NanoBondy Reaction through NeissLock Anhydride Allows Covalent Immune Cell Decoration

Area of Science:

• Bioconjugation Chemistry
• Molecular Engineering
• Immunology

Background:

• Current cell-surface modification methods are often reversible, non-specific, or require genetic manipulation.
• There is a need for precise and covalent methods to modify cell surfaces for therapeutic and research applications.

Purpose of the Study:

• To develop a novel nanobody-based technology, NanoBondy, for covalent cell-surface protein ligation.
• To enable targeted and inducible modification of unmodified cell-surface proteins.

Main Methods:

• Engineered nanobodies (NanoBondies) using NeissLock chemistry from *Neisseria meningitidis*.
• Utilized a disulfide clamp to position a self-processing module for proximity-directed ligation.
• Calcium-induced anhydride formation at the NanoBondy C-terminus for covalent linkage to surface amines on CD45.
• Optimized reaction conditions and validated specificity using tandem mass spectrometry on immune cells.

Main Results:

• NanoBondy successfully achieved covalent ligation to CD45, a cell-surface marker on hematopoietic cells.
• The ligation reaction was rapid (within 2 minutes), robust to varying buffer conditions, pH, and temperature.
• Demonstrated specificity for endogenous CD45 on NK cells and T cells, confirming targeted modification without genetic engineering.

Conclusions:

• NanoBondy technology offers a modular, targeted, and inducible approach for covalent cell-surface modification.
• This method enables precise labeling of immune cells without genetic modification, opening avenues for cell-based therapies and diagnostics.