(W1330-02-07) Enhancing Antibody Detection for PABAD-Based Neutralizing Antibody Detection and Validation

Purpose: Effective detection of neutralizing antibodies (NAb) is critical as NAbs reduce drug efficacy and interfere with drug binding to target, and even have the potential to cause life-threatening adverse events. Differentiating between antibodies that bind the drug but do not neutralize and NAbs can be challenging, especially for indications such as cancer where high drug levels in patients can interfere with detection. To detect NAbs and reduce drug interference, we used a novel method previously developed by our group, PEG precipitation, Acid dissociation and Biotin-drug as an Assay Drug (PABAD). The PABAD method uses polyethylene glycol (PEG) precipitation to selectively precipitate the NAb bound to drug by excluding them from solvent. PABAD has several advantages over conventional industry methods including no tedious magnetic bead use, one shorter acid incubation as opposed to two and significantly less Biotin-drug utilization. Most significantly, the PABAD method can tolerate high levels of drug in patient samples, in this method up to 400 µg/mL. This project aimed to understand why certain NAbs were more compatible with the PABAD method than others and how to optimize the method to better detect a broader range of NAbs from patient samples in indications with high probability of drug interference.

Methods: Using a competitive ligand binding assay format, we assessed differences in inhibition of two potential NAb positive controls (PCs) with the PABAD method. LC/MS was used to determine recovery of the NAbs, drug and serum IgGs after PEG precipitation. To better visualize PC interactions with drug, mass photometry provided information on different molecular weight species in the mixture.

Results: Of the potential NAbs tested as positive controls, two demonstrated neutralizing ability by effective inhibition of the drug, with similar binding kinetics and comparable humanized backbones. Both demonstrated stability in acid and effective separation of the PC and drug complex upon treatment. PC 1, however, was not compatible with PEG precipitation while PC 2 demonstrated robust drug tolerance and sensitivity. LC/MS assays were developed to better determine recovery of the PC, drug and IgG carryover after PEG-NaCl precipitation to determine why PC 1 was incompatible with the PABAD assay. Using unique signature peptides targeting the PC isotypes and the drug CDR region, LC/MS confirmed NAb assay results with robust recovery of PC 2 compared to PC 1. LC/MS data further demonstrated precipitation of free drug and serum IgG, dependent on the percentage of PEG-NaCl, with ideal recovery of the PC and drug complex at 5% PEG-NaCl. Mass photometry, a technique that uses light scattering to determine molecular weights of molecules, was used to test the interactions of each of these PCs with drug. Mass photometry demonstrated a difference in molecular weight, with PC 2 around 161 kD while PC 1 was around 172 kd, possibly due to post-translational modifications, which may affect drug interactions. The interactions of each of these NAbs with increasing concentrations of drug also differed, at a 1:1 NAb:drug ratio PC 2 had a higher range of high molecular weight peaks compared to more defined peaks for PC 1. Increasing the ratio of NAb:drug to 1:10 consolidated the peaks for PC 2 compared to PC 1, suggesting increased stability upon interaction with increasing concentrations of drug. Competitive ligand binding assay data confirmed that the interaction of PC with drug, even in the absence of PEG, can lead to differences in measured percent inhibition between both these NAbs. Furthermore, reduction of drug levels for PC 1 led to enhanced recovery after PEG precipitation, implying that further removal of free drug may improve recovery of this PC.

Conclusion: Sample pretreatment to reduce free drug in NAb assays is a requirement for indications with high dosing levels. The PABAD method is compatible with very high levels of system drug and allows removal though PEG precipitation. We continue to use tools and cutting-edge technology to further optimize PABAD conditions to improve recovery of PC 1 and other NAbs that are less compatible with PEG precipitation. Doing so expands the breadth of NAb detection from high dose treated patients.

References: Wickramarachchi D, Wagner J, Woo T, Ferrari F, Steinmetz T, Helmy R, Xu W. A Novel Neutralization Antibody Assay Method to Overcome Drug Interference with Better Compatibility with Acid-Sensitive Neutralizing Antibodies. AAPS J. 2023 Jan 25;25(1):18. doi: 10.1208/s12248-023-00783-9. PMID: 36697923.

Acknowledgements: The authors declare no conflicts of interest