Chemical Probes Targeting CTLA-4: Building the Foundation for Non-Antibody Immune Modulattors 2310508

Abstract Introduction CTLA-4 is a critical inhibitory receptor that maintains immune tolerance by restricting CD28 co-stimulation. Clinical biologics have demonstrated the therapeutic value of modulating this pathway in autoimmune disease. However, antibody-based therapies have limitations that constrain mechanistic studies and limit precision in tuning immune responses. This work aims to develop small-molecule chemical probes that target CTLA-4 or its ligands, enabling a non-antibody approach to modulating immune regulation Methods Recombinant human CTLA-4, CD80, and CD86 extracellular domains were expressed and purified for biophysical characterization. Candidate small molecules predicted to engage CTLA-4 or the CTLA-4/CD80 interface were evaluated using biolayer interferometry (BLI) to assess preliminary interaction patterns. An allogeneic human T cell—dendritic cell co-culture and a multicolor flow cytometry panel were established to measure early activation markers and CTLA-4 expression dynamics. Results BLI studies identified initial interaction signatures with CTLA-4 and the CTLA-4/CD80 complex, demonstrating successful biochemical engagement. The T cell—dendritic cell assay produced consistent activation and checkpoint-expression readouts, establishing a functional system in which these compounds can be evaluated. Together, these early data support continued development of small-molecule probes to explore CTLA-4—mediated immune regulation. Conclusion This work establishes a chemical-immunology platform for identifying and assessing small-molecule modulators of CTLA-4. By enabling non-antibody engagement of this checkpoint pathway, these tools will support future studies aimed at refining CTLA-4—mediated immune regulation and informing therapeutic approaches for autoimmune disease. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)