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Exploring cellular biochemistry with nanobodies

Open AccessPublished:August 31, 2020DOI:https://doi.org/10.1074/jbc.REV120.012960
      Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain–only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.
      Tools to detect, visualize, and modulate the properties of proteins are essential to understand the function of the targets recognized and the biology that follows. Introduction of exogenous expression vectors and CRISPR/Cas gene-editing tools provide an unprecedented ability to introduce, alter, or eliminate proteins of choice in cells or intact organisms. These approaches are designed to modify biological processes of interest. Introduction of expression vectors allows production of proteins of choice, WT or mutant, including versions fused with fluorescent proteins or other tags for visualization. Expression of proteins from nonnative loci, as in exogenous expression vectors, or as fusion proteins with tags often alters expression levels, subcellular localization, and biological function. The development of antibody fragments that can interact with and perturb endogenous proteins in cells and organisms without the need for genomic modification would be useful. Nanobodies have unique qualities that make them well-suited for this goal.
      Nanobodies, like full-size conventional antibodies, show the affinity and antigen specificity required for specific targeting of molecules of interest, even though they comprise only a single variable region. Nanobodies have several useful features not regularly found in conventional antibodies. These include their small size, the capacity to bind and stabilize specific receptor conformations, and their availability in high yield from bacterial expression systems. Nanobodies have been widely used to target soluble protein antigens or those found at the surface of cells (e.g. for structural studies and imaging applications (for reviews see Refs.
      • Chanier T.
      • Chames P.
      Nanobody engineering: toward next generation immunotherapies and immunoimaging of cancer.
      and
      • Manglik A.
      • Kobilka B.K.
      • Steyaert J.
      Nanobodies to study G protein-coupled receptor structure and function.
      ). Similar to full-sized antibodies, nanobodies are suitable for flow cytometry, immunoprecipitation, affinity purification, and microscopy (
      • Beghein E.
      • Gettemans J.
      Nanobody technology: a versatile toolkit for microscopic imaging, protein-protein interaction analysis, and protein function exploration.
      ,
      • Braun M.B.
      • Traenkle B.
      • Koch P.A.
      • Emele F.
      • Weiss F.
      • Poetz O.
      • Stehle T.
      • Rothbauer U.
      Peptides in headlock–a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.
      ,
      • Traenkle B.
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      • Maier J.
      • Kaiser P.D.
      • Scholz A.M.
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      • Buchfellner A.
      • Romer T.
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      Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.
      ,
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      • Kilisch M.
      • Martínez-Carranza M.
      • Sograte-Idrissi S.
      • Rajavel A.
      • Schlichthaerle T.
      • Engels N.
      • Jungmann R.
      • Stenmark P.
      • Opazo F.
      • Frey S.
      The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications.
      ,
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      • McNaughton B.R.
      Evaluation of nanobody conjugates and protein fusions as bioanalytical reagents.
      ). Although nanobodies are often applied in settings that could just as well use standard, full-size antibodies, we emphasize scenarios where the use of a nanobody provides advantages. In this review, we cover topics including methods for the identification of target-specific nanobodies, functionalization of nanobodies using chemical and enzymatic methods, and the use of nanobodies that engage targets inside or at the surface of the cell as well as viral targets. We cover the development of nanobody-epitope tag pairs and the use of nanobodies in synthetic biology. This review may serve as an accessible resource for scientists looking to identify nanobodies useful for their system of interest. We focus on areas such as nanobody functionalization and synthetic biology, in which methods and use of nanobodies are rapidly evolving.

      Screening platforms

      Conventional antibodies (Igs) consist of two identical heavy (H) and light (L) chains that pair to form a stably folded protein, with an antigen-binding site to which the two variable (V) domains, VH and VL, contribute. Both interchain and intrachain disulfides and N-linked glycosylation are needed for effective assembly of Igs. These requirements preclude the proper assembly of full-size antibodies in the reducing environment of the cytoplasm. Single-chain variable fragments (scFvs) consist of the variable domains from the heavy and light chains, connected by a linker. Although some scFvs can function in the cytoplasm, many scFvs require intrachain disulfides to afford stability and appropriate heavy-light chain pairing. Heavy chain–only antibodies from camelids fold and function in the absence of light chains. These camelid immunoglobulin heavy chains can be shrunk to just their variable domains (Fig. 1) to yield VHHs or nanobodies, which can retain antigen binding in the absence of disulfide bond formation. They can thus be used in the cytosol of live cells, as discussed below. This feature of nanobodies is one of the signature advantages of their application, relative to more conventional alternatives, as discussed below.
