(W0930-06-33) Receptor Occupancy Assay Development Challenges for Rare Targets of Bispecific Drugs

Purpose: Receptor occupancy (RO) assays allow for the quantification of binding of therapeutics to their intended targets on the surface of different cell subsets. Flow cytometry-based RO assays are a strong tool for preclinical and clinical development, providing rapid, high-throughput and quantitative measure for PK/PD modelling. Research challenges remain however when RO assays are developed and validated for rare, low expressing targets. Herein we present an example of such an assay, its challenges and the steps taken to resolve performance issues with impact on data and RO calculations.

Methods: We launched development activities for a 6-marker (CD3, CD4, CD8, CD45, CD34, Anti-Human IgG Fc𝛾-PE) flow cytometry panel designed to measure bound drug and total receptor density for a bispecific drug that is a CD3+ T-cell redirector to CD34+ cells in patients with myeloproliferative diseases. During development, a CD34+ cell line was spiked into normal whole blood to mimic the disease state population. Cell line models for rare phenotypes ensure processes are working prior to testing intended use samples. Development included determination of background gating reagents (either FMO, MsIgG1 or MsIgG2b isotypes), drug specific secondary detection reagents, titrations and assessment of the impact of cell autofluorescence on occupancy readout. The three-tube panel design included: markers for population gating, T cell subsets, and CD34+ cells; Isotype or FMO for background staining (Tube 1), anti-human detection reagent to quantify either bound drug (Tube 2), or total receptor density using a drug pre-saturated condition followed by anti-human detection reagent (Tube 3). The objectives of this assay development were: 1) achieve close to 0% binding for the undrugged condition, 2) achieve complete saturation, and 3) observe a robust signal window in a model system prior to moving into intended use samples

Results: Anti-human secondary detection reagent titrations (5-point, 2-fold serial dilution) were performed in the presence and absence of drug saturating conditions. For Bound RO assays in the absence of drug, the secondary detection reagents should mimic the same intensity and frequencies measured by the background determination method (Tube 1). Likewise, the saturated pre-drug spike (Tube 3) should have a significant delta frequency compared to the un-drugged control, mimicking a pre-dose sample (Tube 2, un-drugged). This outcome was not observed, as anti-human secondary binding was upwards of 8.63% for undrugged conditions (Fig. 1) when using the FMO for gate placement. In the first attempt to resolve the background issue, both MsIgG1 and MsIgG2a were evaluated. The recommended isotype for the secondary detection should have been an MsIgG1, but MsIgG2b, the historically higher background option, was attempted, increasing it to 2 x neat. Neither isotype at any titration point reflected the expected match, however MsIgG2b brought the undrugged %Anti-Human IgG Fc𝛾 for the CD34+ population from 87.29% with FMO to 2.14% and was chosen for further experimentation. Optimizing compensation and spillover values are critical factors for background resolution particularly when monitoring biomarkers expressed at low levels. BD’s Comp beads and ThermoFisher’s AbC Comp beads were tested but did not resolve the need for manual compensation adjustments. Reconfiguration of the fluors in the panel was reevaluated. The CD45 fluorophore was changed from APC-H7 to BV510, decreasing manual compensation adjustments. Using the MsIgG2b and CD45 BV510, a panel confirmation was completed consisting of undrugged and drugged conditions to mimic pre-dose and dosed samples, respectively. The CD3+ arm of the drug had successful binding and low background, but re-introduction of the CD34+ cultured cell line for Target Arm maintained poor binding and high background (Fig. 2). As drug binding on the Target Arm was critical to the assay, it was hypothesized that low level apoptosis of the cell line was impacting background autofluorescence. An Annexin V/7-AAD assay (Fig. 3A) was performed demonstrating ~50% viable cells. Selecting for the viable CD34+ population did not allow for robust delta of drugged to undrugged signal (only ~10% delta detected) (Fig. 3B-C). Fresh disease sample blood was used in the redefined panel and recapitulated the findings of increased background of ~15%.

Conclusion: Ultimately, we determined that the drug itself does not bind robustly for a flow cytometry assay in whole blood. We then shifted our focus to other means of PD readout. Despite these findings, our troubleshooting was invaluable in demonstrating the many challenges and potential solutions that can be encountered with RO assays for bispecific drugs targeting rare populations. While background issues with the CD3+ arm of the drug were resolved, the Target Arm remained problematic. We hope that the challenges, attempted solutions, and lessons learned can be beneficial to other studies involving RO assays.

References: Liang, et al., Cytometry B Clin Cytom, 90, 2, 117-27 (2016).


Anti-Human IgG Fc𝛾 titration data. Drugged condition is gated based on population cluster due to the population spread. The 1:8 titer call had the best balance between background and binding with undrugged and drugged 9.14% and 76.04%, respectively.


Panel confirmation for all 3 tubes. A. Drug binding and background looked acceptable for CD3+ T cells. For the undrugged condition, Tube 2 was only 2.49%, demonstrating improved background. For the drugged condition, Tube 2 and Tube 3 were 43.97% and 99.61%, respectively, showing successful binding for the panel. B. While CD34+ binding was low for the undrugged condition at 1.99%, for drugged condition Tube 2 and Tube 3 were 7.03% and T 12.33%, respectively. These findings represented poor binding and high background for this arm of the bispecific drug.


Annexin V assay to detect cell autofluorescence. A. Panel setup to include Annexin V to stain for apoptosis and 7-AAD to stain for necrosis. B. Selection of the CD34+ population of cells and selection of CD34+ Viable cell population, double-negative for Annexin V and 7-AAD. C. Partial Panel Confirmation to investigate background and binding. Removing apoptotic population did not resolve background and binding issues; we observed that CD34+ binding was very poor, with a difference between drugged and undrugged of only 10%, and almost no difference between the Bound (Tube 2) and Saturated (Tube 3) tubes.