There is a high demand for agonist biomolecules such as cytokine surrogates in both biological and medicinal research fields. These are typically sourced through natural ligand engineering or affinity-based screening, followed by individual functional validation. However, efficient screening methods for identifying rare hits within immense libraries are very limited. In this research article, we introduce a phenotypic screening method utilizing biological receptor activation-dependent cell survival (BRADS). This method offers a high-throughput, low-background, and cost-effective approach that can be implemented in virtually any biochemical laboratory setting. As a proof-of-concept, we successfully identified a surrogate for human leptin following a two-week cell culture process, without the need for specialized high-throughput equipment or reagents. This surrogate effectively emulates the activity of native human leptin in cell validation assays. Our findings not only underscore the effectiveness of BRADS but also suggest its potential applicability to a broad range of biological receptors, including Notch and GPCRs.

Leptin receptor (LepR) signaling contributes to energy homeostasis and inflammatory responses, making LepR antagonism a potential anti-inflammatory strategy. Although H6-derived antibody ligands exhibit LepR agonistic activity, the pharmacological properties of H6 in a full-length immunoglobulin G format (H6-IgG) remain unclear. We evaluated H6-IgG binding, competition with leptin, and signaling activity, examined NanoBiT reporter responses and structural models, and assessed its activity in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute cutaneous inflammation model in C57BL/6 mice. H6-IgG bound human and mouse LepR and interfered with leptin binding. H6-IgG showed no appreciable agonist activity but inhibited leptin-induced STAT3/STAT5 signaling and NanoBiT reporter responses. Structural modeling suggested distinct membrane-proximal LepR arrangements associated with H6-IgG and leptin. In female mice, H6-IgG (5 mg/kg) significantly reduced TPA-induced ear swelling. These findings identify H6-IgG as a functional LepR antagonist with anti-inflammatory activity in an acute cutaneous inflammation model.