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Screening in phenotypic assays is an important strategy for the discovery of innovative drugs and novel targets. Here we present key strategies for developing successful phenotypic screens and prosecuting phenotypic drug discovery (PDD) programs. Successful screens incorporate physiological relevance through the use of human cell types and assay designs that have (1) strong mechanistic connection to clinical outcomes and (2) strong biological justification for both efficacy and safety. In addition to guidance for designing successful screens, we also propose incorporation of specific counterscreens at an early point in the program. The suggested counterscreens are based on analysis of 1000s of drugs and drug candidates profiled through a large set of human-based phenotypic assays. These assays include cytotoxicity in human primary vascular endothelial cells, proliferation of endothelial cells, and proliferation of lymphocytes, all under specific activation conditions. These counterscreens form a generic screening funnel to triage a large fraction of early-stage hits, binning compounds into those with undesirable mechanisms (associated with acute toxicity), mechanisms with utility for oncology indications, and mechanisms useful for autoimmune indications. The application of this screening funnel offers a standardized and more predictable path for prosecuting PDD programs, reducing the risk of failure, and improving program timelines.

Approved immuno-oncology (IO) drugs that have expanded the arsenal of available treatment options for recalcitrant cancers include antibodies to PD-(L)1 checkpoints–a relatively new class of immunotherapies. While the clinical success reported for these drugs is compelling, efficacy is limited to a small cohort of patients. The need for more IO therapeutic options has prompted the pursuit of next-generation candidates that can achieve better patient outcomes. One approach is to screen compounds for novel targets that elicit an immunorestorative response within the tumor microenvironment (TME), consistent with approved IO drugs. Ideally, a small molecule therapeutic would represent a distinct advantage over existing biologics, both in terms of administration as well as ability to optimize chemistry. In order to efficiently identify molecules with IO potential, a high throughput, physiologically relevant screening approach that recapitulates the complexity of the TME is needed. BioMAP Oncology systems are co-cultures of human primary cell types (endothelial cells, fibroblasts, and PBMCs) and cancer cells that model immune suppressed TME biology. Compounds can be screened in these in vitro TME models to look for impacts on protein biomarkers relevant for immune responses, inflammation, matrix modulation and for impacts on cancer cells. These systems were validated for immune restorative activities using anti PD-(L)1 therapeutics such as pembrolizumab, nivolumab, and durvalumab. Specifically, increased pro-inflammatory cytokine levels served as hallmark activities indicating a reversal of the immune suppressive influence of the cancer cell. In one of the BioMAP Oncology systems, we screened a library of 72 compounds targeting different molecules, the vast majority of which had not previously been identified as potential immuno-oncology candidates. Several small molecules that increased one or more cytokines were identified, indicating immune restorative potential. Other small molecules inhibited immune responses and/or demonstrated anti-inflammatory activities. Additionally, modulation of matrix biomarkers on tissue and tumor cells indicate impacts on distinct compartments of the TME. This approach using human cell-based in vitro TME models represents a rapid and cost-effective manner to rapidly screen libraries of small molecules (both novel and repurposed) for immuno-oncology potential. Citation Format: Alison O'Mahony, Sheryl P. Denker, Sharlene Velichko. Phenotypic screening of small molecules for novel immuno-oncology potential using BioMAP® Early Screening Services [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 6289.

Small molecule inhibitors of Janus kinases (Jakinibs) represent a promising treatment for myeloproliferative disorders including polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF). Currently multiple JAK2 inhibitors (ruxolitinib, fedratinib, momelotinib, baricitinib) are either approved or in clinical development for the treatment of MF. The primary target of Jakinibs, the JAK-STAT pathway, plays a critical role in multiple cellular processes relevant for myelofibrosis, including survival, differentiation, and proliferation. However, all approved Jakinibs are reportedly promiscuous in that they inhibit kinases beyond the JAK family. Their overall clinical impact, therefore, reflects the sum of these target activities, as well as downstream secondary pharmacology relevant to both efficacy and safety. We evaluated these four Jakinibs using phenotypic profiling with the BioMAP® Diversity PLUS Panel to identify common versus differential activities. The BioMAP platform consists of human primary cell-based assay or systems designed to recapitulate key aspects of tissue and disease states. Distinct activity profiles were generated for each of the tested Jakinibs based on their impacts on 148 different biomarkers in 12 different model systems. Comparison of these profiles at concentrations relevant for clinical exposure levels revealed common activities including inhibition of T and B cell proliferation and decreased levels of inflammation and immune activation biomarkers. Together these activities represent a phenotypic signature related to shared primary JAK targets. In contrast, multiple differentiating activities were observed between the four drug profiles: baricitinib increased TNFα levels whereas momelotinib decreased TNFα in the LPS system modeling monocyte activation; fedratinib and momelotinib, but not ruxolitinib or baricitinib, inhibited endothelial cell proliferation; only momelotinib inhibited macrophage responses and decreased PGE2 levels. These data indicate that even within the specific JAK2 inhibitor drug class, individual entities have distinct modes of action relevant for clinical outcomes. Citation Format: Sharlene Velichko, Sheryl P. Denker, Alison O'Mahony. Phenotypic evaluation and discrimination of clinical JAK inhibitors for myeloproliferative disorders [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 1847.