Assay development considerations to improve drug tolerance in direct competitive ligand binding neutralizing antibody assays, pretreatment strategies.

Neutralizing anti-drug antibodies (ADAs) may affect safety, efficacy, and pharmacokinetic profile of a biotherapeutic drug and thus their assessment is of particular importance during immunogenicity testing. Neutralizing antibody (NAb) assays typically rely on NAbs ability to block the drug-target interaction. Higher NAb concentration and/or higher binding affinity of NAb to the drug, lowers the drug-target binding interaction. However, in the presence of high concentrations of residual circulating drug, as often seen for drugs with longer half-lives or in repeat-dose studies, NAbs may exist as drug bound complexes. In direct NAb assay formats, the NAb-drug complexes present in the sample could result in the NAb being unable to block the drug-target interaction eventually leading to a false negative response. The residual free circulating drug present in the sample may bind to the target in the NAb assay thereby competing with the drug used in the assay and inhibiting the assay signal, leading to a false positive response. For traditional ADA assays, multiple approaches involving acid treatment have been described to mitigate circulating drug interference issue. Here, we report two acid-treatment approaches that utilize the Dynabeads extraction with acid dissociation and Affinity Capture Elution (ACE) principle to improve drug tolerance in NAb assays.

Avelumab internalization by human circulating immune cells is mediated by both Fc gamma receptor and PD-L1 binding.

Avelumab is an IgG1 anti-programmed death ligand 1 (anti-PD-L1) monoclonal antibody that has been approved as a monotherapy for metastatic Merkel cell carcinoma and advanced urothelial carcinoma, and in combination with axitinib for advanced renal cell carcinoma. Avelumab is cleared faster and has a shorter half-life than other anti-PD-L1 antibodies, such as atezolizumab and durvalumab, but the mechanisms underlying these differences are unknown. IgG antibodies can be cleared through receptor-mediated endocytosis after binding of the antibody Fab region to target proteins, or via Fcγ receptor (FcγR)-mediated endocytosis. Unlike other approved anti-PD-L1 antibodies, avelumab has a native Fc region that retains FcγR binding capability. We hypothesized that the rapid clearance of avelumab might be due to the synergistic effect of both FcγR-mediated and PD-L1 target-mediated internalization. To investigate this, we performed in vitro and in vivo studies that compared engineered variants of avelumab and atezolizumab to determine mechanisms of cellular internalization. We found that both FcγR and PD-L1 binding contribute to avelumab internalization. While FcγR binding was the dominant mechanism of avelumab internalization in vitro, with CD64 acting as the most important FcγR, studies in mice and cynomolgus monkeys showed that both FcγR and PD-L1 contribute to avelumab elimination, with PD-L1 binding playing a greater role. These studies suggest that the rapid internalization of avelumab might be due to simultaneous binding of both PD-L1 and FcγR in trans. Our findings also provide a basis to alter the clearance and half-life of monoclonal antibodies in therapeutic development.