Biotherapeutics have the potential to elicit an immune response in patients resulting in the formation of anti-drug antibodies (ADA). The presence of ADA can impact the pharmacokinetics, efficacy, and safety profile of the drug and critically, ADA may cross-react and neutralize non-redundant endogenous counterparts of the drug (Ridker et al., 2017; Casadevall et al., 2002; Bartelds et al., 2007; van Schouwenburg et al., 2013; Kalden and Schulze-Koops, 2017). As such, regulatory agencies require monitoring and characterization of immunogenicity, which is often accomplished using a tiered approach. Samples are first screened and confirmed for the presence of binding antibodies, followed by estimation of antibody titers. Finally, assessment of the neutralizing ability of the ADA is determined where appropriate (FDA, 2019).

Neutralizing antibodies (NAb) are a subset of ADA. NAbs bind epitopes within or near the active site of the therapeutic drug, thereby preventing the drug from binding its target and resulting in inhibition or neutralization of pharmacological activity, and therefore reduced efficacy (Shankar et al., 2014; Group PS, the University of British Columbia MSMRIAG, 2001; Jolicoeur and Tacey, 2012; Kimball et al., 2020). It is imperative to develop assays that consistently and adequately detect NAbs. There are two categories of NAb assays accepted by regulatory agencies: cell-based assays and competitive ligand-binding assays (CLBA). Cell-based NAb assays are functional assays that measure a cellular response (eg, phosphorylation of receptor or downstream target, cellular proliferation, or apoptosis) resulting from activation or inhibition of a signal transduction pathway by the therapeutic drug. The presence of NAbs disrupts this activity resulting in an abrogated response. In the second approach, the CLBA relies on competition between NAb and drug target for binding to the labeled drug. Labeled drug is inhibited from binding its target when NAbs are present, resulting in a change in assay signal. Selection of assay format is based on a variety of factors including therapeutic mechanism of action, assay performance, and expected drug levels (Wu et al., 2016).

Cell-based assays have sometimes been preferred by regulatory agencies for the detection of NAbs. Because these assays utilize cellular response as a measurement to detect inhibition of therapeutic drug activity by NAbs, they provide a direct assessment of the biological function and may thus reflect what occurs in the patient (Wu et al., 2016; Hu et al., 2015; Zhang et al., 2021). However, cell-based assays can have many technical challenges, not limited to operational complexity, poor sensitivity, minimal drug tolerance, high variability, and low serum tolerance (Jolicoeur and Tacey, 2012; Zhang et al., 2021; Finco et al., 2011; Gunn 3rd et al., 2016; Gupta et al., 2007). Therefore, the use of a CLBA may be an alternative option when suitable justifications can be made, such as lack of appropriate cell line, inability to develop a sensitive cell-based assay, or when the drug's mechanism of action is neutralization of a soluble target (Wu et al., 2016; Hu et al., 2015; Zhang et al., 2021; Finco et al., 2011). Several comparative studies of cell-based assays and CLBA for a variety of drug products representing different mechanisms of action have been published demonstrating the feasibility of using CLBA for detection of neutralizing antibodies (Hu et al., 2015; Finco et al., 2011; Wu et al., 2018).

Insulin efsitora alfa (efsitora, basal insulin Fc, LY3209590) is a novel fusion protein designed for once-weekly administration for the treatment of patients with Type 1 diabetes or Type 2 diabetes (Heise et al., 2023). Efsitora is comprised of a novel single-chain variant of insulin fused to a human immunoglobulin (Ig) G2 fragment crystallizable (Fc) domain via a peptide linker (Moyers et al., 2022). Efsitora has been shown to have an in vitro pharmacological profile similar to that of native insulin (Heise et al., 2023; Moyers et al., 2022). To support clinical development of efsitora, we developed a CLBA for detection of anti- efsitora NAbs in human serum. In our current study, we describe the assay method as well as the key development parameters (including sensitivity, drug tolerance, and precision).