1,5 ml para este volume para contabilizar transbordamento (16,5 ml TMZ ). </li><li> Multiplicar este volume total de 3 mg / ml para determinar o peso do liofilizado TMZ necessário (por exemplo, 16,5 x 3 = 49,5 mg TMZ). Pese isso em um cônico de 50 ml. CUIDADO: TMZ é tóxico por inalação, ingestão e contato com os olhos, pele e membranas mucosas. Consulte com a segurança da instituição ocupacional e / ou de saúde ambiental e do departamento de bem-estar para recomendações sobre como reduzir o risco de exposição. </li><li> Multiplicar o volume total de 0,15 para determinar o volume de dimetil sulfóxido (DMSO) necessário (por exemplo, 16,5 x0,15 = 2,475 ml de DMSO). Filtrar esterilizar este volume de DMSO (mais um adicional de 2 ml para explicar transbordamento) através de um filtro de 0,2 um para um tubo estéril. Adicionar 2,475 mL de DMSO estéril para o pó TMZ. </li><li> Coloque a solução TMZ / DMSO num copo de água sobre uma placa quente a 65 ° C durante 10-15 min. Uma vez TMZ estiver completamente dissolvido, a solução deve ficar de cor amarelo pálido; se a solução torna-se cor de rosa, em seguida, a TMZ é degradada, e uma nova solução deve ser preparada com um lote fresco de TMZ. </li><li> Calcula-se o volume apropriado de solução salina estéril através da multiplicação necessária 0,85 pelo volume total (0,85 x 16,5 ml = 14,025 ml de solução salina). Adicionar 15 ml de solução salina estéril para uma cónico de 50 ml. Enquanto TMZ é dissolução em DMSO, a solução salina de calor a 65 ° C. </li><li> Vortex a solução / DMSO TMZ para garantir que o pó TMZ é totalmente dissolvido e adiciona-se lentamente solução salina aquecida até a solução / DMSO TMZ. </li><li> Imediatamente após a preparação, carregue totalmente 15 x 1 ml syring tuberculinaes com solução TMZ para administração a quatro dias camundongos com tumores estabelecidos. </li><li> Repetir este procedimento nos dias 1 a 4 para um total de cinco administrações TMZ. </li></ol> 2. cérebro inteiro de Irradiação de portadores de tumor Ratos Dias 2 a 4: <ol><li> Ligue o irradiador de raios-X e deixe-o aquecer-up de tensão total. Certifique-se de que o filtro apropriado está no lugar e entrada a tensão adequada e as configurações atuais (Figura 2A). NOTA: A dosimetria da irradiador deve ser estabelecido de antemão para determinar as configurações de tensão e layout de grade (Figura 2A, B) apropriado. </li><li> Calcula-se a duração do tempo que é necessário para resultar em 5,5 Gy de irradiação de raios-X. Por exemplo, se os raios X são entregues a uma taxa de 2 Gy / min, em seguida, 2,75 min é necessário para fornecer um total de 5,5 Gy. Introduza o tempo de duração apropriada. NOTA: A Tabela 2 apresenta rato WBI doses and seus equivalentes clínicos em seres humanos. No contexto de gliomas de alto grau, de 60 Gy WBI fraccionado (fracções de 2 Gy, 5 dias / semana, durante 6 semanas) é o padrão de cuidado clínico, e o equivalente murino aqui utilizado é de 16,5 Gy (5,5 Gy x 3). </li><li> Prepara-se uma solução fresca de cetamina / xilazina na anestesia sistémica, através da adição de 2 ml de cetamina e xilazina 1 ml a 17 ml de solução salina de modo a que a solução final é de 10 mg / ml de cetamina e 1 mg / ml de xilazina. </li><li> Pesar ratinhos e administrar 10 uL de cetamina / xilazina intraperitonealmente por grama de peso corporal de modo a que os animais recebem uma dose de cetamina a 100 mg / kg e 10 mg / kg de xilazina. Ratos deve ser totalmente sedado e respirando visivelmente dentro de 2 min de cetamina administração / xilazina. </li><li> Para manter a umidade oftálmica em animais sedados, esfregue suavemente uma pequena quantidade de pomada lágrimas artificiais em cada olho. </li><li> Coloque ratos sedados na grelha de tal modo que as cabeças estão posicionadas na área que recebe o raio-X em maiorintensidade (Figura 2B, C). Corpos Protetor de pescoço para baixo com tamanho adequado tubo de chumbo para bloquear a entrega de raios-X sistêmica (Figura 2D). </li><li> Coloque ratinhos posicionado sob o feixe de