      Figure thumbnail gr1
      Figure 1Structures of human and camelid Igs and fragments. Conventional human Igs (i.e. IgG) have been truncated to provide functional fragments (Fab and scFv) that contain variable regions from the light and heavy chains. In the case of the scFv, a linker is required to facilitate appropriate pairing of heavy- and light-chain variable regions. A subset of antibodies from camelids consists of only the heavy chains. Expression of the isolated variable region from heavy chain–only antibodies provides functional single-domain antibodies (VHHs/nanobodies).
      Methods to identify nanobodies that bind to targets of interest are essential for their effective deployment (
      • Liu W.
      • Song H.
      • Chen Q.
      • Yu J.
      • Xian M.
      • Nian R.
      • Feng D.
      Recent advances in the selection and identification of antigen-specific nanobodies.
      ). Target-binding nanobody clones are usually isolated from screening highly diverse pools of nanobodies. Such pools must be sufficiently large to contain appropriately specific nanobodies, a suitable screening method must be at hand to identify specific binders, and such binders should retain their properties in the relevant contexts, as in the case of cytoplasmic expression or when dealing with membrane proteins. Screening methods that provide nanobodies with desirable functional properties (receptor antagonism, agonism), selectivity for specific target conformations (structural studies, biosensors), and functionality in different subcellular localization (cytoplasmic, cell surface) are in short supply and constitute an area of emphasis for future exploration. Both immunization and screening strategies ought to be designed with the final application(s) of the resulting nanobodies in mind. For example, immunization with unfolded, denatured proteins is more likely to yield reagents that are useful in immunoblotting or immunohistochemistry on fixed samples.
      For library construction, B cells from naive or immunized camelids can serve as the point of departure, as can cultured camelid B cells exposed to antigens of interest (
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      • Jiménez-Munguía I.
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      • Potocnakova L.
      • Kanova E.
      • Bhide M.
      Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis.
      ). Purified proteins (
      • Pardon E.
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      • Triest S.
      • Rasmussen S.G.F.
      • Wohlkönig A.
      • Ruf A.
      • Muyldermans S.
      • Hol W.G.J.
      • Kobilka B.K.
      • Steyaert J.
      A general protocol for the generation of Nanobodies for structural biology.
      ), cells or cell lysates containing antigens of interest (
      • Jähnichen S.
      • Blanchetot C.
      • Maussang D.
      • Gonzalez-Pajuelo M.
      • Chow K.Y.
      • Bosch L.
      • De Vrieze S.
      • Serruys B.
      • Ulrichts H.
      • Vandevelde W.
      • Saunders M.
      • De Haard H.J.
      • Schols D.
      • Leurs R.
      • Vanlandschoot P.
      • et al.
      CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells.
      ,
      • Itoh K.
      • Reis A.H.
      • Hayhurst A.
      • Sokol S.Y.
      Isolation of nanobodies against Xenopus embryonic antigens using immune and non-immune phage display libraries.
      ), or recombinant DNA to induce antigen expression in the host (
      • Koch-Nolte F.
      • Reyelt J.
      • Schössow B.
      • Schwarz N.
      • Scheuplein F.
      • Rothenburg S.
      • Haag F.
      • Alzogaray V.
      • Cauerhff A.
      • Goldbaum F.A.
      Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo.
      ,
      • Rossotti M.A.
      • Henry K.A.
      • van Faassen H.
      • Tanha J.
      • Callaghan D.
      • Hussack G.
      • Arbabi-Ghahroudi M.
      • MacKenzie C.R.
      Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling.
      ) can serve as immunogens. DNA-based immunization has been particularly valuable for the generation of nanobodies against properly folded membrane proteins (
      • Eden T.
      • Menzel S.
      • Wesolowski J.
      • Bergmann P.
      • Nissen M.
      • Dubberke G.
      • Seyfried F.
      • Albrecht B.
      • Haag F.
      • Koch-Nolte F.
      A cDNA immunization strategy to generate nanobodies against membrane proteins in native conformation.
