Engineered Novel CD155 and Nectin-4 Homodimer Proteins Dramatically Enhance Binding Affinities to Their Receptor TIGIT Compared to Respective Monomers
Poster Abstract
Understanding immune checkpoint receptor and ligand interactions is crucial for immuno-oncology. Nectin-4 and CD155 are part of the Nectin/Nectin-like family ligands of TIGIT (an inhibitory receptor on lymphocytes), can form homodimers on the cell membrane, and are often over-expressed on tumor cells. Nectin-4 and CD155 are type 1 integral membrane proteins. The ectodomain (ECD) containing 3 Ig-like domains (1 Ig-V followed by 2 Ig-C) is responsible for TIGIT interaction. To better mimic the native dimer conformation and quaternary structure, we designed novel soluble Nectin-4 and CD155 cis-homodimers with the ECD 3 Ig-like domains (ECD-3D) fused to a dimer motif at the C-terminus. These dimeric proteins expressed/purified from HEK293T cells demonstrated potent binding to the specific antibodies. Very interestingly, these Nectin-4 and CD155 dimeric proteins dramatically enhanced the binding to their receptor TIGIT by >50 fold compared to the respective monomer proteins, as measured by ELISA. The Nectin-4 dimer DNA immunization scheme elicited potent antibody responses in mice. AlphaFold 2 predicted that the Nectin-4 ECD-3D dimeric protein structure was well-superimposed with the known ECD-2D crystal structure from the RCSB Protein Data Bank. The findings support that the engineered novel Nectin-4 and CD155 cis-dimer proteins mimic native conformations and can be potentially used as advanced bioactive antigens for immuno-oncology research and anti-tumor drug discovery.
Conclusions
- The novel designs of CD155 and Nectin-4 ectodomain homodimer proteins to mimic the native dimer structures are well-superimposed with the known CD155 and Nectin-4 crystal structures.
- The recombinant CD155 homodimer and Nectin-4 homodimer proteins expressed from HEK293T cells were purified with high purity and demonstrated specific potent binding to the respective antibodies.
- The CD155 homodimer and Nectin-4 homodimer significantly increased the binding potencies to their receptor TIGIT-Fc dimer with >50 and >100 fold compared to CD155 monomer and Nectin-4 monomer, respectively.
- These novel CD155, Nectin-4 and TIGIT-Fc homodimer proteins can be used as antigens and immunogens for research and drug discovery.
- The high affinity and wider receptor/ligand binding windows can serve as more sensitive tools for therapeutic molecule evaluations.
