NanoBondy reacting through NeissLock anhydride allows covalent immune cell decoration

Cell-surface conjugation has enormous therapeutic and research potential. Existing technologies for cell-surface modification are usually reversible, non-specific, or rely on genetic editing of target cells. Here we present the NanoBondy, a nanobody modified for covalent ligation to an endogenous protein target at the cell-surface. The NanoBondy utilizes the 20 naturally occurring amino acids, harnessing NeissLock chemistry engineered from Neisseria meningitidis . We evaluated binding and specificity of a panel of nanobodies to CD45, a long-lived surface marker of nucleated hematopoietic cells. We demonstrated conversion of existing nanobodies to covalently reacting NanoBondies using a disulfide clamp to position the self-processing module of FrpA close to the nanobody antigen-binding site. Addition of calcium induces anhydride formation at the NanoBondy C-terminus and proximity-directed ligation to surface amines on CD45. We optimized NanoBondy reaction by fine-tuning linkers and disulfide clamp sites to modulate anhydride positioning. Tandem mass spectrometry mapped reaction sites between the NanoBondy and CD45. NanoBondy ligation was robust to buffer, pH and temperature and was detected within 2 minutes. We established reaction specificity of NanoBondies to endogenous CD45 at the surface of NK cells and T cells. NanoBondy technology provides a modular approach for targeted, inducible and covalent cell-surface modification of immune cells.