Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

CD28 is a key T cell co-stimulatory receptor implicated in antitumor immunity and immune-related disorders, yet no small molecule modulators of CD28 have reached clinical development. Here, we report the discovery and characterization of small molecule CD28 antagonists identified through affinity selection-mass spectrometry (AS-MS). Subsequent catalog-based structure-activity relationship (SAR) optimization led to the identification of two lead compounds, 5MS-5 and 19MS-5, which exhibit direct CD28 binding and potent inhibition of CD28-B7 interactions in cellular reporter assays. In vitro pharmacokinetic profiling demonstrated favorable solubility, metabolic stability, and permeability, alongside low off-target liabilities. Functionally, both compounds suppressed cytokine production in primary human T cells co-cultured with tumor spheroids and human epithelial tissues, validating their ability to inhibit CD28-driven immune activation in physiologically relevant models. These findings establish 5MS-5 and 19MS-5 as promising CD28 inhibitors and provide a foundation for developing orally bioavailable immunomodulators targeting T cell co-stimulation. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/667814v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@2d0ab7org.highwire.dtl.DTLVardef@11a985forg.highwire.dtl.DTLVardef@109a13corg.highwire.dtl.DTLVardef@1388451_HPS_FORMAT_FIGEXP M_FIG C_FIG