Figure 6.

CD20-expressing NKG2D CAR-T cells can be removed through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity

(A) Schematic representation of CD20-expressing piggyBac transposon. NKG2Dbp CAR and full-length CD20 are expressed in a polycistronic transcript separated by the P2A self-cleaving peptide. (B) Expression of CAR and CD20 on 21-day NKG2D CAR-T and NKG2D-CD20 CAR-T cells. Flow cytometry data shown are representative of three independent experiments. (C) Expansion of NKG2D-CD20 CAR-T cells and NKG2D CAR-T cells is achieved with the same methodology described in Figure 2A. Expansion folds shown are single measurements from three healthy donors on day 21 post-activation. (D) The cytotoxicity of NKG2D-CD20 CAR-T cells was assessed in parallel with donor-matched NKG2D CAR-T cells against CAOV3 and HCT-116. Data shown are mean ± SD of triplicates from a representative experiment (n = 3). (E) ADCC assay was performed with 21-day CAR-T cells and 17-day NK cells. Data shown are representative of three independent experiments. (F) CDC assay was performed by incubating CAR-T cells with baby rabbit complement and anti-CD20 antibody (rituximab). Data shown are pooled from two independent experiments.