Supplementary Fig.S1: Anti-CD20 CAR enhances NK92 cells in-vitro cytolytic activity against CD20+ B-NHL cells by retroviral transduction. (A) Schematic representation of the anti-CD20 chimeric receptor in an RD114-pseudotyped retroviral vector. LTR indicates long terminal repeat; AMP, ampicillin resistance. (B)-(C) Expression of anti-CD20 CAR was confirmed in infected Jurkat cells by flow cytometry after staining with a goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin followed by streptavidin PE (y-axes); expression of GFP is also shown (x-axes) (B) and it was also confirmed in infected PBMC by western blot analysis with an anti-human CD3� antibody (C). (D) NK92 cells expressing anti-CD20 chimeric receptors or expression GFP only were incubated with BATDA labeled targets: CD20+ NK sensitive Ramos, CD20+ NK resistant Daudi and the CD20- pre-B-ALL cell line RS4;11 at the indicated E/ T ratios for 4 hours. Europium release assays were performed according to the manufacturer's instructions. Each data point represents the mean (SEM; n=3). Average values are reported as the mean {plus minus} SEM. p values using unpaired student t test were noted in (D).
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.
