(M1330-01-07) A Translational Approach for Measuring Bi-specific Anti-Drug Antibodies in Non-Clinical Studies Using a Drug Tolerant Generic Plug and Play Format

Purpose: Biotherapeutic monoclonal antibody drugs have the potential to affect product safety and efficacy by eliciting an immune response. Understanding unexpected drug clearance and/or adverse safety events through the use of valid, sensitive, specific, and selective anti-drug antibody (ADA) assays is a key aspect of mitigating risk and allowing progression from the non-clinical drug development to clinical drug development. Drug-drug bridging assays are the current state of the art for the detection of ADA. However, these types of assays bear the risk of interference from the high concentrations of circulating drug in non-clinical studies and often require study extension to evaluate ADA formation even when acid dissociation pretreatments are applied. Preclinical safety studies with signs of immune complex disease and without the detection of ADA resulting from high serum drug concentrations is often an issue when attempting to de-risk loss in drug exposure and/or safety findings. To address this issue, a generic drug tolerant translational immunoassay has been developed that can measure ADA in the presence of high circulating drug concentrations at early dosing timepoints and can distinguish between complexed and free ADA.

Methods: A therapeutic bi-specific mAb drug (composed of the target binding regions of Antibody A and Antibody B) was administered to cynomolgus monkeys on Day 1 and Day 8 at a dose of 10 mg/kg intravenously. Monkey serum samples were analyzed for ADA using a stepwise, generic plug and play ADA immunoassay method (Figure 1). ADA were measured using the generic immunoassay by capturing therapeutic mAb drug complexed with ADA using biotinylated anti-human IgG that did not cross react with cynomolgus IgG. ADA bound to the therapeutic mAb were detected using ruthenylated anti-monkey IgG that did not cross react with human IgG. Circulating ADA not complexed to the therapeutic mAb were measured by the addition (spike) of therapeutic mAb drug to the sample and subsequently measuring ADA via the formed mAb drug:ADA complex. By comparing the results from unspiked and drug spiked sample, the complexed or free form of the ADA is translatable. Domain specificity of the ADA were determined by spiking in the therapeutic mAb drug, Human IgG, Antibody A, and Antibody B to unspiked samples that yielded a complexed or free ADA response.

Results: ADA were detected in three of three monkeys given 10 mg/kg using the drug tolerant generic immunoassay. Monkey 1 demonstrated complexed ADA one week after the second dose (Table 1). All subsequent time points for this monkey elicited a free ADA response and demonstrated a broad specificity against the Human IgG, Antibody A, and Antibody B components (Table 2). Monkey 2 demonstrated complexed ADA one week after the second dose. The response remained complexed until the 2016 hour time point, at which time a free ADA response was observed. ADA domain specificity results demonstrated a roughly 3-times higher specific response for Antibody A versus Antibody B. Domain specificity for Human IgG was measurable and near the sensitivity of the analytical method. Monkey 3 demonstrated complexed ADA one week after the second dose. All subsequent time points elicited a free ADA response. ADA domain specificity demonstrated similar results between Antibody A and Antibody B with no specificity observed for Human IgG.

Conclusion: All monkeys dosed with a bi-specific mAb therapeutic at 10 mg/kg intravenously elicited an ADA response. Two of three monkeys elicited domain specific responses. Domain specificity for a bi-specific therapeutic may be ascertained by spiking positive samples with the appropriate negative control (Human IgG), therapeutic mAb drug, and the two parent compounds that comprise the bi-specific therapeutic mAb drug. Using the drug tolerant generic plug and play method for the measurement of ADA in non-clinical species offers the ability to evaluate study samples with high circulating drug concentrations, often immediately after dosing, and provides translatable insight into the form of the ADA. The assay can be applied to any human mAb therapeutic in a non-clinical matrix allowing for a flexible approach to the measurement of immunogenicity study samples.


Figure 1


Table 1


Table 2

Acknowledgements: All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.