Comparison of the mIgM-binding and apoptosis-inducing properties of a large panel of bivalent anti-IgM MoAbs with differing intrinsic affinity and differing IgM domain specificity. The capacity of the bivalent MoAb to bind Ramos mIgM was assessed by indirect immunofluorescence following 30 minutes of incubation of Ramos cells with 10 μg/mL of MoAb at 4°C (MoAb:cell ratio = 1 μg/105 cells). Data are indicated as the MFI (□). The MoAbs are listed (left to right) in descending order of their mIgM binding potential by this assay. With the notable exception of MoAb XG9, there was a relatively good correspondence between relative binding potential of the bivalent MoAb and the previously established Ka values for binding of MoAb Fab′.30 The capacity of these ligands to induce apoptosis was assessed by culturing Ramos cells with 10 μg/mL of each MoAb (MoAb:cell ratio = 2 μg/105 cells) for 42 hours and subsequent staining with FITC-annexin. The later results are shown as MFI FITC-annexin bound (•) as well as the percentage of annexin-positive cells (○); (mean ± SD from 2 experiments). A further replicate experiment in which all the MoAbs, with the exception of Mu18, were evaluated for MoAb binding or capacity to induce annexin-positive cells upon in vitro culture yielded similar results to those shown above.
