(M1030-01-06) Investigating the Impact of a Soluble Target on ADA Measurements in a Bispecific Antibody Clinical Study

Purpose: Anti-A/B is a bispecific antibody therapeutic being evaluated in an early dose-escalation clinical study. It is designed to target membrane antigens A and B in order to elicit anti-tumor activities. In addition to the membrane antigen format, soluble target A (sA) is also expected in circulation as a dimer. Levels of sA in drug naïve subjects are at the low pg/mL level, however elevated levels of sA post-treatment are expected due to the formation of anti-A/B and sA complexes. Therefore, the elevated levels of sA has the potential to interfere with the ADA assay used for anti-A/B immunogenicity monitoring, which is a homogeneous ELISA utilizing biotin- and digoxigenin (DIG) - labeled anti-A/B to capture ADAs in patient samples. During ADA method validation, interference of sA was not observed at the tested level (100-fold of the baseline levels). However, since the exact post-treatment levels of sA are unknown, patient samples in this early clinical phase trial are closely monitored to ensure that the positive ADA results are not due to false signals caused by sA. Here we describe the post-treatment levels of sA in patients and the investigation of sA interference in the anti-A/B ADA measurements.

Methods: Soluble target sA was measured using SMCxPRO (MilliporeSigma), an ultrasensitive fluorescence immunoassay platform. Soluble target sA in samples, controls, and standards is captured with an in-house generated rabbit monoclonal antibody against sA coated on a microtiter plate, and detected with an AlexaFluor 647 conjugated rabbit monoclonal antibody against one arm of anti-A/B. Fluorescence was measured using an SMCxPRO Instrument. The ADAs were measured in a homogeneous ELISA using biotin- and DIG- labeled anti-A/B to capture ADAs from patient samples. Signal is detected with an horseradish peroxidase (HRP)-conjugated mouse anti-DIG antibody. The immunogenicity testing for anti-A/B follows a tiered testing approach consisting of screening, confirmatory, and titering assays. The assay parameters were evaluated during assay validation according to previously described recommendations¹.

Results: In screening samples (collected at baseline), all patients showed low levels of sA, with a median sA level of 22.3 pg/mL. Following treatments of anti-A/B, patients showed an increase in sA levels in a dose-dependent manner, with high within-cohort variability in the highest dosed cohort. Out of 12 patients in this early phase trial who were evaluable for both sA and ADA status, 8 (75%) were determined to have ADAs against anti-A/B following the screening and confirmatory assays. The titers for the 8 ADA positive patients ranged from < 1.70 to 4.71. In these patients, ADA titer was not determined to be correlated to with treatment dose level. In addition, patients in the higher dose cohorts had the higher sA levels (ranging from 1305.0 pg/mL to 9394.2 pg/mL) but without clear correlation to the ADA responses. Therefore, sA interference was precluded.

Conclusion: Elevated levels of sA  are expected post anti-A/B treatments as a result of target engagement by anti-A/B, which could potentially cause false positive readouts in the ADA measurements.   Monitoring the sA levels in anti-A/B treated patients  revealed that sA levels became upregulated in a dose-dependent manner following anti-A/B treatments, however a correlation between sA levels and ADA status or ADA titers was not observed. Our investigations suggest that while sA became elevated after anti-A/B treatments, it did not interfere with our immunogenicity assessments.

References: 1. U.S. Department of Health and Human Services Food and Drug Administration (FDA), Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER). (2019). Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection.