A fc-engineered NKG2D × B7-H3 bispecific antibody enhances the antitumor activity by orchestrating cytotoxic lymphocytes.

B7-H3, an immune-checkpoint molecule that is overexpressed in several cancer types, has been identified as a promising immunotherapy target. However, most immunotherapy approaches against B7-H3 tumor cells focus on manipulating the T cells. Natural killer (NK) cells, another important part of the cellular immune system, also exhibit anti-tumor properties and play complementary roles in tumor eradication with T cells. In this study, natural killer group 2D (NKG2D), an activating receptor in most cytotoxic immune cells, was selected for engaging NK cells. We obtained specific anti-NKG2D nanobodies via phage display and developed a series of B7-H3 × NKG2D bispecific antibodies (bsAb) with different formats to fight against the B7-H3 tumor cells. Through functional comparison of candidate antibodies in vitro, B1-C53 was selected and further modified with the optimized Fc fragment (known as FC-C53) to enhance anti-tumor immunity by antibody-dependent cell-mediated cytotoxicity (ADCC). Stronger tumor lysis mediated by FC-C53 was demonstrated both in vitro and in vivo when simultaneously directed at both NK cells and CD8 T cells, particularly after the additional administration of a B7-H3 × CD3 bispecific T cell engager that targets B7-H3 with another epitope. Overall, we provided a strategy based on the B7-H3 × NKG2D antibody to improve the anti-B7-H3 immunotherapy approaches by orchestrating cytotoxic lymphocytes.