An Integrated Biophysical Fragment Screening Approach Identifies Novel Binders of the CD28 Immune Receptor.

CD28 is an essential costimulatory receptor required for full T cell activation, and its dysregulation contributes to multiple immune-mediated pathologies. Despite its central immunological role, CD28 remains largely unexplored as a target for small-molecule modulation, primarily due to the shallow and large interface of its ligand-binding site. Here, we applied a fragment-based high-throughput screening (HTS) strategy to identify low molecular weight chemotypes capable of engaging with human CD28. A 3,200-member library composed of structurally diverse fragments, enriched for scaffolds designed to target protein-protein interaction (PPI) interfaces, was screened in single-dose format using temperature-related intensity change (TRIC) technology, yielding 36 primary hits (1.13% hit rate). Follow-up surface plasmon resonance (SPR) validation confirmed two fragments as direct CD28 binders. Molecular docking analysis revealed a plausible binding orientation for PPIF3 within the CD28 extracellular domain, suggesting potential interaction hotspots that may be exploited during future optimization. Together, these findings provide the first demonstration that fragment-based screening can successfully identify chemotypes capable of engaging with the CD28 PPI interface. This work establishes a scalable, biophysics-driven workflow for CD28 ligand discovery and lays the foundation for subsequent hit-to-lead development of small molecule CD28 modulators.