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 costimulatory 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 validated hits, 5MS-5 and 19MS-5, which exhibit direct CD28 binding and potent inhibition of CD28-B7 interactions in cellular reporter assays. Pharmacokinetic profiling demonstrated favorable solubility, metabolic stability, permeability, and oral exposure in vivo. Functionally, both compounds suppressed cytokine production in primary human T cells cocultured 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 costimulation.