Indirect assessment of neutralizing anti-drug antibodies utilizing pharmacokinetic assay data.

Neutralizing anti-drug antibodies (NAbs) can adversely impact efficacy and safety of biologic therapeutics. However, current assay formats to detect NAbs are limited in their use during the dosing phase due to interference by circulating drug, resulting in low drug tolerance. To improve the drug tolerance for NAb detection, an alternative approach for indirect NAb (iNAb) assessment was developed and qualified that uses a combination of pharmacokinetic (PK) assays to measure the serum concentrations of free and total drug. It is demonstrated that the ratio of free to total drug concentrations, referred to as F/T ratio, is a novel PK parameter that can indicate neutralizing activity in test samples. The iNAb assessment correctly identified NAb-positive samples with high drug concentrations that led to false negative results in a conventional NAb assay. Moreover, iNAb reliably distinguished between NAbs and non-neutralizing anti-drug antibodies over a wide range of concentrations. A proposal on how to deploy iNAb assessment within a broader immunogenicity testing strategy is presented.

A signaling-enhanced chimeric receptor to activate the ICOS pathway in T cells.

Activation of the inducible costimulator (ICOS) signaling pathway in T cells is difficult to assess with bioassays, because most T cell lines do not constitutively express ICOS. Additionally, engagement of ICOS by its natural ligand B7 related protein 1 (B7RP1) is insufficient to elicit ICOS signaling, but requires simultaneous costimulation of the T cell receptor (TCR) to be effective. Here we describe a genetically engineered human T cell line that expresses a chimeric receptor (ICOS-CD3) consisting of full-length human ICOS fused at its C-terminal end to the cytoplasmic domain of human CD3 zeta. When engaged by B7RP1, ICOS-CD3 initiated signaling independently of TCR costimulation and induced substantially more IL-2 secretion in Jurkat T cells compared to wildtype ICOS. We demonstrate that this signaling-enhanced chimeric receptor can be used in simple and sensitive bioassays to detect bioactive B7RP1, anti-B7RP1 drugs, and the presence of corresponding neutralizing anti-drug antibodies.