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Immune checkpoint (IC) receptors play critical roles in maintaining immune homeostasis and have emerged as proven therapeutic targets for the treatment of various cancers and autoimmune disorders. Monoclonal antibodies (mAbs) designed to block immune inhibitory receptors PD-1 and CTLA-4 have shown unprecedented efficacy in cancer treatment. This led to rapid expansion of mAb discovery programs to many more IC receptors including members of the T cell immunoreceptor with Ig and ITIM domains (TIGIT) - CD112R - CD96 Axis. TIGIT binds to its ligands CD155 (PVR) and CD112 (PVRL2) while CD112R (PVRIG) binds to CD112 and CD96 (TACTILE) binds to CD155. Binding of TIGIT/CD112R to CD155/CD112 inhibits the co-stimulatory activity of the paired receptor CD226. Human CD96 can have co-stimulatory or co-inhibitory activity based on the cell type and context. Thus, therapeutic targeting of this axis can modulate T cell and NK cell activation and unleash anti-cancer immunity. One of the bottlenecks of immunotherapy mAb development is the lack of quantitative and reproducible bioassays for functional analysis and potency determination. Existing methods rely heavily on primary immune cells, which is labor- and cost-intensive and highly variable. To address this need, we developed a panel of cell-based reporter bioassays that can quantitatively measure the potencies of mAbs targeting TIGIT/CD155, TIGIT/CD112, CD112R/CD112, and CD96/CD155. Owing to the clinical combination of TIGIT blockade with PD-1/PD-L1 blockade, we further developed a potency bioassay for bispecific antibodies simultaneously targeting TIGIT and PD-1/PD-L1. These bioassays each consist of an engineered T effector cell line that expresses T cell receptors and IC receptors of interest, and an engineered artificial antigen presenting cells (aAPC) expressing the cor responding ligands for the IC receptors. The T effector cell lines also express a luciferase reporter driven by a promoter specifically responding to TCR activation which can be inhibited by signaling from the IC receptors. Additionally, we developed bioassays that can measure potencies of agonist or antagonist mAbs targeting CD226. These bioassays are designed to reflect the mechanisms of action for the drug candidates designed for each IC receptor and the assay signals are specific. Most are prequalified according to ICH guidelines and show the precision, accuracy and linearity required for cGMP environments. Therefore, these MoA-based bioassays can serve as valuable tools for early drug discovery, potency determination and stability studies throughout the development of mAbs targeting the TIGIT/CD112R/CD96 axis. Citation Format: Steven Edenson, Jamison Grailer, Denise Garvin, Frank Fan, Jim Harnett, Mei Cong. MoA-based potency bioassays for immunotherapy programs targeting the TIGIT/CD112R/CD96 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6651. |
