A CRITICAL COMPARISON OF 3 INTERNALIZATION ASSAYS APPLIED TO THE EVALUATION OF A GIVEN MAB AS A TOXIN-CARRIER CANDIDATE

In the attempt to define a strategy for screening new monoclonal antib odies (mAb) that could be appropriate for clinical application in onco logy, we evaluated the suitability of three methods: a direct internal ization assay (DIA), an indirect internalization assay (IIA) and an in -direct cytotoxicity assay (ICA), by applying them to already selected mAb. The latter were directed against three antigenic systems [38-kDa glycoprotein (gp38), epidermal growth factor receptor, and the neu on cogene product], which, according to their tumor selectivity, could be considered suitable for mAb-guided therapy. The dose-dependent and ti me-dependent binding, as well as the low intra-assay variability, demo nstrated the reliability of the three tests. However, a certain degree of inter-assay variability was observed in each one, the highest valu e being that found when IIA was applied. Furthermore, the degree of va riability, as well as the predictability, seemed to be more related to the mAb/antigen (Ag) combination used rather than to the test applied . From the overall data we suggest a procedure to be applied for scree ning purposes. As a first approach applied to the raw material, ICA is only suitable for screening in the case of an already selected toxin whereas IIA may be helpful to eliminate the true negative mAb. After p urification of the relevant mAb a repeated analysis using DIA could al low the selection of true internalizing mAb. However, this second scre ening should be followed by a further analysis of the fate of the Ag-A b complex after internalization.