Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

CD28 is the prototypical costimulatory receptor that integrates with TCR signaling to sustain T-cell activation, proliferation, and survival. While indispensable for adaptive immunity, persistent CD28 signaling drives autoimmunity, graft-versus-host disease, and inflammatory pathology. Despite its therapeutic relevance, CD28 has long been regarded as an undruggable target due to its flat, solvent-exposed dimer interface, restricting modulation to biologics. Here, we describe a structure–activity relationship (SAR) campaign to optimize a small molecule CD28 inhibitor. Guided by biophysical profiling and functional assays, derivatives of the 8VS and 22VS chemotypes were evaluated, leading to the identification of BPU11 as a chemically tractable lead with improved pharmacokinetic stability, aqueous solubility, and plasma persistence. BPU11 consistently disrupted CD28–B7 interactions across biochemical and cellular systems, and potently suppressed T-cell activation in both a tumor–PBMC co-culture and a human PBMC– mucosal tissue model, functionally mimicking the biologic antagonist FR104. Molecular docking and dynamics simulations revealed engagement of the lipophilic canyon of CD28 through stabilizing hydrogen-bonding and hydrophobic interactions. These findings expand the pharmacological space of immune checkpoint blockade beyond antibodies and position BPU11 as a foundation for next-generation immunotherapies.