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dc.contributor.authorKueberuwa, Gray
dc.contributor.authorZheng, W
dc.contributor.authorKalaitsidou, Milena
dc.contributor.authorGilham, David E
dc.contributor.authorHawkins, Robert E
dc.date.accessioned2018-12-10T11:48:51Z
dc.date.available2018-12-10T11:48:51Z
dc.date.issued2018en
dc.identifier.citationKueberuwa G, Zheng W, Kalaitsidou M, Gilham DE, Hawkins RE. A syngeneic mouse B-cell lymphoma model for pre-clinical evaluation of CD19 CAR T cells. J Vis Exp. 2018 Oct 16(140).en
dc.identifier.pmid30394400en
dc.identifier.doi10.3791/58492en
dc.identifier.urihttp://hdl.handle.net/10541/621387
dc.description.abstractThe astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.en
dc.language.isoenen
dc.relation.urlhttps://dx.doi.org/10.3791/58492en
dc.titleA syngeneic mouse B-cell lymphoma model for pre-clinical evaluation of CD19 CAR T cells.en
dc.typeArticleen
dc.contributor.departmentManchester Cancer Research Centre Building, Department Cancer Sciences, University of Manchesteren
dc.identifier.journalJ Vis Expen


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