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Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. Screens for protein–protein interactions and for drugs that disrupt them typically use in vitro assays which fail to capture the complexity of the cell’s interior. By fixing proteins to distinct cellular locations, Herce et al. demonstrate a fluorescent-three-hybrid approach to probe such interactions in their cellular contexts.

GFP fluorescence can be modulated in mammalian cells by binding to single-chain antibodies (nanobodies), selected to make GFP brighter or dimmer; these changes are explained by the crystal structures of the GFP-nanobody complexes. The applications of such nanobodies to monitor protein expression and subcellular localization in real time are explored. Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP–nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals , combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability.

Chromobodies have recently drawn great attention as bioimaging nanotools. They offer high antigen binding specificity and affinity comparable to conventional antibodies, but much smaller size and higher stability. Chromobodies can be used in live cell imaging for specific spatio-temporal visualization of cellular processes. To date, functional application of chromobodies requires lengthy genetic manipulation of the target cell. Here, we develop multifunctional large-pore mesoporous silica nanoparticles (MSNs) as nanocarriers to directly transport chromobodies into living cells for antigen-visualization in real time. The multifunctional large-pore MSNs feature high loading capacity for chromobodies and are efficiently taken up by cells. By functionalizing the internal MSN surface with nitrilotriacetic acid-metal ion complexes, we can control the release of His 6 -tagged chromobodies from MSNs in acidified endosomes and observe successful chromobody-antigen binding in the cytosol. Hence, by combining the two nanotools, chromobodies and MSNs, we establish a new powerful approach for chromobody applications in living cells.

Purpose Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Methods Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. Results The initial oligomer screen identified two cationizable four-arm structured oligomers ( 734, 735 ) which mediate intracellular nanobody delivery in a receptor-independent ( 734 ) or folate receptor facilitated ( 735 ) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Conclusion Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, distribution, modification, and interaction of all cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not about endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications, and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (V H H) from Camelidae . These extremely stable V H H domains can be produced in bacteria, coupled to matrices, and used for affinity purification and proteome studies. Alternatively, these V H H domains can be fused with fluorescent proteins and expressed in living cells. These fluorescent antigen-binding proteins called “chromobodies” can be used to detect and trace proteins and other cellular components in vivo. Chromobodies can, in principle, detect any antigenic structure, including posttranslational modifications, and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analysis. Depending on the epitope chosen, chromobodies can also be used to modulate protein function in living cells. Figure Detection of the nuclear lamina with lamin chromobody in living cells.

TUB-010 is a next-generation antibody-drug conjugate (ADC) targeting CD30, which is expressed on various hematopoietic malignancies such as Hodgkin lymphoma and anaplastic large cell lymphoma. Patients with refractory and relapsed CD30-positive cancers often lack effective and tolerable therapy options. Among the therapeutic options for these patients is brentuximab vedotin (Adcetris), a monomethyl auristatin E (MMAE)-delivering anti-CD30 ADC with a mean drug-to antibody ratio (DAR) of 4. Adcetris exhibits a high response rate at the cost of significant toxicities, among which neutropenia and peripheral neuropathy are the most prevalent adverse events, which are likely driven by the payload MMAE and instability of the maleimide conjugation chemistry. TUB-010 uses the same antibody brentuximab and payload MMAE as Adcetris, but instead of maleimide chemistry, TUB-010 is based on the Tub-tag conjugation strategy, which enables the generation of a homogenous and site-specific DAR 2 ADC with unique biophysical properties. This new technology stably attaches MMAE to the hydrophilic Tub-tag peptides on the light chains via chemoenzymatic conjugation using the enzyme tubulin tyrosine ligase. TUB-010 demonstrates similar binding affinity, internalization and lysosomal release characteristics as Adcetris in CD30-positive cells. When normalized to the MMAE concentration, TUB-010 shows comparable in vitro cytotoxic efficacy as well as similar bystander activity compared to Adcetris on established cancer cell lines. Importantly, TUB-010 exhibits higher stability with neglectable premature deconjugation in circulation and reduced high molecular weight species formation as well as lower non-specific cytotoxicity on target-negative cells compared to Adcetris. As a consequence, TUB-010 induces superior tumor control compared to Adcetris when dosed at equal MMAE concentrations in vivo and also shows lower toxicity and higher tolerability in rodents and non-human primates. Taken together, TUB-010 is a novel and potential best-in-class anti-CD30 ADC with improved biophysical properties designed to deliver the cytotoxic payload with higher precision and with a wider therapeutic window than Adcetris using Tub-tag conjugation technology. Therefore, TUB-010 may increase the clinical benefit of ADC therapies for patients with CD30-positive malignancies.

Chromobodies have recently drawn great attention as bioimaging nanotools. They offer antigen binding specificity and affinity comparable to conventional antibodies, but much smaller size and higher stability. Importantly, chromobodies can be used in live cell imaging for highly specific spatio-temporal visualization of cellular processes. To date, functional application of chromobodies requires lengthy genetic manipulation of the target cell. Here, we developed multifunctional large-pore mesoporous silica nanoparticles (MSNs) as nanocarriers to directly transport chromobodies into living cells for antigen-visualization in real time. The multifunctional large-pore MSNs feature high loading capacity for chromobodies, and are efficiently taken up by cells. By functionalizing the internal MSN surface with nitrilotriacetic acid-metal ion complexes, we could control the release of His6-tagged chromobodies from MSNs in acidified endosomes. When chromobodies escape from the endosomes through the proton sponge effect generated by their built-in His6-tags, co-localization of signals from fluorescent endogenous antigen and organic dye-labeled chromobodies can be detected. Hence, by combining the two nanotools, chromobodies and MSNs, we established a new powerful approach for chromobody applications in living cells.