Small Molecule-Based Blockade of CD28 Suppresses T Cell Costimulation Across Cellular and Mucosal Co-culture Models

Background and PurposeCD28 is a pivotal costimulatory receptor that governs T cell activation through interaction with B7 ligands (CD80/CD86). While antibody-based inhibitors of CD28 signaling have advanced clinically, the development of small molecule modulators remains limited due to the receptors shallow, flexible surface. We sought to discover small-molecule modulators with favorable pharmacokinetic properties capable of disrupting CD28-B7 interactions in translational models of T cell activation. Experimental ApproachStructure-based virtual screening was conducted using pharmacophore filtering and consensus docking against the human CD28 ectodomain. Hit compounds were validated using temperature-related intensity change (TRIC) and microscale thermophoresis (MST). Functional antagonism was assessed through ELISA, NanoBit luciferase complementation, and a CD28 Blockade Bioassay. In vitro ADME and safety pharmacology profiling were performed, and immunosuppressive activity was evaluated in tumor-PBMC and mucosal-PBMC co-culture assays. Key ResultsLead compound 22VS bound CD28 in biophysical screening, targeting a lipophilic canyon anchored by K24, Q25, and P27. 22VS inhibited CD28-CD80/CD86 interactions in ELISA and cell-based assays with submicromolar potency. 22VS robustly suppressed T cell activation markers in both tumor- PBMC and human mucosal epithelial-PBMC co-culture models, phenocopying the anti-CD28 biologic FR104. It showed no cytotoxicity up to 300 {micro}M and exhibited high solubility, low clearance, strong membrane permeability, and minimal off-target effects in pharmacokinetic screens. Conclusion and ImplicationsThis study identifies a novel druggable site on CD28 and validates 22VS as a selective, non-toxic small molecule inhibitor with translational potential for immune modulation in autoimmunity, transplantation, and cancer. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICD28 is a key T cell costimulatory receptor essential for immune activation. C_LIO_LISmall-molecule inhibitors of CD28 are largely unexplored compared to biologics. C_LI What this study addsO_LIIdentifies a novel druggable pocket on CD28 via structure-based virtual screening. C_LIO_LIDiscovers 22VS, a selective small molecule CD28 inhibitor with cellular activity. C_LIO_LIDemonstrates that 22VS suppresses T cell activation in tumor-PBMC and mucosal-PBMC co- culture assays, phenocopying a benchmark biologic (FR104). C_LIO_LIEstablishes 22VS as a drug-like compound with favorable in vitro pharmacokinetic properties, including metabolic stability, permeability, and low off-target toxicity. C_LI Clinical significanceO_LIHighlights the potential of 22VS as a lead for immunomodulatory therapeutic development. C_LIO_LISupports small-molecule targeting of CD28-B7 interactions in T cell-driven diseases. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/665260v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f1d2forg.highwire.dtl.DTLVardef@451c98org.highwire.dtl.DTLVardef@158446corg.highwire.dtl.DTLVardef@1f42ef1_HPS_FORMAT_FIGEXP M_FIG C_FIG