raios-X de tal forma que o laser denotando o ponto focal do feixe de raios-X está na coordenada (0,0) sobre a disposição da grade. Comece a entrega de raios-X. </li><li> Quando a entrega de raios-X é completo, remover animais do irradiador e coloque sobre uma almofada de aquecimento quente. Não abrigar animais sedados com animais conscientes até animais recuperou a consciência suficiente para manter decúbito externo. Uma vez que os animais podem manter decúbito externo, coloque animais conscientes de volta em suas gaiolas apropriadas. </li><li> Repita este procedimento nos dias 3 e 4 de um total de três doses fracionadas de radiação. </li></ol> 3. Transfection Dia -1: <ol><li> Prepare meios D10 pela adição de 50 ml de soro fetal bovino (FBS) a 500 ml de Dulbecco Modified Eagle Médium (DMEM). </li><li> Prepare meios de células T (MTC) por adição de 5,5 ml de L-glutamina, 5,5 ml de piruvato de sódio, 5,5 ml de aminoácidos não essenciais, e 5,5 ml de penicilina / estreptomicina (pen / strep) em 500 ml de meio RPMI-1640. Em seguida, adicione 550 mL de 2-mercaptoetanol e gentamicina 550 mL. Finalmente, adicione 50 ml FBS. </li><li> Colheita in vitro de células em cultura e HEK293T levar a uma concentração de 7,5 x 10 6 células / ml em meio D10. </li><li> Placa 10 ml de meio de D10 e adicionar 1 ml de suspensão de células HEK293T em cada um de 16 x 10 cm poli-D-lisina (PDL) placas revestidas. </li><li> Incubar durante a noite a 37 ° C com 5% de CO 2. </li></ol> Dia 0: <ol start="6"><li> Substituir meios com 10 ml D10 fresco, pelo menos, 30 min antes da transfecção de células. </li><li> Determinar a quantidade de vector de plasmídeo, pCL-Eco vector, e o reagente de transfecção lipossómica necessária para transfecção através da multiplicação do número total de placas + 1 (16 + 1 = 17) de 14,1 ugvector plasmídico (14,1 x 17 = 239,7 g), 9,9 ug pCL-Eco vector (9,9 x 17 = 168,3 g), e 60 ul de reagente de transfecção lipossómica (60 x 17 = 1020 mL). Todos os reagentes devem estar à temperatura ambiente antes de preparar soluções. </li><li> Rotular dois tubos A e B. No tubo A, adicionar 239,7 g vector plasmídeo e 168,3 mg PCL-Eco vector de (1,5 x 17) ml de soro reduzida meio de Eagle modificado (RS-MEM). No tubo B, adicionar 1,020 mL de reagente de transfecção lipossómica de (1,5 x 17) ml de RS-MEM. </li><li> Incubar A e B separadamente, durante 5 min à temperatura ambiente. </li><li> Misturar A e B, em conjunto com cuidado (vortex durante 1-2 segundos ou gire várias vezes) e incubar durante 20 min à temperatura ambiente para formar complexos lípido / ADN. </li><li> Adicionar lípido / DNA do complexo gota a gota às células HEK293T em placa de 10 cm. </li><li> Incubar a 37 ° C durante 6-8 horas ou durante a noite (não exceder 24 h). </li><li> Depois de 6-8 horas de incubação, substitua médio, com 12 ml de TCM fresco para a produção viral. Este chapeados mediaserão utilizados no Dia 2 como sobrenadante viral para a transdução de células T. </li></ol> 4. A esplenectomia e Preparação de Células T Dia 0: <ol><li> Despeje 10 ml de TCM em um cônico de 50 ml e colocar no gelo para a coleta de baço. </li><li> Sacrificar o número adequado de animais por asfixia com CO2 e decapitação secundário: Colocar os animais em uma gaiola de recepção de CO 2 a um caudal de 10-30% do volume da gaiola / minuto, por orientações American Veterinary Medical Association (não mais do que 5 animais podem ser sacrificado simultaneamente) até termina respiração e durante dois minutos depois. Remover animais da câmara de CO 2 e decapitar. NOTA: Um baço de um 6-12 semanas de idade do sexo feminino C57BL / 6 do mouse irá produzir cerca de 4,5-5 x 10 7 esplenócitos. Aqui, 4 baços serão colhidas para aproximadamente 200 x 10 6 células. </li><li> Coloque o rato de tal forma que seu lado direito está virado para cima, epulverizar com etanol a 70%. Com a pinça, pegue uma dobra fina de pele abaixo da caixa torácica esquerda e cortou uma pequena incisão com a tesoura. Retire a pele, pegue cuidadosamente uma dobra fina do