A new bioassay for the immunocytokine L19-IL2 for simultaneous analysis of both functional moieties

Currently, cancer directed new biological entities (NBEs) in the pharmaceutical R&D pipelines are derived from monoclonal antibodies in various formats, such as immunocytokines. Generally, immunocytokines are bi-functional molecules that consist of a specific targeting antibody-based portion and a linked cytokine. To confirm the quality of the drug product both moieties have to be characterized using appropriate techniques. Until now, the binding capacity of antibodies is usually examined by ligand binding assays whereas the biological activity of the linked cytokine is determined by cell-based potency assays. However, the simultaneous analysis of both functional moieties in a single assay format has not been described so far. In this paper we present a newly designed bioassay format for the anti-cancer immunocytokine L19-IL2, comprising of the human vascular targeting single-chain Fv L19 and human interleukin 2 (IL2). This new potency assay allows simultaneous analysis of both moieties, thus specific L19 binding capacity and the ability of IL2 to induce the proliferation of the detector cytotoxic T-cell line CTLL-2. Assay development was performed with special focus on application of different fitting models for the sigmoid dose-response curves to evaluate the influence of model optimization on the validity of assay results. For assay validation generally accepted characteristics were determined. Assay specificity was shown by testing L19-IL2 related compounds. All other validation parameters were derived from 25 batch runs using five nominal L19-IL2 concentrations, covering a range from 60% to 140% of the standard's potency. Accuracy ranged from -3.4% to -6.9% relative error (%RE). Interbatch precision ranged from 6.1% to 10.6% coefficient of variation (%CV). For assay linearity a coefficient of determination (R(2)) of 0.9992 was found. Assay robustness was shown with L19-IL2 samples after three freeze-thaw cycles and also with different cell passages of the used cytotoxic T-cell line. Based on the data, we conclude that this assay is valid for potency estimation of the immunocytokine L19-IL2. Moreover, this format represents a major improvement compared to other approaches which only allow potency evaluation of both functional moieties in separate assays. In general the underlying assay principle described seems suitable for potency determination of other immunocytokines.