Abstract
Nectin-4, also known as PVRL-4, is a type 1 integral membrane glycoprotein that can form a homo-cis dimer on the cell membrane. The extracellular region contains 3 domains, a distal immunoglobulin-like (Ig-like) V-type domain followed by two Ig-like C2-type domains. Nectin-4 is overexpressed on various tumor cells. It is involved in cell-cell junctions and plays a role in cancer progression by influencing cell adhesion, migration, and proliferation. Nectin-4 has been recently identified as a novel ligand of T cell immunoglobulin and ITIM domain (TIGIT) and interaction between Nectin-4 and TIGIT inhibits NK cell cytotoxicity thus suppressing immune responses, allowing cancer cells to evade immune surveillance. Nectin-4 and Nectin-1 can also interact in a heterophilic manner. As an emerging target of cancer therapeutics, a recombinant protein mimicking the Nectin-4 native dimer conformation can be crucial for therapeutic antibody and vaccine discovery.
The goals of this study are to: (a) create a novel soluble bioactive Nectin-4 homodimer molecule mimicking its native dimer quaternary structure on the cell surface and (b) evaluate the recombinant Nectin-4 dimer binding potency to its receptor vs the monomer form. The dimer can be used as an immunogen and an antigen to increase the potential of cancer therapeutic antibody and vaccine discovery when targeting quaternary structural epitopes.
To better mimic the native dimer conformation and quaternary structure, we designed a novel soluble Nectin-4 cis-dimer with 3 extracellular Ig-like domains fused to a dimer motif at the C-terminus. The Nectin-4 dimer protein was expressed and purified from HEK293 cells and confirmed by SDS-PAGE and Western blot analyses. We characterized the Nectin-4 dimer protein binding potency to TIGIT and Nectin-1 by ELISA. We also studied Nectin-4 dimer immunogenicity using a Nectin-4 dimer DNA immunization protocol to induce potent antibody responses in mice.
Very interestingly, the results demonstrated that the Nectin-4 dimer binding potency to TIGIT and Nectin-1 increased dramatically compared to the Nectin-4 monomer protein. The Nectin-4 dimer DNA immunization generated a high titer antibody response in mice. This novel Nectin-4 cis-dimer is not only bioactive but also has increased functional activities in vitro. It can be a very useful immunogen and antigen to advance Nectin-4 targeted drug discovery.
