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

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

Este protocolo se basa en un diseño experimental donde 10 los ratones se tratan con 10 7 CAR células T cada uno. Esto significa que se necesitarán 10 8 células CAR T; el rendimiento debe ser sobrestimada por 5 x 10 7 -1 x 10 8 a la cuenta de la pérdida de viabilidad. El siguiente protocolo se escala para generar aproximadamente 200 x 10 6 células. Las células se administran por vía intravenosa a la hembra C57BL / 6 ratones con 9 días tumores intracraneales ...

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La línea de tiempo de tratamiento descrito aquí fue diseñado para modelar estándar clínico de atención y potenciar sus efectos para la terapia adoptiva CAR. CAR dosis de células T, los regímenes de TMZ, y la administración de la radioterapia pueden ser modificados para mejorar la actividad in vivo de células T, lymphodepletion, y el asesinato del tumor en. Regímenes de TMZ se puede aumentar para producir mielosupresión de host y una mayor expansión de las células transferidas adoptivament...

El glioblastoma (GBM) es el tumor cerebral maligno primario más común y es invariablemente fatal. La resección quirúrgica junto con estándar no específica de la quimioterapia y la radioterapia cuidado de no eliminar completamente las células malignas, que resulta en un pronóstico sombrío de menos de 15 meses en los pacientes con esta enfermedad 1. En contraste, la inmunoterapia ofrece un enfoque preciso para apuntar específicamente a las células tumorales, y por lo tanto tiene el potencial para servir como una plataforma de tratamiento altamente eficaz con menor riesgo de toxicidad colateral 2-4. Células T diseñadas ex vivo para expresar receptores de antígenos quiméricos (CAR) ofrecen una estrategia versátil para la inmunoterapia tumoral. CARs se generan mediante la fusión de la región variable extracelular de un anticuerpo con la molécula de señalización de una o más células T intracelular (s), en lugar de una de longitud completa complejo mayor de histocompatibilidad (MHC) -restringido receptor de la célula T 5. Este modo de recono antígeno-anticuerpo-comoOn permite para las células T específicas de antígeno reactivos para reconocer y responder a los antígenos tumorales en ausencia de MHC y se pueden adaptar para un repertorio de antígeno virtualmente infinito. Células T CAR ingeniería contra una variedad de antígenos tumorales han demostrado eficacia preclínica y la promesa excepcional en la clínica 6-9. En concreto, en el contexto de GBM, una variante de crecimiento epidérmico receptor del factor de CAR plataforma de células T focalización III (EGFRvIII), una mutación específica del tumor expresa en la superficie de la célula 10, se ha demostrado prolongar la supervivencia en los ratones portadores de glioma 11. A pesar de su versatilidad, sin embargo, el beneficio clínico de la terapia adoptiva CAR no se ha realizado plenamente, en parte debido a la inmunosupresión asociada al tumor y la evasión inmune 12-16, así como los desafíos en el establecimiento y mantenimiento de las células T específicas de antígeno in vivo. Aprovechando estándar de cuidado (SOC) con inmunoterapia potencialmente puede superar varios de estos limitaciones, resulta en una mayor eficacia tanto en la configuración preclínica y clínica. SOC para el post-resección GBM consiste en altas dosis de temozolomida (TMZ), un agente alquilante de ADN 17, y la irradiación de todo el cerebro (WBI) 1. Estos tratamientos se presume que en sinergia con vacunas tumorales a través de la regulación positiva de la expresión de MHC tumor 18-20 y el derramamiento de antígenos por las células tumorales muertas 17,19,21,22. De hecho, la adición de TMZ 20,23 o 18,24 WBI conduce a una mayor eficacia antitumoral de los tratamientos de base inmunitaria en el entorno preclínico. Además, como muchos agentes quimioterapéuticos citotóxicos no específicos, TMZ se sabe que causa 25,26 linfopenia sistémica, que puede ser aprovechada como medio de host-acondicionado para las plataformas de terapia adoptiva 27-29. Lymphodepletion mediada por TMZ se ha demostrado para mejorar la frecuencia y la función de las células T específicas de antígeno, lo que aumenta la eficacia de un ADOPplataforma de terapia tiva contra los tumores intracraneales 30. En el contexto de la terapia CAR, lymphodepletion sirve como un medio de host-acondicionado por tanto reducir el número de células T supresoras endógena 31, y la inducción de la proliferación homeostática 32 a través de la competencia reducido para citoquinas 33, mejorando así la actividad antitumoral 11,34. Dada la relación sinérgica entre GBM SOC e inmunoterapia plataformas, evaluar nuevas terapias adoptivas y plataformas de vacunas en el contexto del SOC es crítico para sacar conclusiones significativas respecto a la eficacia. En este protocolo, se describe un método para la generación y administración intravenosa de células T CAR-EGFRvIII murino específico junto a TMZ y WBI en ratones con tumores intracraneales EGFRvIII-positivos (ver Figura 1 para la línea de tiempo de tratamiento). En pocas palabras, se producen las células CAR T ex vivo por transducción retroviral. Riñón embrionario humano (HEK) 293T células se transfectaron usando un complejo de ADN / lípido (que contiene el vector y los plásmidos CAR PCL-Eco) para producir virus, que luego se utiliza para transducir esplenocitos murinos activados que se cosechan y se cultivaron en paralelo. Durante el curso de la generación de CAR, los anfitriones murinos con tumores intracraneales EGFRvIII positivos se administran fraccionado de todo el cerebro de la irradiación de rayos X y el tratamiento sistémico TMZ a dosis comparables a SOC clínica. Las células T CAR se entregan por vía intravenosa a los anfitriones lymphodepleted. El siguiente procedimiento se describe en siete fases separadas: (1) La administración de temozolomida a (3) Transfección Ratones portadores de tumores, (2) la irradiación de todo el cerebro de ratones portadores de tumores,, (4) La esplenectomía y células T Preparación, (5 ) Transducción, (6) Cultura de células T CAR y cosecha, y (7) la administración de células T CAR a ratones portadores de tumores. Estas fases consisten en varias etapas que abarcan 6-7 días y se realizan simultáneamente.