peritônio com uma pinça, e cortou uma pequena cavidade. </li><li> O baço é um órgão alongado pequeno, escuro vermelho que se assemelha a um bean achatada; delicadamente agarrar o baço com a pinça e impostos especiais de consumo por cortar o tecido conjuntivo circundante. Colocar os baços extirpados em cónico contendo 10 ml de TCM em gelo. </li><li> Pour baços mais de um 70 um filtro de células de malha e desagregar por trituração com a extremidade romba do interior de uma seringa de 5 ml para gerar uma suspensão de célula única. Devem ser desagregados Um máximo de dois baços per mesh peneira. </li><li> Use um pequeno volume de TCM para lavar cuidadosamente o filtro seguinte desagregação de recolher quaisquer esplenocitos restantes. Associação Todos os baços desagregados em uma suspensão de uma única célula. Levar o volume final para 50 mL com uma lavagem de TCM para umad girar a 300 xg durante 10 min. </li><li> Prepara-se uma solução de tampão de lise 1x por adição de 5 ml de 10x tampão de lise a 45 ml de água estéril. Para eliminar as células vermelhas do sangue, ressuspender o sedimento em 5 ml de tampão de lise 1x por baço em uma cónico de 50 ml, misturando bem cuidado pipetando para cima e para baixo. Se não superior a 5 baços, utilizar um tubo de centrífuga de 250 ml. Colocar o tubo de centrifugação cónico ou em um banho de água a 37 C ° durante 5 min. </li><li> Remover a reacção de lise a partir do banho de água e adicionar TCM na proporção de 1: 1 com tampão de lise para neutralizar a reacção. Lavar por centrifugação a 300 xg durante 10 min. </li><li> Aspirar o sobrenadante e pellet totalmente ressuspender em TCM (2 ml / baço, 8 ml total para 4 baços) por pipetagem cima e para baixo. </li><li> Contar as células por adição de 10 ul de suspensão de células a 190 ul azul de tripano (diluição de 1:20). Multiplicar o número obtido em um dos quatro quadrados em grade por 20 x 10 4 x volume total (8 mL) para se obter o número total de células (por exemplo, células 125 x 20 x 10 4 </saté&gt; x 8 ml = 200 x 10 6 esplenócitos). </li><li> Dilui-se as células a uma concentração de 2 x 10 6 células / ml em TCM, suplementado com 2 ug / ml de concanavalina A (ConA) e 50 UI / ml de recombinante humano IL-2 (rhIL-2). Assim, para 200 x 10 6 esplenócitos, adicionar 92 ml de meio de células de 8 ml, 200 ug de Con A, e 5000 UI de rhIL-2. </li><li> Adicionar 2 mL de células para cada poço de placas tratadas de cultura de tecidos de 24 poços, de tal modo que 4 x 10 6 células em cada poço são (por exemplo, 100 ml de células vai demorar aproximadamente 4 placas). </li><li> Incubar durante a noite a 37 ° C com 5% de CO 2. </li></ol> 5. Transdução Dia 1: <ol><li> Calcular o número de cultura não-tecido tratado placas de 24 poços necessários para a transdução através da multiplicação do número de baços colhidos por 2 (8 placas são necessários para baços 4). </li><li> Em seguida, calcular o volume requerido de solução fragmento de fibronectina humana recombinante (RHFF)necessária para placas de revestimento através da multiplicação (número total de poços + 3) x 0,5 ml (8 x 24 placas = 192 poços + 3 = 195) x 0,5 mL = 97,5 mL. </li><li> Prepare a 97,5 ml de solução salina tamponada com fosfato (PBS) contendo RHFF a uma concentração de 25 ug / ml, multiplicando o volume total de 25? G (97,5 x 25 = 2437,5 ug). Adicionar esta quantidade de RHFF a 97,5 ml PBS. </li><li> Bata de cultura não-tecido tratado placas de 24 poços por adição de 0,5 ml de solução PBS / RHFF por poço. </li><li> Incubar durante a noite a 4 ° C. </li></ol> Dia 2: <ol start="6"><li> Despejar solução de PBS / RHFF de cultura não-tecido tratado placas de 24 poços. </li><li> Adicionar 1 ml / poço de albumina de soro bovino a 2% (BSA) em PBS e incubar à temperatura ambiente durante 30 min. </li><li> Remover BSA por firmemente upending placa. Lavar por adição de 2 ml de PBS. </li><li> Recolher o sobrenadante virai por transferência de meios de comunicação a partir de cada placa HEK293T PDL para um tubo de centrífuga de 250 ml e girar 