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.

Photocaged antibody fragments,t ermed photobodies,h ave been developed that are impaired in their antigenbinding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of as ingle tyrosine in an anobody (a single-domain antibody fragment). Tyrosine is af requently occurring residue in central positions of the paratope region. o-Nitrobenzyl-protected tyrosine variants were incorporated into four nanobodies,i ncluding examples directed against EGFR and HER2, and photodeprotection restores the native sequence.Ananti-GFP photobody exhibited an at least 10 000fold impaired binding affinity before photodeprotection compared with the parent nanobody.Abispecific nanobodyphotobody 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.

We have recently reported photocaged nanobodies, termed photobodies, that can be light‐activated in their antigen binding properties due to the removal of the photolabile protection group. We aimed to establish the delivery and activation of photobodies inside mammalian cells, because intracellular protein manipulation and detection by using antibodies and antibody fragments like nanobodies is highly attractive, however, the spatial and temporal control of their application in cells is extremely limited. AntiGFP photobodies with ortho‐nitrobenzyltyrosine in the paratope region were delivered into HeLa cells using the bead‐loading technique. Light‐activation was carried out with spatial control in single cells to trigger binding to a GFP tag fused to endogenously expressed target proteins. By means of its fused nuclear localization sequence, an activated photobody dragged the GFP fusion protein into the nucleus, the kinetics of which could be monitored using live cell microscopy. These results show the potential of photobodies for intracellular applications.

Nanobodies against short linear peptide‐epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra‐ and intracellular applications. We here report the design of a photocaged variant of the ultra‐high affinity ALFA‐tag nanobody, also termed ALFA‐tag photobody. ortho‐Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000‐fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light‐dependent binding of the ALFA‐tag photobody to HeLa cells presenting the ALFA‐tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.

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