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.
Highlights
Chimeric antigen receptor (CAR) T-cell therapy has shown astonishing clinical success in the treatment of CD19+ malignancies leading to the approval of tisagenlecleucel for relapsed acute lymphoblastic leukaemia[1] and axicabtagene ciloleucel for progressive large B-cell non-Hodgkin lymphoma[2] in 2017.The importance of Interactions between cancer and the immune system in both disease progression and therapeutic mechanisms is becoming increasingly recognized[3,4,5]
We showed that the secretion of IL-12 from within CAR T cells could lead to eradication of established lymphoma with a success rate of ~ 25%17
Syngeneic mouse models allow the testing of disease progression and therapy while maintaining an intact immune system
Summary
Chimeric antigen receptor (CAR) T-cell therapy has shown astonishing clinical success in the treatment of CD19+ malignancies leading to the approval of tisagenlecleucel for relapsed acute lymphoblastic leukaemia[1] and axicabtagene ciloleucel for progressive large B-cell non-Hodgkin lymphoma[2] in 2017. An important caveat of CAR T-cell therapy is that lymphodepleting pre-conditioning is required for therapeutic success[13,14] This is typically achieved in patients by administering chemotherapy prior to infusion of CAR T cells[15,16]. To create a test system that represents the patients ineligible for lymphodepletion, we established a lymphoreplete syngeneic mouse model in which we model CAR T-cell therapy of lymphoma In this model, we showed that the secretion of IL-12 from within CAR T cells could lead to eradication of established lymphoma with a success rate of ~ 25%17.
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