A general approach for stabilizing nanobodies for intracellular expression

Abstract

Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.

Development of a multifunctional toolkit of intrabody-based biosensors recognizing the V5 peptide tag: highlighting applications with G protein-coupled receptors

“…As for the TANGO, we compared both β-arrestin-1 and β-arrestin-2 fused at the N- or C-terminus with the V5-tag; the ALFA-tag was also included as a reference because the corresponding NbALFA intrabody is already characterized and was shown to work well with all placements (N-term, C-term, and internal) of the tag. As shown in Fig.4b-d , all configurations produced a robust signal, but C-terminally tagged NbV5 (NbV5-GFP2) gave a better BRET 2 ratio, while the NbALFA is less affected. As shown in Ext.…”

“…Conventional antibodies have proven to be robust workhorses in various cell biology research fields 1 . Notwithstanding their indispensability, their large size and complex architecture preclude them from functioning within living cells 2 . These limitations spurred advances in engineering novel recombinant antibody constructs, termed intracellular antibodies or intrabodies.…”

NLR immune receptor–nanobody fusions confer plant disease resistance

“…We reasoned that nanobody aggregation or misfolding upon intracellular expression ( 32 ) could explain the observed autoactivity (Fig. 1C and fig.…”

“…S1). We introduced previously described stabilizing nanobody mutations ( 32 ) in LaG-24, LaM-2, LaM-3, and LaM-6 and found that they abolished Pikobody autoactivity (fig. S2).…”

“…We focused on the minimal antigen-binding fragment of single-domain heavy-chain antibodies (known as VHHs or nanobodies) of camelid mammals ( 28 – 31 ) because they are small, soluble 10- to 15-kDa domains, which tend to correctly fold intracellularly and have many useful properties for biotechnological applications. To test our idea, we generated orthogonal Pik-1 sensors in which the integrated HMA domain is swapped with nanobodies that bind either green fluorescent protein (GFP) or mCherry ( 32 – 34 ) (Fig. 1, A and B, and table S1).…”