Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Abstract

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.

Nanobody-Based Probes for Subcellular Protein Identification and Visualization

“…This process can be reversed to activate the nanobody binding (Farrants et al, 2020). The light-dependent nanobody, termed photobody, uses a genetic photocaged tyrosine variant that results in the inactivation of the antigen-binding site (Arbely et al, 2012;Mootz et al, 2019). The photocaged tyrosine is photo-labile, and upon light induction (365 nm) the antigen-binding properties of the chromobodies are restored.…”

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

“…Photocaged ncAAs can be used to regulate protein functions by exposure to light, which makes them useful cell-biological tools. For instance onitrobenzyl-Tyr (103) was used to construct light-activatable nanobodies (Jedlitzke et al, 2020) and antibody fragment (Bridge et al, 2019), o-nitrobenzyl-4-hydroxyphenylalanine (105) was used for generating a photo-controlled variant of wet adhesive protein (Hauf et al, 2017), a caged diaminopropionic acid (106) was used for catalytic mechanism characterization of valinomycin synthetase (Huguenin-Dezot et al, 2019), a photocaged Sec, 4,5-dimethoxyl-2-nitrobenzyl-Sec (107), was used for production of selenoproteins as well as for site specific alkylation (Welegedara et al, 2018).…”

Photocaged Nanobodies Delivered into Cells for Light Activation of Biological Processes

“…Importantly, activated photobodies retain the small size of nanobodies and removal of the photocage releases the native sequence. We reported four photobodies, [8a] each with a single tyrosine residue replaced with genetically encoded ortho‐nitrobenzyltyrosine (ONBY), [9] whose protection group could be removed with 365 nm. The photobodies were produced in Escherichia coli and functionally characterized in various extracellular binding applications, for example, dependent upon photoactivation, they bound to epitopes presented on mammalian cells and a bispecific photobody‐nanobody fusion construct could be used to trigger protein dimerization on a cell surface [8a] …”

“…However, this would require a significant amount of genetic manipulation of the mammalian cells for genetic code expansion [11] . We therefore chose to use the efficient photobody production in E. coli [8a] . For the intracellular delivery of purified photobodies, we pursued the bead‐loading technique, because it is easily and quickly performed without the need for any specialized equipment [12] .…”

“…To test photobody delivery and observe its regain of antigen binding upon light activation we followed the scheme shown in Figure 1A. An antiGFP photobody generated from the enhancer GFP nanobody [14] with a Y37ONBY substitution was previously biophysically characterized and exhibited a ≥10,000‐fold increase in binding affinity following a few seconds of photoactivation with restoration of the single digit nanomolar binding affinity [8a] . For this study, we prepared an antiGFP photobody ( 1 ) with a single appended cysteine for dye bioconjugation, and verified photodecaging of the purified protein by mass spectrometry (MS) (Figure S1).…”