(W1230-02-07) EpiScreen®2.0 Time Course Assay: A Sensitive and Data Rich Tool for Pre-Clinical Immunogenicity Testing of Biologics

Purpose: The purpose of this study was to develop a next-generation immunogenicity assay that does not compromise sensitivity, while delivering data on specificity and mechanism of action. The result is an improved assay which increases confidence in pre-clinical immunogenicity risk assessment data and lead selection.

Methods: PBMC were isolated from healthy community donor leukopaks. A cohort of 50 donors was selected and HLA typed. Each test sample (Herceptin, Abciximab, Atezolizumab, ATR-107, CEFT, KLH) were added to the PBMCs, and cultures were incubated for a total of 8 days at 37°C with 5% CO2. Cells were analyzed by flow cytometry at 3 time points. Cells were stained for CD3, CD4, CD25, CD134 (OX-40) and viability, while EdU uptake was readout for proliferation (Figure 1).

Results: EpiScreen 2.0 specifically assessedCD4+ T-cell proliferation via flow cytometry, providing more relevant information than a bulk readout. Proliferation assessment via EdU incorporation has a wider assay window than the traditional [3H]-thymidine method, offering better ranking of immunogenicity. Furthermore, the activation-induced markers CD25 and OX-40 help to pinpoint samples with higher risk of immunogenicity (Figure 2 and Table 1). The new assay further allows the monitoring of other cell populations, such as CD8+ activation, thereby providing insights into the mechanism of action – particularly useful for gene therapy where vectors can enter an indirect antigen processing pathway.

Conclusion: This assay presents a new and much improved pre-clinical immunogenicity testing method for biologics, which is sensitive, specific, data-rich, MoA-reflective and customizable. 

References: Stempels, F.C. et al., 2022. A sensitive and less cytotoxic assay for identification of proliferating T cells based on bioorthogonally-functionalized uridine analogue. Journal of Immunological Methods 502: 113228

Poloni C. et al., 2023. T-cell activation-indiced marker assays in health and disease. Immunology and Cell Biology 101: 491-503

Figure 1. Schematic of EpiScreen® 2.0 assay

Figure 2. EpiScreen® 2.0 assay performance. Graphs show average stimulation index (SI) for positive responding donors vs % positive donors.

Table 1. EpiScreen® 2.0 assay proliferation and activation responses.