Abstract 1308: Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151
Authors: Cardenas Juan M. , Kotha Jayaprakash , Kotha Sridevi , Jennings Lisa K. , Graham Michelle , Lambert Stacie , Hew Kinjal 
Published: July 2021

Abstract
Introduction: GARP is a cell surface protein expressed post activation on regulatory T-cells (Treg), monocytes, B cells, and platelets. GARP binds to latent transforming growth factor beta (TGF-β1) and regulates the release of active TGF-β1. TGF-β1 modulates Treg-mediated immunosuppression and represses effector T cell responses. ABBV-151 is a first-in-class monoclonal antibody that binds to the GARP-TGF-β1 complex and blocks active TGF-β1 release. Here we describe the validation of a TE assay for ABBV-151 using human platelets to measure receptor occupancy (RO) and binding saturation, that will inform dosing decisions during a first-in-human Phase I dose escalation study.
Procedure: A competing (LHG10.6 that recognizes the GARP-TGF-β1 complex) and non-competing (1E7 that recognizes total GARP) anti-GARP antibodies were used to measure TE on platelets. Human whole blood was incubated with increasing concentrations of ABBV-151, followed by platelet-rich plasma (PRP) preparation and storage at -80°C. Platelets were thawed, washed, and stained with a fluorescently labeled antibody cocktail (LHGH10.6, 1E7 and CD61) and analyzed on a flow cytometer. The analytical measurement range, inter-operator and inter-assay precision and sample stability at -80°C (up to 12 months) for this TE assay was determined.
Results: ABBV-151 TE assay demonstrated a dose response relationship over a range of ABBV-151 concentrations (0.08 to 400 ng/mL) that corresponded to 0-80% occupancy, with an EC50 of 6.2 ± 2.1 ng/mL, and 0-100% saturation with an EC50 of 6.7 ± 1.9 ng/mL. Inter-assay precision demonstrated ≤3% CV at low ABBV-151 doses, and ≤14% in samples at high doses. Inter-operator precision for untreated and treated samples was between 3 to 9% CV, respectively. The stability data indicated frozen platelets could be stored up to 12 months with highly reproducible assay performance.
Conclusions: This TE assay for ABBV-151 on platelets demonstrates desirable analytical performance characteristics. The percent saturation (PS) showed higher precision than the RO measurements for this assay and PS was determined to be a reliable metric for measuring TE. Frozen platelets samples were suitable for the TE assay for up to a year allowing for batch processing of samples at a central lab and for minimizing the variability in test results. This novel TE assay overcomes key limitations of implementing TE assays in clinical trials, and quantification of PS using this TE assay in clinical trials is currently ongoing to assess the pharmacodynamic/pharmacokinetic relationship of ABBV-151 therapy.
Citation Format: Juan M. Cardenas, Jayaprakash Kotha, Sridevi Kotha, Lisa K. Jennings, Michelle Graham, Stacie Lambert, Kinjal Hew. Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1308.