A syngeneic mouse B-cell lymphoma model for pre-clinical evaluation of CD19 CAR T cells.
AffiliationManchester Cancer Research Centre Building, Department Cancer Sciences, University of Manchester
MetadataShow full item record
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.
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).
JournalJ Vis Exp
- Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor.
- Authors: Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler-Stevenson M, Yang JC, Phan GQ, Hughes MS, Sherry RM, Raffeld M, Feldman S, Lu L, Li YF, Ngo LT, Goy A, Feldman T, Spaner DE, Wang ML, Chen CC, Kranick SM, Nath A, Nathan DA, Morton KE, Toomey MA, Rosenberg SA
- Issue date: 2015 Feb 20
- CD20-CD19 Bispecific CAR T Cells for the Treatment of B-Cell Malignancies.
- Authors: Martyniszyn A, Krahl AC, André MC, Hombach AA, Abken H
- Issue date: 2017 Dec
- Chimeric Antigen Receptor-Engineered T Cell Therapy in Lymphoma.
- Authors: Strati P, Neelapu SS
- Issue date: 2019 Mar 27
- Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.
- Authors: Kochenderfer JN, Rosenberg SA
- Issue date: 2013 May
- Treatment of patients with relapsed or refractory CD19+ lymphoid disease with T lymphocytes transduced by RV-SFG.CD19.CD28.4-1BBzeta retroviral vector: a unicentre phase I/II clinical trial protocol.
- Authors: Schubert ML, Schmitt A, Sellner L, Neuber B, Kunz J, Wuchter P, Kunz A, Gern U, Michels B, Hofmann S, Hückelhoven-Krauss A, Kulozik A, Ho AD, Müller-Tidow C, Dreger P, Schmitt M
- Issue date: 2019 May 19