Choice of labeling and cell line influences interactions between the Fab fragment AbD15179 and its target antigen CD44v6

Medical imaging by use of immunotargeting generally relies on a labeled molecule binding to a specific target on the cell surface. It is important to utilize both cell-based and time-resolved binding assays in order to understand the properties of such molecular interactions in a relevant setting. In this report we describe the detailed characterization of the interaction properties for AbD15179, a promising CD44v6-targeting antibody fragment for radio-immunotargeting. Influence of labeling and cell-line model on the protein interaction kinetics was assessed using three different labeling approaches ((111)In, (125)I and FITC) on three different squamous carcinoma cell lines. Interactions were measured using time-resolved assays on living cells, and further analyzed with Interaction Map®. Results demonstrated a general biphasic appearance of a high- and a low-affinity binding event in all cases. The relative contribution from these two interactions differed between conjugates. For (125)I-Fab, the population of low-affinity binders could be significantly increased by extending the chloramine T exposure during labeling, whereas the (111)In-labeling predominantly resulted in a high-affinity interaction. Interactions were also shown to be cell line dependent, with e.g. SCC-25 cells generally mediating a faster dissociation of conjugates compared to the other cell lines. In conclusion, we report both cell line dependent and labeling associated variations in interaction kinetics for AbD15179 binding to CD44v6. This has implications for cell-based kinetic assays and applications based on labeled conjugates in general, as well as in a clinical setting, where each individual tumor may create different kinetic profiles for the same conjugate.