Figure 4. Specificity and safety in vitro. (A) Cytokine release in PBMC cultures in the presence of the CD30/CD16A TandAb. 5 × 10⁵ human PBMC with and without 1 × 10⁴ CD30⁺ KARPAS-299 cells, or 1 × 10⁴ KARPAS-299 cells alone were cultured in the presence of increasing concentrations of the CD30/CD16A TandAb (denoted by TandAb; at 0.001–10 µg/mL), 10 µg/mL OKT3, or without antibody (denoted by 0). The concentrations of secreted TNF and IFN-γ were quantified using multiplexing after 24 h incubation. Results from one representative experiment are shown. (B and C) Assessment of bystander cell killing in a cytotoxicity assay with mixed target cells. 1 × 10⁴ calcein-labeled CD20^-/CD30⁺ KARPAS-299 target cells, combined with 1x10⁴ unlabeled CD20⁺/CD30^- Raji target cells (B) or 1 × 10⁴ unlabeled KARPAS-299 combined with calcein-labeled Raji cells (C), were incubated for 3 h, together with increasing concentrations of the CD30/CD16A TandAb or rituximab (MabThera, Roche, Hertfordshire, UK), in the presence or absence of 5 × 10⁴ enriched human NK cells as effectors for 3 h in a cytotoxicity assay. The percentage of specific target cell lysis was calculated and the mean and SD from duplicates were plotted.