<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.

Las células T CAR son generados por la transducción con el vector retroviral EGFRvIII CAR 11. Este vector, MSGV1, se desarrolló a partir del vector SFGtcLuc_ITE4 35, que contiene el virus de células madre murinas (MSCV) repeticiones terminales largas, la extendida región gag y sitio de empalme sobre (donador de empalme, sd, y aceptor de empalme, sa), y viral señal de empaquetamiento (ψ). El CAR EGFRvIII que contiene el fragmento humano anti-EGFRvIII de una sola cadena variable (scFv) 139, en...

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.

Generación de células T CAR para realizar la terapia adoptiva en el contexto de Glioblastoma estándar de cuidado

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

Research

JoVE Journal

Immunology and Infection

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

Transducción retroviral de receptores de células T en ratones las 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 ...

Investigación

Inmunología e Infección

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

Generación de células T Multivirus específicos para prevenir / tratar las infecciones virales después de trasplante alogénico de células madre hematopoyéticas

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

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

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

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de 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

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposón modificación del sistema de células T humanas primarias

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

Péptido: MHC tetrámero basado en el enriquecimiento de células T específicos para el epítopo

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

Tipo 1 Diabetes inducción acelerada en ratones por transferencia adoptiva de células CD4 + células T diabetogénico

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

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

Evaluar el desarrollo de las células murino plasmocitoide dendríticas en las placas de Peyer Usando la transferencia adoptiva de Progenitores Hematopoyéticas

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Generación de células T humanas aloantígeno-específicos de la sangre periférica

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

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

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

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

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

Enriquecimiento de la célula automatizada de citomegalovirus células T específicas para aplicaciones clínicas utilizando el Sistema de citoquinas de captura

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