CD28-IN-2 is a selective inhibitor of the CD28-B7 interaction (IC50: 22.4 μM) that directly binds to CD28, with a Kd value of 24.1 μM. It inhibits CD28-driven immune activation and blocks T cell costimulation. CD28-IN-2 is applicable in the research of anti-tumor immunity and immune-related diseases.

CD28-IN-2 (5MS-5) effectively inhibits CD28-dependent cytokine release and T cell activation markers (IFN-γ, IL-2, and soluble CD69 (sCD69)) in a 3D co-culture model composed of IFN-γ-pretreated A549 tumor spheroids and human PBMC when administered at concentrations of 10-50 μM for 48 hours. In a co-culture of CD28-expressing Jurkat T cells (bearing a TCR/CD3-CD28 responsive luciferase reporter gene) and Raji antigen-presenting cells, CD28-IN-2 exhibits a dose-dependent CD28-mediated luminescence inhibition with an IC50 of 10.3 μM. The compound shows weak inhibition of the hERG potassium channel (IC50 > 50 μM) and displays minimal inhibition of major cytochrome P450 isoforms at a concentration of 10 μM.