10 min a 500 x g. </li><li> Transferir cuidadosamente su viralpernatant em um 250 ml fresco tubo de centrifugação, tendo a certeza de não perturbar o sedimento de células que podem ter se formado. </li><li> Adicionar TCM fresco ao sobrenadante virai, tal que o volume final de 3 ml é mais do que a quantidade necessária para poços RHFF-revestidos. Por exemplo, para 192 poços RHFF-revestidos, trazer ao volume de sobrenadante virai a 192 + 3 ml = 195 ml. </li><li> Remover esplenócitos cultivadas na incubadora e ressuspender por suavemente pipetando para cima e para baixo 2 - 3 vezes em cada poço. Transfira para uma cónico de 250 ml. Se utilizando uma pipeta multicanal, utilizando um reservatório estéril antes de serem transferidos vai ajudar a acelerar esta etapa. Contagem como anteriormente descrito e centrifugação a 300 xg durante 10 min. </li><li> Adicionar rhIL-2 a sobrenadante viral a uma concentração de 50 Ul / ml. Por exemplo, adicionar 50 x 195 = 9.750 UI a 195 ml de sobrenadante viral-2 rhIL. </li><li> Ressuspender esplenócitos em sobrenadante virai a 1 x 10 6 células / ml </li><li> Adicionar 1 ml / poço de uma suspensão de esplenócitos em placas de 24 poços revestidas com RHFF. </li><li>Rotação por 90 min de acordo com as seguintes configurações: 770 xg, aceleração = 4, freio / desaceleração = 0, 32 ° C. </li><li> Prepara-se uma solução de TCM com 50 UI / ml de rhIL-2. Por exemplo, preparar uma solução de 200 TCM por adição de 10.000 UI de rhIL-2. Adicionar 1 ml de rhIL-2 / TCM a cada poço, após a centrifugação. </li><li> Cultura durante a noite a 37 ° C em 5% de CO 2. </li></ol> 6. CAR célula T Cultura e colheita Dias 3 e 4: <ol><li> Se as células T obter&gt; 80% de confluência, as células podem ser divididas (isto normalmente ocorre no dia 3 ou dia 4). Para dividir as células, pipetar suavemente para cima e para baixo em cada poço de 2-3 vezes, e mover-se 1 ml de cada poço em novos poços de uma placa de 24 poços de cultura de tecidos tratados fresco. Em seguida, adicionar 1 ml de TCM fresco com 50 UI / ml de IL-2 a cada cavidade de tal modo que o volume final é de 2 ml em todos os poços. </li><li> Se as células não atingem&gt; 80% de confluência, realizar uma mudança de metade meios por pipetagem fora lentamente 1 ml de meios de comunicação a partir do topo de cada cavidade. Evite disturbing células se instalaram no fundo durante a remoção da mídia. Adicionar 1 ml de TCM fresco contendo 50 UI / ml de rhIL-2. </li></ol> Dia 5: <ol start="3"><li> Suspenda as células T CAR cuidado pipetando para cima e para baixo 3 vezes em cada poço e transferir para um tubo de centrífuga de 250 ml. Girar células a 300 xg durante 10 min. </li><li> Sobrenadante completamente aspirado sem perturbar pellet. Lavar uma vez com PBS, a contagem de células como descrito anteriormente, e lavar com PBS uma segunda vez. Um rendimento típico de células T CAR é de aproximadamente 1 x 10 6 células por poço (8 placas de células x 24 poços = 192 x 10 6 CAR T). </li><li> Ressuspender as células em PBS a uma concentração de 5 x 10 7 / ml para uma injecção de 1 x 10 7 num volume de 200? G (por exemplo, para 192 x 10 6 células T CAR, ressuspender o sedimento lavado em 3,84 ml de PBS). </li></ol> 7. CAR T Administration celular para portadores de tumor Ratos Dia 5: <ol><li> Transport células sobre gelo para instalações de animais por injecção intravenosa. Carga 500-1.000 ul em seringa de insulina com 27-31 G agulha, assegurando que todas as bolhas são expulsos. </li><li> Pegue um rato na base da cauda e colocar em restrainer tubo. </li><li> Puxar a cauda esticada com a veia virada para cima, e deslizar a agulha aproximadamente 1-2 mm sob a pele para dentro da veia, inserindo-o paralelo na veia da cauda. Lentamente expelir 200 ul na veia. Se a agulha estiver correctamente introduzida na veia, o volume vai fluir facilmente in; caso contrário, haverá resistência e da agulha terá que ser removido a partir da cauda e re-inserido correctamente. </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.

