SENSORS AND PROBES

Precision control of intrabodies in live cells

New methods from the Heo and Johnsson labs enable light- and drug-inducible control of intrabody binding in cells.

Antibodies are indispensable biological tools. In nature, they are the foot soldiers of the immune system’s surveillance apparatus, and in the laboratory they are sensitive tracking devices that we use to locate and enumerate our favorite biomolecules. But despite their many applications, conventional antibodies are limited by difficulties associated with producing functional binders within the cytoplasm. ‘Intrabodies’, however, are antibody-derived or antibody-like proteins that have been optimized for the reducing conditions of the cell interior. As a result, these molecules can be used to immunolabel proteins within intact and living specimens. In two independent Nature Methods publications, the Heo1 and Johnsson2 labs now introduce inducible strategies for controlling binding of intrabodies to their antigen targets.

Several types of intrabodies have been developed, but in recent years one class in particular has proven to be exceptionally susceptible to engineering: the camelid-derived ‘nanobodies’ (Nbs). Antibodies from camelid organisms offer the advantage of containing small single-chain variable regions (VHH) that can be readily converted into Nb form. The recent use of display libraries has led to an impressive expansion of the Nb repertoire3,4,5, and engineering strategies have been used to install generalized features into the Nb scaffold6. Yet, despite these advances, controlling when and where Nbs bind their targets has remained challenging.

Unlike conventional immunolabeling methods, intrabody labeling is achieved via gene expression and recombinant DNA. This approach permits users to bypass the destructive and invasive measures that are typically needed to get conventional antibodies inside a cell, such as fixation and permeabilization, or microinjection. But this mode of labeling can also make using intrabodies tricky, especially in situations where tight regulation is required. For example, when using inhibitory intrabodies to block the activity of endogenous proteins, users may want to precisely control when and where antigen binding occurs. While inducible expression strategies can be used to define the timing and extent of intrabody expression, regulation at the transcriptional level is slow and difficult to spatially control within cells.

Divide and conquer

In a report from the Heo lab, Yu et al. describe an approach in which intrabodies are bisected into polypeptide fragments that can combine to reconstitute a functional antigen-binding complex. The authors identified a split-site in the Nb framework that separates complementary determining regions (CDRs) 1 and 2 from CDR 3, producing fragments that can be conditionally reconstituted into a functional binder. To exert control over Nb formation, the authors fuse the fragments to inducible heterodimerization domains, such as the FKBP/FRB or the Magnet7 systems. The resulting fusions produce Nb segments that can form functional binders in response to rapamycin or blue light, respectively. The light-inducible version of these tools, called ‘optobodies’, offers the advantage of achieving spatial and subcellular control over antigen binding.

Using optobodies based on Nbs that bind fluorescent proteins, the authors validated their approach by showing that light could be used to trigger the relocalization of cytoplasmic optobody components to the surface of antigen-decorated mitochondria (Fig. 1). In addition to tag-directed systems, optobodies against endogenous proteins, including actin, gelsolin (an actin regulator) and the β2 adrenergic receptor were also generated. In elegantly designed experiments, the authors showed that these recombinant tools could be used to bind and modulate the activity of endogenous antigens. In the case of the gelsolin optobody, downstream cell migration properties could be controlled. This technique demonstrates an ability to optogenetically manipulate endogenously expressed proteins produced at natural levels and in their native (unmodified) forms.

Fig. 1: Conditional antigen binding using optobodies and LAMAs.
figure1

Optobody components are able to reconstitute a functional antigen-binding complex following the light-induced heterodimerization between fused domains (left). The interaction between LAMAs and their antigens can be modulated through the binding of TMP to the integrated cpDHFR drug receptor (right).

The probes generated by Yu et al. represent powerful tools for directing the assembly of Nb:antigen complexes against both recombinant and endogenous epitopes. The successful bisection of Nbs against diverse antigens suggests that the optobody approach might be generalizable across a broad set of Nbs. Although the characterized optobodies appear to have limited reversibility, the technique will have important applications in the study of dynamic subcellular processes where long-lasting changes can be advantageous.

Nudging nanobodies on and off targets

In a complementary strategy, the Johnsson lab describes a method by which the antigen binding capability of nanobodies can be rapidly and reversibly controlled using small molecules. In this approach, ‘ligand-modulated antibody fragments’, or LAMAs, are generated by inserting circularly permuted Escherichia coli dihydrofolate reductase (cpDHFR) into the Nb scaffold. Using the GFP enhancer Nb as an initial model, the authors showed that inserting cpDHFR in a critical CDR results in a chimera that retains its ability to tightly bind GFP but also rapidly dissociates from the antigen in the presence of the DHFR-binding ligands NADPH and trimethylprim (TMP). The strategy could also be extended to other Nbs, as demonstrated by the generation of LAMAs against the minimal ALFA peptide epitope8 and the HIV capsid protein p24.

Structural data suggested that the ligand-induced stabilization of cpDHFR serves to sterically hinder the fused Nb from interacting with GFP. Kinetic analyses demonstrated that this molecular displacement and occlusion occur within minutes of TMP addition and can be reversed within minutes after TMP removal. Taking their LAMAs into cells, Farrants et al. demonstrated that intracellular antigens can be reversibly manipulated in response to TMP. Using cells coexpressing a GFP fusion of Mad2L1 (a mitotic spindle checkpoint regulator) and a mitochondrially localized anti-GFP LAMA (Fig. 1), the authors showed that sequestration of the Mad2L1-GFP to mitochondria in the absence of TMP results in effects resembling those of Mad2L1 knockdown. TMP treatment to release Mad2L1-GFP restored proper mitotic checkpoint regulation.

Reversibility is a powerful feature, and interestingly, a LAMA generated using a LAP1-binding Nb exhibited ‘drug-on’ behavior, in contrast to the ‘drug-off’ activity of the other LAMAs. In addition, the authors noted challenges in generating a LAMA using a G-associated kinase-binding Nb. Together these results suggest that the manner in which Nbs will respond to cpDHFR insertion will be difficult to predict a priori. A solution to this challenge would be to screen diversified LAMA libraries against antigens in the presence and absence of TMP, which in principle would allow users to discover LAMAs exhibiting desired drug responses.

The road ahead

Optobodies and LAMAs represent important and complementary additions to the Nbs toolbox, and together these new tools highlight the versatility of Nbs as highly engineerable molecules. Both the Heo and Johnsson labs’ papers underscore how the availability of structural information can facilitate the reconfiguration of these proteins to take on new and useful functions. As the number of available Nbs continues to grow, it will be interesting to see how end users implement these tools to dissect biological functions. Given their genetically encoded nature, the use of optobodies and LAMAs to interrogate endogenous antigen targets in a cell-specific manner (via restricted expression in select cell types, for example) represents an enticing avenue for the implementation of these tools in vivo.

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Correspondence to John T. Ngo.

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Marzilli, A.M., McMahan, J.B. & Ngo, J.T. Precision control of intrabodies in live cells. Nat Methods 17, 259–260 (2020). https://doi.org/10.1038/s41592-020-0767-2

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