# NanoBondy Reaction through NeissLock Anhydride Allows Covalent Immune Cell Decoration

## TL;DR

NanoBondy is a new tool that allows covalent modification of immune cell surfaces without genetic editing, using a chemical reaction inspired by bacteria.

## Contribution

NanoBondy introduces a covalent, inducible, and modular method for cell-surface modification using NeissLock chemistry.

## Key findings

- NanoBondy covalently reacts with CD45 on immune cells within 2 minutes.
- The reaction is robust to changes in buffer, pH, and temperature.
- Tandem mass spectrometry confirmed specific ligation sites between NanoBondy and CD45.

## Abstract

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.