A linha do tempo de tratamento descrito aqui foi projetado para modelar padrão clínico de cuidado e alavancar os seus efeitos para a terapia adotiva CAR. T doses carro de célula, regimes TMZ, administrativos e radioterapia podem ser modificados para aumentar a actividade in vivo de células T, lymphodepletion e assassinato tumor. Os regimes de TMZ pode ser aumentada para se obter mieloablação hospedeiro e aumentar a expansão das células adoptivamente transferidos 30. Além disso, os efeitos ly...

Glioblastoma (GBM) é o tumor cerebral maligno primário mais comum e é invariavelmente fatal. A ressecção cirúrgica associada com o padrão não específico de quimioterapia e radioterapia cuidado para não eliminar completamente as células malignas, resultando em um mau prognóstico de menos de 15 meses, em pacientes com esta doença 1. Em contraste, a imunoterapia oferece uma abordagem precisa para visar especificamente células tumorais, e portanto tem o potencial de servir como uma plataforma de tratamento altamente eficaz, com um risco reduzido de toxicidade colateral 2-4. As células T engenharia ex vivo para expressar receptores de antigénios quiméricos (CARs) oferecem uma estratégia versátil para imunoterapia de tumor. CARs são gerados por fusão da região variável extracelular de um anticorpo com uma ou mais das células T intracelular molécula (s) de sinalização, em vez de um de histocompatibilidade principal de comprimento completo complexo (MHC) -restricted receptor de células T 5. Este modo de anticorpo-like reconhecimen antígenosobre permite células específicas para o antigénio T reactivas para reconhecer e responder aos antigénios tumorais na ausência de MHC e podem ser adaptados para um repertório antigénio praticamente infinito. CARRO células T modificadas contra uma variedade de antigénios de tumor demonstraram eficácia pré-clínica e excelente na promessa clínica 6-9. Especificamente, no contexto de GBM, uma variante de t CARRO plataforma de direccionamento celular de crescimento epidermal do receptor do factor III (EGFRvIII), uma mutação específica do tumor expressos na superfície da célula 10, foi mostrado prolongar a sobrevivência de ratinhos portadores de glioma 11. Apesar de sua versatilidade, no entanto, o benefício clínico da terapia adoptiva CAR ainda não foi plenamente realizado, em parte devido à imunossupressão associada ao tumor e evasão imune 12-16, bem como desafios na criação e manutenção de células T específicas de antigénio in vivo. Aproveitando padrão de atendimento (SOC) com a imunoterapia pode potencialmente superar vários destes limitações, resultando num aumento da eficácia tanto na configuração de pré-clínica e clínica. SOC para pós-ressecção GBM consiste de temozolomida em altas doses (TMZ), um agente de alquilação de ADN 17, e a irradiação do cérebro inteiro (WBI) 1. Presume-se que estes tratamentos a sinergia com vacinas tumorais via regulação positiva de tumor MHC expressão 18-20 eo derramamento de antígenos pelas células tumorais mortas 17,19,21,22. Com efeito, a adição de TMZ 20,23 ou 18,24 WBI leva ao aumento da eficácia antitumoral de tratamentos de base imunológica na configuração pré-clínico. Além disso, como muitos quimioterápicos citotóxicos não específicos, TMZ é conhecido por causar 25,26 lymphopenia sistêmica, que pode ser aproveitado como um meio de host-condicionado para as plataformas de terapia adotivos 27-29. Lymphodepletion TMZ mediada tem demonstrado aumentar a frequência e função de células T específicas de antigénio, que conduz a um aumento da eficácia de um adopplataforma terapia tiva contra tumores intracranianos 30. No contexto da terapia de CAR, lymphodepletion serve como um meio de acolhimento-condicionado tanto pela redução do número de células T supressoras endógenas 31, e induzindo a proliferação homeostática 32 através de competição reduzida para citocinas 33, aumentando assim a actividade anti-tumoral 11,34. Dada a relação sinérgica entre SOC e imunoterapia plataformas GBM, avaliando terapias adotivos novos e plataformas de vacina no contexto do SOC é fundamental para tirar conclusões significativas a respeito da eficácia. Neste protocolo, que descrevem um método para a geração e administração intravenosa de células de murino CAR T específicas para EGFRvIII ao lado TMZ e WBI em camundongos com tumores intracranianos EGFRvIII-positivos (veja a Figura 1 para a linha do tempo de tratamento). Resumidamente, as células T são feitos CAR ex vivo por transdução retroviral. Rim embrionário humano (HEK) 293T células são transfectadas utilizando um complexo de ADN / lípido (contendo o vector CAR e plasmídeos pCL-Eco) para produzir vírus, que é então utilizado para transduzir os esplenócitos de murideo activadas que são colhidas e cultivadas em paralelo. Durante o curso da geração CAR, hospedeiros murinos com tumores intracranianos EGFRvIII-positivos são administrados fraccionado em todo o cérebro de irradiação de raios X e TMZ tratamento sistémico com doses comparáveis ​​às SOC clínica. Células carro T são então entregues por via intravenosa aos anfitriões lymphodepleted. O procedimento que se segue é descrita em sete fases distintas: (1) administração de temozolomida para (3) Transfecção Ratos, (2) Irradiação Cérebro Total de Ratos portadores de tumor, que possui o tumor, (4) e esplenectomia Preparação de células T, (5 ) Transdução, (6) Cultura de células T CAR e colheita, e (7) a administração de células T CAR para portadores de tumor Ratos. Estas fases consistem em várias etapas que se estendem por 6-7 dias e são realizadas simultaneamente.

