Engineering chain-branched bispecific VHH complexes ("Dendrobodies") using SpyCatcher and SnoopCatcher pairings

Bispecific antibodies (bsAbs) that direct immune cells toward tumor cells have substantial therapeutic potential. However, bsAbs produced by tandemly linking single-domain antibodies (VHHs) often exhibit reduced activity because the N-terminal region of each VHH, which contains the complementarity-determining regions (CDR) loops, becomes sterically hindered by the adjacent domain. To address this issue, we developed a branched VHH complex architecture, termed Dendrobody, in which VHHs are joined through their C-terminal sides using orthogonal SpyCatcher/SpyTag and SnoopCatcher/SnoopTag systems. This design enables site-specific covalent assembly of multiple VHHs while preserving accessibility of their antigen-binding regions. We generated a bispecific anti-CD3/anti-EGFR Dendrobody (Dendrobody-CD3/EGFR) through a one-pot ligation of three recombinant components. The purified complex displayed correct folding with a thermal transition temperature (Tm) of 56 °C. Biolayer interferometry showed strong EGFR binding, and the Dendrobody triggered selective T-cell-mediated cytotoxicity toward EGFR-positive cancer cells. No cytotoxicity was detected in EGFR-negative cells, confirming antigen specificity. In addition, the platform allowed straightforward production of another variant, Dendrobody-CD16/EGFR, demonstrating its modularity. The Dendrobody framework thus offers an alternative strategy for constructing bispecific and multispecific VHH-based therapeutics with minimal steric interference and high functional adaptability.