Figure 4.

Internalization and cytotoxicity of aTCRA6 ADC variants

(A) Schematic representation of aTCRA6 ADCs. In aTCRA6 DAR2 (left), the anti-TCRA6 heavy chains are C-terminally modified with DBCO-PEG₄-Val-Cit-PAB-MMAE. In aTCRA6 DAR5_2-8 (right), the interchain cysteines of anti-TCRA6 mAb are conjugated to maleimide-PEG₄-Val-Cit-PAB-MMAE. DBCO, dibenzocyclooctyne; PEG, polyethylene glycol; Val, valine, Cit, citrulline; PAB, p-aminobenzyl alcohol; MMAE, monomethyl auristatin E. (B) Cytotoxicity was assessed by exposition of Jurkat TCRA6 target cells and Jurkat WT off-target cells to aTCRA6 DAR2 (0.06–400 nM) and aTCRA6 DAR5_2-8 (0.005–30 nM) for 72 h. Cell proliferation was normalized to untreated control cells (0 nM). Internalization was studied by application of pHAb-conjugated aTCRA6 antibody in varying concentrations (0.04–250 nM) to Jurkat TCRA6 target cells and Jurkat WT off-target cells and incubation overnight. Fold internalization was calculated by the ratio of relative fluorescence units (RFU) of the respective antibody sample and the untreated sample without antibody (0 nM). EC₅₀s were determined using variable slope four-parameter fit. Results are shown as mean, and error bars represent standard deviation derived from experimental triplicates. Data are representative of at least two independent experiments.