<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.

Células carro T são gerados por transdução com o vector retroviral EGFRvIII CAR 11. Este vector, MSGV1, foi desenvolvido a partir do vector SFGtcLuc_ITE4 35, que contém o vírus de células estaminais de murino (MSCV) repetições terminais longas, a região gag estendida e local de splicing do envelope (dador de splicing, sd, e aceitador de splicing, sa), e viral sinal de empacotamento (ψ). O CARRO EGFRvIII contendo o fragmento de anticorpo humano anti-EGFRvIII de cadeia única variável (sc...

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.

Geração de células T para CAR adoptivo Therapy no Contexto do Glioblastoma Padrão de Atendimento

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 visualizações.  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. 

Vídeo: 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

Transdução retroviral de receptor de células T no rato células 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 ...

Imunologia e Infecção

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.

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

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.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

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 modificação do sistema Transposon de células T primárias Humanos

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.

Péptido: Enriquecimento tetrâmero com base MHC das células T específicas para epitopos

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 Diabetes Tipo 1 Indução em Ratos por transferência adotiva de diabetogênicos TCD4 +

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.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

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.

As células T de enriquecimento de células automatizado de citomegalovírus específico-para aplicações clínicas usando o Sistema de captura de citocinas

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.

Estereotáxica transferência adotiva de citotóxicas células imunes em modelos murino de humanos ortotópico 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 ...

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.