PBMC Cytokine Release Assay (CRA) – T Cell Immunogenicity Risk Panel

• The development of anti-drug antibodies (ADA) towards peptide drugs can alter their bioavailability and pharmacokinetics in vivo resulting in a reduction in overall drug efficacy.¹
• The presence of ADA also negatively impacts the safety of these therapeutics by increasing the likelihood of adverse reactions being observed in patients to whom the drug is administered.²
• While ADA generation can occur via B-cell dependent pathways,³ the immunogenic response of biotherapeutics is predominantly T-cell mediated and governed by epitopes found in their primary sequences.⁴
• Plate-based approaches centring on evaluating the immunogenic risk posed by therapeutic monoclonal antibodies (mAbs) have identified IL-2 as the most predictive cytokine readout,⁵ although IFN-γ was also shown to have appreciable utility in this regard.^5-6
• Our standard T-cell immunogenicity risk panel measures the release of IL-2 & IFN-γ released by PBMC upon exposure to test articles 6 days post challenge.
• We have demonstrated the utility of this panel by screening a selection of mAbs whose immunogenicity risks have been widely documented in the literature (Table 1).
• Our standard assay uses Phytohemagglutinin-L (PHA-L) as a positive control to induce the release of cytokines associated with T-cell activation. When screened as part of our CRA, cellular exposure to PHA-L resulted in an increase in the levels of both IL-2 and IFN-γ (Fig 1).

_{1) De Groot AS & Scott DW, (2007). Immunogenicity of protein therapeutics. Trends Immunol., 28; 482–90.
2) Hansel TT et al., (2010). The safety and side effects of monoclonal antibodies. Nat. Rev. Drug Discov., 9; 325-38.
3) Vaisman-Mentesh et al., (2020). The Molecular Mechanisms That Underlie the Immune Biology of Anti-drug Antibody Formation Following Treatment With Monoclonal Antibodies. Front. Immunol., 11, 1951.
4) Jawa V et al., (2020). T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation–Updated Consensus and Review. Front. Immunol., 11:1301.
5) Joubert MK et al., (2016). Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics. PLoS One. 11 (8); e0159328.
6) Rombach-Riegraf V et al., (2014). Aggregation of human recombinant monoclonal antibodies influences the capacity of dendritic cells to stimulate adaptive T-cell responses in vitro. PLoS ONE. 9; e86322.}