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.

This protocol is based on an experimental design where 10 mice are treated with 107 CAR T cells each. This means that 108 CAR T cells will be needed; the yield should be overestimated by 5 x 107-1 x 108 to account for loss in viability. The following protocol is scaled to generate approximately 200 x 106 cells. The cells are then administered intravenously to female C57BL/6 mice with 9 day established syngeneic EGFRvIII-positive intracranial tumors, developed from th...

<ol>
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The treatment timeline described here was designed to model clinical standard of care and leverage its effects for CAR adoptive therapy. CAR T cell doses, TMZ regimens, and radiotherapy administration can be modified to enhance in vivo T cell activity, lymphodepletion, and tumor killing. TMZ regimens can be increased to yield host myeloablation and increased expansion of adoptively transferred cells30. Furthermore, the lymphodepletive effects of TMZ can be recapitulated by low-dose (4 - 6 Gy) single-f...

Glioblastoma (GBM) is the most common primary malignant brain tumor and is invariably fatal. Surgical resection coupled with non-specific standard of care chemotherapy and radiotherapy fails to completely eliminate malignant cells, resulting in a dismal prognosis of less than 15 months in patients with this disease1. In contrast, immunotherapy offers a precise approach for specifically targeting tumor cells, and thus has the potential to serve as a highly effective treatment platform with reduced risk of collateral toxicity2-4. T cells engineered ex vivo to express chimeric antigen receptors (CARs) offer a versatile strategy for tumor immunotherapy. CARs are generated by fusing the extracellular variable region of an antibody with one or more intracellular T cell signaling molecule(s), in lieu of a full-length major histocompatibility complex (MHC)-restricted T cell receptor5. This mode of antibody-like antigen recognition allows for reactive antigen-specific T cells to recognize and respond to tumor antigens in the absence of MHC and can be adapted for a virtually infinite antigen repertoire.
CAR T cells engineered against a variety of tumor antigens have shown preclinical efficacy and outstanding promise in the clinic6-9. Specifically, in the context of GBM, a CAR T cell platform targeting epidermal growth factor receptor variant III (EGFRvIII), a tumor-specific mutation expressed on the cell surface10, was shown to prolong survival in glioma-bearing mice11. Despite their versatility, however, the clinical benefit of CAR adoptive therapy has not been fully realized, due in part to tumor-associated immunosuppression and immune evasion12-16 as well as challenges in establishing and maintaining antigen-specific T cells in vivo. Leveraging standard of care (SOC) with immunotherapy can potentially overcome several of these limitations, resulting in enhanced efficacy in both the preclinical and clinical setting.
SOC for post-resection GBM consists of high-dose temozolomide (TMZ), a DNA alkylating agent17, and whole brain irradiation (WBI)1. These treatments are presumed to synergize with tumor vaccines via upregulation of tumor MHC expression18-20 and the shedding of antigens by dead tumor cells17,19,21,22. Indeed, the addition of TMZ20,23 or WBI18,24 leads to enhanced antitumor efficacy of immune-based treatments in the preclinical setting. Furthermore, like many non-specific cytotoxic chemotherapeutics, TMZ is known to cause systemic lymphopenia25,26, which can be leveraged as a means of host-conditioning for adoptive therapy platforms27-29. TMZ-mediated lymphodepletion has been shown to enhance the frequency and function of antigen-specific T cells, leading to increased efficacy of an adoptive therapy platform against intracranial tumors30. In the context of CAR therapy, lymphodepletion serves as a means of host-conditioning by both reducing the number of endogenous suppressor T cells31, and inducing homeostatic proliferation32 via reduced competition for cytokines33, thus enhancing antitumor activity11,34. Given the synergistic relationship between GBM SOC and immunotherapy platforms, evaluating novel adoptive therapies and vaccine platforms in the context of SOC is critical for drawing meaningful conclusions regarding efficacy.
In this protocol, we outline a method for the generation and intravenous administration of murine EGFRvIII-specific CAR T cells alongside TMZ and WBI in mice bearing EGFRvIII-positive intracranial tumors (see Figure 1 for treatment timeline). Briefly, CAR T cells are made ex vivo by retroviral transduction. Human embryonic kidney (HEK) 293T cells are transfected using a DNA/lipid complex (containing the CAR vector and pCL-Eco plasmids) to produce virus, which is then used to transduce activated murine splenocytes that are harvested and cultured in parallel. During the course of CAR generation, murine hosts bearing EGFRvIII-positive intracranial tumors are administered fractionated whole-brain X-ray irradiation and systemic TMZ treatment at doses comparable to clinical SOC. CAR T cells are then delivered intravenously to lymphodepleted hosts.
The following procedure is described in seven separate phases: (1) Administration of Temozolomide to Tumor-bearing Mice, (2) Whole Brain Irradiation of Tumor-bearing Mice, (3) Transfection, (4) Splenectomy and T cell Preparation, (5) Transduction, (6) CAR T cell Culture and Harvest, and (7) CAR T cell administration to Tumor-bearing Mice. These phases consist of several steps that span 6-7 days and are performed concurrently.

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

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

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

CAR T cells are generated by transduction with the EGFRvIII CAR retroviral vector11. This vector, MSGV1, was developed from the SFGtcLuc_ITE4 vector35, which contains the murine stem cell virus (MSCV) long terminal repeats, the extended gag region and envelope splice site (splice donor, sd, and splice acceptor, sa), and viral packaging signal (&#968;). The EGFRvIII CAR containing the human anti-EGFRvIII single-chain variable fragment (scFv) 139, in tandem with murine CD8TM, CD28, 4-1BB, and CD3&#950...

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.

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. 