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    A syngeneic mouse B-cell lymphoma model for pre-clinical evaluation of CD19 CAR T cells.

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    Authors
    Kueberuwa, Gray
    Zheng, W
    Kalaitsidou, Milena
    Gilham, David E
    Hawkins, Robert E
    Affiliation
    Manchester Cancer Research Centre Building, Department Cancer Sciences, University of Manchester
    Issue Date
    2018
    
    Metadata
    Show full item record
    Abstract
    The 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.
    Citation
    Kueberuwa 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).
    Journal
    J Vis Exp
    URI
    http://hdl.handle.net/10541/621387
    DOI
    10.3791/58492
    PubMed ID
    30394400
    Additional Links
    https://dx.doi.org/10.3791/58492
    Type
    Article
    Language
    en
    ae974a485f413a2113503eed53cd6c53
    10.3791/58492
    Scopus Count
    Collections
    All Paterson Institute for Cancer Research

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