Poster: Immune Checkpoint CD28 and CTLA-4 Homodimer Proteins Demonstrate Potent Binding  

CD28 and CTLA-4 are immune checkpoint proteins on the T cell surface as homodimers and bind to the same ligands CD80 (B7-1) and CD86 (B7-2) on antigen presenting cells. However, CD28 and CTLA-4 play opposite roles in T cell activation. CD28 acts as a co-stimulatory receptor to promote T cell activation while CTLA-4 acts as a co-inhibitory receptor to suppress T cell activation. To better mimic the native dimer conformation and quaternary structure, we designed novel soluble CD28 and CTLA-4 cis-homodimer proteins with the ectodomain (ECD) fused to a dimer motif at the C-terminus, and the CD80-Fc dimer with the ECD fused with a dimeric Fc-Tag at the C-terminus. These dimeric proteins expressed/purified from HEK293T cells demonstrated potent binding to the specific antibodies. We further investigated the binding abilities of the CD28 homodimer and the CTLA-4 homodimer to the CD80 ligand. The results demonstrated that both CD28 and CTLA-4 dimeric proteins have high binding potencies to the CD80 homodimer as measured by ELISA, localized surface plasmon resonance (LSPR), and Bio-Layer Interferometry (BLI). AlphaFold 2 predicted that the CD28 and CTLA-4 ECD dimeric protein structures were well-superimposed with the known crystal structure from the RCSB Protein Data Bank. The findings support that the engineered novel CD28, CTLA-4 and CD80 dimeric proteins mimic native conformations and can be
potentially used as advanced bioactive antigens for immuno-oncology research and drug discovery