Construction of a novel B7H3 VHH-Fc antibody with potent antitumor activity and NK/T cell activation

B7-H3 is a tumor-specific antigen capable of regulating both NK and T cell activities. However, current B7-H3 antibodies in clinical or preclinical development lack immunoregulatory functions.

A camelid nanobody phage immune library targeting human 4Ig B7-H3 was constructed to screen out VHH antibodies (variable domain of heavy chain of heavy-chain-only antibody). NK cell killing and T cell proliferation assays were used to test the ability of VHH to activate NK and T cells. To leverage Fc-mediated effector functions, we humanized the VHH and fused it to an enhanced human IgG1 Fc domain, generating a VHH-Fc fusion protein. We then performed an antibody-dependent cellular cytotoxicity (ADCC) assay to confirm the functionality of the Fc domain. The in vivo antitumor activity was subsequently evaluated in PBMC-humanized NSG mice bearing U-87 MG tumors.

We identified a Camelus-derived B7-H3-targeting VHH, termed 2A9, and its humanized variant, 75-16. In vitro, both 2A9 and 75-16 promoted NK cell-mediated killing and increased CD4⁺ T cell proliferation, even without an IgG Fc domain. The VHH-Fc fusion protein, 75-16-G1-2, incorporating an engineered IgG1 Fc domain, demonstrated enhanced FcγR affinity and ADCC in vitro. Furthermore, 75-16-G1-2 exhibited antitumor activities in U-87 MG xenograft tumor model established in human PBMC-reconstructed immunodeficient NSG mice.

Together, our results suggest that the B7-H3-targeting VHH-Fc fusion protein 75-16-G1-2 is a potentially superior treatment for B7-H3-positive tumors by a mechanism of both VHH-induced activation of NK and T cells and Fc-mediated ADCC.