Site-specific C-terminal dinitrophenylation to reconstitute the antibody Fc functions for nanobodies

Abstract

Nanobodies are a class of camelid-derived single-domain antibodies that have many potential advantages over conventional antibodies and have been utilized to develop new therapeutic strategies for cancer and other diseases. However, nanobodies lack the Fc region of a conventional antibody, which possesses many functions, <i>e.g.</i>, eliciting antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), essential for effective immunotherapy. The small molecular size of nanobodies also leads to poor pharmacokinetics, such as short <i>in vivo</i> half-life. To address these deficiencies, an endogenous antibody-based strategy to reconstitute the Fc functions for nanobodies was developed. As a proof-of-principle, an anti-human EGFR nanobody, 7D12, was selected to conduct C-terminal modification with the dinitrophenyl (DNP) hapten through Sortase A-mediated site-specific ligation. It was expected that the DNP motif would recruit endogenous human <i>anti</i>-DNP antibodies to indirectly reinstate the Fc functions. The resultant nanobody-DNP conjugates were shown to exhibit specific and high affinity binding to human EGFR expressed on target cancer cells. It was further proved that in the presence of <i>anti</i>-DNP antibody, these conjugates could mediate potent ADCC and CDC <i>in vitro</i> and exhibit significantly elongated half-life <i>in vivo</i>. Ultimately, it was proven in severe combined immunodeficiency (SCID) mice that treatment with the nanobody 7D12-DNP conjugate and <i>anti</i>-DNP mouse serum could inhibit xenograft tumor growth efficiently. In view of the abundance of <i>anti</i>-DNP and other endogenous antibodies in the human blood system, this could be a generally applicable approach employed to reconstitute the Fc functions for nanobodies and develop nanobody-based cancer immunotherapy and other therapies.