Abstract 6308: Development of a pharmacological platform to study in real time immune checkpoints signaling pathways: validation with therapeutic mAbs and small molecules

Abstract

Abstract Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therapeutics that block inhibitory receptors or activate immunostimulatory checkpoint receptors have proven to be powerful agents for the restoration of anti-tumor immune responses. While some antibodies targeting these checkpoint receptors have been tested and approved for use in man, many others are in development. For the same targets, there are also small molecules being tested in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, we have successfully developed and validated immune checkpoint pharmacological assays based on our proprietary BRET technology, bioSensAllTM. In these assays, the activation or blockade of the signaling of checkpoint receptors are assessed. PD-1 is expressed on T-cells, while it's ligands, PD-L1 or PD-L2, are expressed on the surface of tumor cells or antigen presenting cells. Like many other immunoglobulin receptors, PD-1 harbors immunoreceptor tyrosine inhibitory motifs (ITIMs) in its cytoplasmic tail that are important signaling motifs. When its ligand, e.g. PD-L1, binds to PD-1, Src family kinases phosphorylate the ITIM motif, resulting in the recruitment of SH2-domain containing phosphatases, SHP-1 and SHP-2, which are involved in inhibiting the T-cell response. As a translation of this concept, we present PD-1 signaling assays using our proprietary BRET technology. For each inhibitory co-receptor, two types of assays were generated. In the first assay, cells expressing the PD-1 and SHP-2 proteins, fused respectively to rGFP and rLucII, are co-incubated with ligand-presenting cells. This results in PD-1 activation and SHP-2 recruitment to the PD-1 receptor, generating a specific BRET signal. In the second assay design, PD-1 positive cells are transfected with SHP-2 and membrane anchor protein, fused respectively with rLucII and rGFP. Upon target engagement following co-culture with cells expressing PD-L1, translocation of SHP-2 close to the membrane triggers a BRET response. Interestingly, the BRET signal is inhibited with either neutralizing antibodies (against PD-L1 or PD-1) or inhibitory small molecules. Further validation was obtained for PD-L2/PD-1 and CD86/CTLA-4. The Immune Checkpoint Platform shows a good accuracy and robustness. These spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Based on their complementarity, those assays can be used in tandem for primary and secondary screenings and offer a strong and reliable platform for drug discovery. Citation Format: Alice Gentil Dit Maurin, Christel Franchet, Xavier Leroy, Stephane Schann. Development of a pharmacological platform to study in real time immune checkpoints signaling pathways: validation with therapeutic mAbs and small molecules [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6308.