Dual targeting of CD244 and CD2 by a viral-immunoevasin-guided engineered CD48-Fc mutant for T- and NK-cell modulation.

Viruses encode immunoevasins that subvert host co-signaling pathways. CD244:CD48 and CD2:CD58 interactions are critical for NK- and T-cell effector responses. We previously identified a herpesvirus-encoded soluble CD48 homolog that functions as a decoy ligand for CD244 and dampens NK-cell activation. Here, we show that this viral protein, A43, also recognizes CD2, and we used its structural features to engineer human CD48-Fc variants with dual CD244/CD2 specificity. The optimized mutein, C5-Fc, which incorporates twelve A43-guided substitutions predicted by structure-based modeling to enhance interface packing and electrostatic complementarity, resulted in high-avidity binding and competitively displacement of native ligands. Fc-silenced C5-Fc (C5-Fc#) disrupted NK- and T-cell conjugate formation and reduced CD244- and CD2-dependent cytotoxicity. It also interfered with the maturation of the antigen-dependent immunological synapse and inhibited T-cell proliferation and the secretion of pro-inflammatory cytokines. These data establish C5-Fc# as a rationally engineered dual-specificity inhibitor with translational potential for autoimmune diseases.