NanoBondy Reaction through NeissLock Anhydride Allows Covalent Immune Cell Decoration

Journal article

Cell-surface conjugation has enormous therapeutic and research potential. Existing technologies for cell-surface modification are usually reversible, nonspecific, or rely on genetic editing of target cells. Here, we present the NanoBondy, a nanobody modified for covalent ligation to an unmodified protein target at the cell surface. The NanoBondy utilizes the 20 naturally occurring amino acids, harnessing NeissLock chemistry engineered from Neisseria meningitidis. We evaluated the binding and specificity of a panel of nanobodies to CD45, a long-lived surface marker of nucleated hematopoietic cells. We demonstrated the 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. The addition of calcium induces anhydride formation at the NanoBondy C-terminus, enabling proximity-directed ligation to surface amines on CD45. We optimized the NanoBondy reaction by fine-tuning linkers and disulfide clamp sites to modulate anhydride positioning. Tandem mass spectrometry mapped reaction sites between NanoBondy and CD45. NanoBondy ligation was robust to buffer, pH, and temperature and was detected within 2 minutes. We established the 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 without their genetic modification.

Authors: Vankayala, LR; Adoni, KR; Lim, SYT; Dam, T; Dushek, O

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Chemical Society
• Journal: Bioconjugate Chemistry
• Volume: 37
• Issue: 2
• Pages: 281-292
• Publication date: 2026-01-24
• Acceptance date: 2025-12-15
• DOI: 10.1021/acs.bioconjchem.5c00519
• EISSN: 1520-4812
• ISSN: 1043-1802
• Language: English
• Keywords: Covalent bond; Chemical ligation; Ligation; Bioconjugation; Surface modification; Tandem; Protein engineering; Click chemistry