(T1230-01-04) Method Development and Validation of an Antidrug Antibody Assay for a Novel Bispecific Biotherapeutic – Overcoming Challenges from Pre-Existing Antidrug Antibody and Target Interferences

Purpose: Antibody-based biotherapeutics have evolved in recent years from conventional monoclonal antibodies (mAb) to more complex structures containing more than one functional domain to achieve advanced therapeutic effects. These novel multi-domain biotherapeutics not seen in nature may potentially hold greater risk for immunogenicity and present unique challenges for immunogenicity assessment due to their more bioengineering modifications and complex manufacturing process as compared to conventional mAb. BI X is a novel tetravalent bispecific antibody with two targets A and B, developed for the treatment of wet age-related macular degeneration. It was required to develop and validate a plasma antidrug antibody (ADA) assay to evaluate immunogenicity of BI X after intravitreal injection into monkeys. During the method development, both pre-existing ADA (PE-ADA) and target interferences were observed in negative control (NC) samples, which significantly impacted the assay’s key parameters, such as cut point (CP), sensitivity and drug tolerance. This work aimed to develop and validate a monkey plasma ADA method through overcoming the challenges of PE-ADA and target interferences to support the intravitreal monkey study.

Methods: Reference standard BI X, anti-BI X antibody (positive control), and other critical reagents were prepared in house. Drug-bridging MSD^® Electrochemiluminescence (ECL) method was used. The ADA assay has two tiers, i.e., screening and confirmatory. Briefly, in the screening step, study samples, positive controls, and negative controls are co-incubated with biotin-labeled BI X and ruthenium-labeled BI X. Antibodies to BI X present in the samples bind to both the biotin- and ruthenium-labeled BI X. Complexes are captured via the binding of biotinylated BI X to streptavidin-coated MSD plate. In the presence of tripropylamine-containing read buffer, ruthenium produces a chemiluminescent signal that is triggered when voltage is applied. The resulting chemiluminescence is measured in relative light units (RLU) which is proportional to the amount of ADA present in the samples. The confirmatory step is run in the same manner, except samples are screened both unspiked and spiked with a level of 20 μg/mL BI X in master mix. To minimize the PE-ADA interference, 80 individual monkey plasma samples were analyzed in both screening and confirmatory and 34 individual samples with a percent inhibition of below 20% were pooled to make custom NCs. Potential false positive assay signals from Targets A and B were investigated and false positive assay signals were observed in the presence of Target A. To remove the target interference, Target A receptor was introduced in the assay. In addition to the use of custom pooled NCs and Target A receptor, the ADA assays were further optimized under different experimental conditions, e.g., labeled-drug conjugate challenge ratios & concentrations, buffers, minimum required dilution (MRD).

Results: The ADA assay was developed by using the custom pooled negative control and the target receptor to eliminate the PE-ADA and target interferences. The ADA assays were further optimized through buffer selection, acid dissociation for sample pre-treatment and reagent quality control to ensure assay robustness and reproducibility. Table 1 lists the key parameters and their optimized conditions.The developed ADA assay was transferred to a CRO and validated in the CRO. The validated ADA assays demonstrated high sensitivity, specificity and ruggedness as shown in Table 2 and used for the ADA sample analysis of the monkey intravitreal study. Note: ADA assays are not quantitative methods and data results reported as positive or negative based on the “Cut Point” assessments.

Conclusion: An ADA assay was successfully developed and validated in monkey plasma for the immunogenicity assessment of BI X for an intravitreal monkey study. The method development succeeded in overcoming the challenges of pre-existing ADA and target interferences by using custom pooled negative control and target receptor to mitigate the impact of the interferences on the assay. The validation results demonstrated that the assay is sensitive, specific, drug tolerant and robust suitable for BI X preclinical immunogenicity assessment.

Acknowledgements: The authors would like to thank Stephanie Kostuk, Steven Anderlot, Andrey Konovalov, Hamid Samareh Afsari, and QPS for their support of this project.