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Background CD40 is a cell surface receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily. CD40 is broadly expressed on many immune cells, mostly by antigen-presenting cells (APCs). On APCs, it plays a central role in stimulating immune synapses during T cells priming, when through its interaction with its ligand CD40 ligand (CD40L) it licenses APCs to activate tumor antigen-specific T cells. This is accomplished through the upregulation of major histocompatibility complex molecules, increased expression of the costimulatory molecules CD86/CD80, and upregulation of TNF superfamily ligands on the APC surface, as well as by secretion of cytokines such as IL-12, which altogether fuel CD8 + T cells activation. The CD40/CD40L axis thus plays a central role in APCs proliferation, as well as antigen presentation, notably in a cancer setting.Methods Generation of peptide-tailored protease-cleavable masks and in vitro characterization in human, mouse, and cynomolgus monkey primary immuno-assays of conditionally active CP-870,893 variants.Results CP-870,893 is a clinically validated anti-CD40 agonist antibody bearing a unique FcγR-independent, soluble agonism mode of action. We previously highlighted at SITC 2024 the crystal structure of CP-870,893 Fab in complex with CD40 receptor, as well as the structure-based generation of CP-870,893 variants achieving a potency equivalent to the cognate CD40L in primary cells immuno-assays. With the intention of addressing the short half-life due to target-mediated drug disposition and the dose-limiting toxicities observed in clinical trials with monospecific anti-CD40 agonist antibodies, we now report for the first time the generation and the in vitro characterization in human, mouse, and cynomolgus monkey immuno-assays of conditionally active CP-870,893 variants featuring a peptide mask covering the paratope and a protease-cleavable linker, where full-fledged agonistic activity is unleashed only after proteases-mediated cleavage of the mask.Conclusions These in vitro cross-species findings suggest a promising path for developing safer anti-CD40 agonist-based therapeutics.