In vitro DC:T assay development and miniaturization for Immunogenicity Risk Assessment automation platform

Immunogenicity refers to the scenario where therapeutic proteins induce an undesired immune response, which has the potential to result in severe side effects including loss of drug efficacy and autoimmunity. In vitro T cell assays allow early evaluation of biopharmaceutical’s capability to induce immunogenicity in a selected human population, and thus is implemented on all large molecule drug candidates.

In this study, we automated an in vitro DC:T assay using frozen human PBMC. The DC:T assay is a complicated, multi-step in vitro T cell assay which includes monocyte isolation & differentiation, dendritic cell (DC) maturation, test molecule loading, DC & CD4+ T cell co-culture and T cell proliferation readout. A multiplexed 7 color flow cytometry panel was developed for mature DC phenotyping. Monocyte-derived DC differentiation and maturation was miniaturized from 6 cell to 96 well to enable assay automation. Several key assay steps were established including cell density, culture media selection, cytokine level titration and DC:T ratio. A robust keyhole limpet hemocyanin -induced response was detected with response rate at 95.8% (SI cutoff=2). Test molecules displayed the expected response rank when compared to a previous study.

The successful development of a PBMC-based automated in vitro DC:T assay will enable early-stage immunogenicity risk assessment of drug candidates at higher capacity, faster turnaround, and lower cost.