<p>Supplementary Fig.S4: In vitro bioluminescence to detect luciferase expression in Raji and Raji-2R cells Raji and Raji-2R cells were electroporated with ffLUCZeo-pcDNA and selected for stable clones expressing luciferase (Luc) with Zeocin. (A) The bioluminescence intensity increased proportionally with increasing Raji-Luc cell numbers. A strong correlation between number of cells and light emission was obtained (R2= 0.99). (B) The bioluminescence intensity increased proportionally with increasing Raji-2R-Luc cell numbers. A strong correlation between number of cells and light emission was obtained (R2= 0.989). (C)-(D) Dynamic change of the bioluminescent signal in Raji-Luc and Raji-2R-Luc after over 2 months selection. Signal intensities are shown as a function of time after addition of D-luciferin.</p>
ARTICLE ABSTRACT
The prognosis is very dismal for patients with relapsed CD20+ B-cell non-Hodgkin lymphoma (B-NHL). Facilitating the development of alternative novel therapeutic strategies is required to improve outcomes in patients with recurrent/refractory CD20+ B-NHL. In this study, we investigated functional activities of anti-CD20 CAR-modified, expanded peripheral blood NK cells (exPBNK) following mRNA nucleofection against CD20+ B-NHL in vitro and in vivo. CAR+ exPBNK had significantly enhanced in vitro cytotoxicity, compared with CAR− exPBNK against CD20+ Ramos (P < 0.05), Daudi, Raji, and two rituximab-resistant cell lines, Raji-2R and Raji-4RH (P < 0.001). As expected, there was no significant difference against CD20− RS4;11 and Jurkat cells. CD107a degranulation and intracellular IFNγ production were also enhanced in CAR+ exPBNK in response to CD20+ B-NHL–specific stimulation. In Raji-Luc and Raji-2R-Luc xenografted NOD/SCID/γ-chain−/− (NSG) mice, the luciferase signals measured in the CAR+ exPBNK-treated group were significantly reduced, compared with the signals measured in the untreated mice and in mice treated with the CAR− exPBNK. Furthermore, the CAR exPBNK-treated mice had significantly extended survival time (P < 0.001) and reduced tumor size, compared with those of the untreated and the CAR− exPBNK-treated mice (P < 0.05). These preclinical data suggest that ex vivo–exPBNK modified with anti-CD20 CAR may have therapeutic potential for treating patients with poor-risk CD20+ hematologic malignancies. Cancer Immunol Res; 3(4); 333–44. ©2014 AACR.
