Identification of a Mirror-Image VHH Antibody with VEGF-Inhibitory Activity and Characterization of Its Immunogenicity

Abstract Immunogenicity remains a major challenge in antibody therapeutics. Antibodies can be recognized as foreign by immune cells, leading to the induction of antidrug antibodies that ultimately compromise therapeutic efficacy. This response is primarily driven by proteolytic processing within antigen-presenting cells and the subsequent presentation of peptide fragments to T cells via major histocompatibility complex molecules. As mirror-image proteins are expected to exhibit resistance to proteolytic degradation, we have been developing mirror-image VHH antibodies composed entirely of d-amino acids. Previously, we generated a d-VHH antibody with specific binding activity to vascular endothelial growth factor A (VEGF-A) and demonstrated that it did not induce antidrug IgG antibodies in a mouse model. However, this d-VHH antibody did not inhibit the VEGF–VEGF receptor interaction, and the immunological processes leading to the lack of IgG antibody induction had not been experimentally demonstrated. In this study, we constructed a new phage-displayed library to improve the hit rate and enable the efficient chemical synthesis of d-VHH antibodies. Using this library, mirror-image screening against d-VEGF identified new VHH antibody sequences with specific binding. The synthesized d-VHH antibody inhibited the VEGF–VEGF receptor interaction. Next, we investigated the immunogenicity of d-VHH antibody using a conventional l-VHH antibody as a reference. The d-VHH antibody resisted enzymatic degradation and, in a mouse model, did not induce inflammatory cytokine production, T-cell responses, or the formation of antidrug antibodies against the administered antibody. Collectively, these findings highlight the potential of inhibitory d-VHH antibodies as a promising approach for developing low-immunogenic therapeutic antibodies.