Resurfaced cell-penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery

Abstract

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromolecules that present a challenge to small-molecule reagents. Additionally, high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies-a small and stable protein that can be evolved to recognize virtually any disease-relevant receptor-are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP-binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell-penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.

Keywords: cell-penetrating; nanobody; polycationic resurfacing; supercharging.

Figure 1

(A) A previously reported nanobody that binds Green Fluorescent Protein (GFP), PDB: 3OGO. This nanobody is referred to as NB1 in this work. Complementarity‐determining region (CDR) loops are highlighted in purple. (B) Residues on NB1 that were mutated to either arginine or lysine to generate the resurfaced polycationic nanobody (pcNB1) are highlighted with blue spheres.

Figure 2

(A) Sequence of wild‐type nanobodies (NB1‐3) and resurfaced polycationic nanobodies (pcNB1‐3) described in this work. (B) PAGE analysis of wild‐type and resurfaced polycationic nanobodies described in this work. (C) Circular dichroism spectra of wild‐type (NB1‐3) and resurfaced polycationic nanobodies (pcNB1‐3) described in this work.

Figure 3

(A–C) Flow cytometry data that supports concentration‐dependent uptake of resurfaced polycationic nanobody‐GFP fusion proteins, but not GFP alone (black line) or wild‐type nanobody‐GFP fusion (gray line). Red line = 10 nM treatment; green line = 250 nM treatment; blue line = 500 nM treatment. (D–F) Fluorescence microscopy images of 3T3 cells following treatment with 250 nM resurfaced nanobody‐GFP fusions. (G) Western blot analysis of digitonin cell lysate for Erk1/2 (cytosolic marker), GFP (internalized resurfaced nanobody‐GFP fusion protein), or Rab5 (endosome marker). Lane 1 = no treatment; lane 2 = wild‐type GFP; lane 3 = wild‐type NB1‐GFP fusion; lane 4 = NB2‐GFP fusion; lane 5 = NB3‐GFP fusion; lanes 6–8 = polycationic resurfaced nanobody‐GFP fusions analogous to lanes 3–5. (H) Western blot analysis of digitonin cell lysate for Erk1/2 (cytosolic marker), His₆ (internalized resurfaced nanobody), or Rab5 (endosome marker). Lane 1 = no treatment; lane 2 = wild‐type NB1; lane 3 = NB2; lane 4 = NB3; lanes 5–7 = polycationic resurfaced NB1, NB2, or NB3, respectively. (I) Western blot showing no Rab5 (endosome marker) in cell lysate following digitonin lysis, but in extract following RIPA lysis. For all figures, experiments were run in triplicate and representative data are shown.

Figure 4

(A) Lane 1: His₆‐NB1; Lane 2: co‐purification of untagged GFP with His₆‐NB1 from E. coli cell lysate; Lane 3: co‐purification of untagged GFP with His₆‐pcNB1; Lane 4: His₆‐GFP; Lane 5: His₆‐pcNB1. (B) Tube 1: His₆‐NB1; Tube 2: His₆‐pcNB1; Tubes 3–4: His₆‐NB1 and co‐eluted GFP; Tubes 5–6: His₆‐pcNB1 and co‐eluted GFP; Tube 7: His₆‐GFP; Tube 8: untagged GFP. For all figures, experiments were run in triplicate and representative data are shown.