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

ABSTRACT 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. Competing Interest Statement The authors have declared no competing interest. ABBREVIATIONS - AS-MS - affinity selection–mass spectrometry - CD - cluster of differentiation - CLint - intrinsic clearance - DEL - DNA-encoded library - FaSSIF - fasted-state simulated intestinal fluid - IC50 - halfmaximal inhibitory concentration - IFN-γ - interferon gamma - IL-2 - interleukin-2 - Kd - equilibrium dissociation constant - LogD - logarithm of distribution coefficient - MD - molecular dynamics - MST - microscale thermophoresis - PBMC - peripheral blood mononuclear cell - PBS - phosphate-buffered saline - PK - pharma-cokinetics - PPB - plasma protein binding - sCD69 - soluble cluster of differentiation 69 - SAR - structure–activity relationship - SEC - size-exclusion chromatography - TCR - T cell receptor - TRIC - temperature-related intensity change.