Figure 2.

(A) Schematic representations of different fusion proteins containing the Fab portion of Hc5 anti-Id mIgG in the form of IgD (Fab-IgD), Fab-glycophorin A (Fab-GlycoA), and Fab-glycophosphatidylinositol (GPI) signal sequence isolated from decay accelerating factor (DAF), which could be converted to Fab-GPI anchored attachment on the plasma membrane (Fab-GPI); (B) Flow cytometry analyses on SP2/0 cell lines that were either untreated or transfected with expression vectors for Hc5 Fab-IgD (co-transfection with Igα expression vector) (IgD + Igα), Fab-GlycoA; or Fab-GPI. Surface expression of SM03 binding moiety was evaluated with 0.01, 0.1 and 1 μg/ml of SM03, and revealed with FITC-conjugated goat anti-human Fc-specific antibody. Only marginal surface expression was observed with Fab-IgD, and intense dose response SM03 staining were detected with the Fab-GlycoA and Fab-GPI cells; (C) Cell cytotoxicity studies on: (i) Ramos lymphoma cells induced by different concentrations of immobilized (hyper-crosslinked) SM03. Induction of cell cytotoxicity was not dose-dependent; (ii) Ramos lymphoma cells induced by SM03 (□) and SM09 (anti-CD20) (▪) in a standard CMC assay, in which only SM09 specific for the non-internalizing CD20 exhibited dose-response CMC killing (positive control); and (iii) surrogate cell lines expressing surface anti-SM03 (Hc5 anti-Id) Fab in the form of Fab-IgD (□), Fab-GPI (○), and Fab-GlycoA (•) induced by SM03 in a standard CMC assay, in which significant dose-dependent SM03 mediated CMC activities were detected with Fab-GPI and Fab-GlycoA cells, with marginal killing on Fab-IgD cell. The EC50 for Fab-GPI and Fab-GlycoA were determined to be 0.216 and 0.133 μg/ml, respectively. No CMC activities were detected when these surrogate target cells were treated with irrelevant antibodies, including rituximab (anti-CD20), infliximab (anti-TNF) and cetuximab (anti-EGF receptor) (negative controls) (data not shown).