Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18.

More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.

Evaluation of assay interference and interpretation of CXCR4 receptor occupancy results in a preclinical study with MEDI3185, a fully human antibody to CXCR4.

BACKGROUND: Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell-surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface. METHODS: We developed both a flow cytometry-based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on-cell affinity with the IC50 values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4. RESULTS: We devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose-dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti-drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies.