For highly conserved mammalian protein, chicken is a suitable immune host to generate antibodies. Monoclonal antibodies have been successfully targeted with immunity checkpoint proteins as a means of cancer treatment; this treatment enhances tumor-specific immunity responses through immunoregulation. Studies have identified the importance of B7-H4 in immunoregulation and its use as a potential target for cancer treatment. High levels of B7-H4 expression are found in tumor tissues and are associated with adverse clinical and pathological characteristics. Using the phage display technique, this study isolated specific single-chain antibody fragments (scFvs) against B7-H4 from chickens. Our experiment proved that B7-H4 clearly induced the inhibition of T-cell activation. Therefore, use of anti-B7-H4 scFvs can effectively block the exhaustion of immunity cells and also stimulate and activate T-cells in peripheral blood mononuclear cells. Sequence analysis revealed that two isolated scFv S2 and S4 have the same VH complementarity-determining regions (CDRs) sequence. Molecule docking was employed to simulate the complex structures of scFv with B7-H4 to analyze the interaction. Our findings revealed that both scFvs employed CDR-H1 and CDR-H3 as main driving forces and had strong binding effects with the B7-H4. The affinity of scFv S2 was better because the CDR-L2 loop of the scFv S2 had three more hydrogen bond interactions with B7-H4. The results of this experiment suggest the usefulness of B7-H4 as a target for immunity checkpoints; the isolated B7-H4-specific chicken antibodies have the potential for use in future cancer immunotherapy applications.