      ,
      • Peyrassol X.
      • Laeremans T.
      • Gouwy M.
      • Lahura V.
      • Debulpaep M.
      • Van Damme J.
      • Steyaert J.
      • Parmentier M.
      • Langer I.
      Development by genetic immunization of monovalent antibodies (nanobodies) behaving as antagonists of the human ChemR23 receptor.
      ). The diversity of nanobody sequences available in a given pool can be further expanded through mutagenesis. Both natural diversity mutagenesis, in which residues at positions in a nanobody with high diversity in naturally occurring collections of nanobodies are varied (
      • Tiller K.E.
      • Chowdhury R.
      • Li T.
      • Ludwig S.D.
      • Sen S.
      • Maranas C.D.
      • Tessier P.M.
      Facile affinity maturation of antibody variable domains using natural diversity mutagenesis.
      ), and virus-mediated directed evolution (
      • English J.G.
      • Olsen R.H.J.
      • Lansu K.
      • Patel M.
      • White K.
      • Cockrell A.S.
      • Singh D.
      • Strachan R.T.
      • Wacker D.
      • Roth B.L.
      VEGAS as a platform for facile directed evolution in mammalian cells.
      ) can increase diversity and identify novel nanobodies. Important features in the screening approach include the source of the nanobody pool (synthetic versus naive versus immunized library) (
      • Liu W.
      • Song H.
      • Chen Q.
      • Yu J.
      • Xian M.
      • Nian R.
      • Feng D.
      Recent advances in the selection and identification of antigen-specific nanobodies.
      ,
      • Moutel S.
      • Bery N.
      • Bernard V.
      • Keller L.
      • Lemesre E.
      • de Marco A.
      • Ligat L.
      • Rain J.-C.
      • Favre G.
      • Olichon A.
      • Perez F.
      NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.
      ), the mechanism by which nanobody proteins are produced and displayed (phage display versus yeast display versus bacterial display versus ribosome display versus DNA/RNA display) (
      • Salema V.
      • Fernández L.Á.
      Escherichia coli surface display for the selection of nanobodies.
      ), and the method by which antigens of interest are presented for selection (peptide or protein immobilization on solid support versus display of antigens on the cell surface versus labeled soluble antigen) (
      • Kavousipour S.
      • Mokarram P.
      • Gargari S.L.M.
      • Mostafavi-Pour Z.
      • Barazesh M.
      • Ramezani A.
      • Ashktorab H.
      • Mohammadi S.
      • Ghavami S.
      A comparison between cell, protein and peptide-based approaches for selection of nanobodies against CD44 from a synthetic library.
      ). Given the importance of identifying nanobodies that bind to membrane proteins, a variety of approaches have yielded nanobodies that bind to intact, properly folded membrane targets (
      • Veugelen S.
      • Dewilde M.
      • De Strooper B.
      • Chávez-Gutiérrez L.
      Screening and characterization strategies for nanobodies targeting membrane proteins.
      ,
      • Doshi R.
      • Chen B.R.
      • Vibat C.R.T.
      • Huang N.
      • Lee C.-W.
      • Chang G.
      In vitro nanobody discovery for integral membrane protein targets.
      ,
      • Crepin R.
      • Veggiani G.
      • Djender S.
      • Beugnet A.
      • Planeix F.
      • Pichon C.
      • Moutel S.
      • Amigorena S.
      • Perez F.
      • Ghinea N.
      • de Marco A.
      Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors.
      ) and that either block or induce activation (
      • Ren H.
      • Li J.
      • Zhang N.
      • Hu L.A.
      • Ma Y.
      • Tagari P.
      • Xu J.
      • Zhang M.-Y.
      Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors.
      ).
      The defining feature of a display method is the mechanism by which the biochemical properties of the nanobodies are linked to the genetic information encoding the nanobodies. The type of display method used also dictates the diversity of the library of nanobody sequences used. Phage display, in which nanobodies are fused in frame with viral proteins for display on the surface of phages—typically an M13 derivative—that encapsulate the relevant DNA sequence, is commonly used to pan for nanobodies (
      • Reader R.H.
      • Workman R.G.
      • Maddison B.C.
      • Gough K.C.
      Advances in the production and batch reformatting of phage antibody libraries.
      ,
      • Romao E.
      • Morales-Yanez F.
      • Hu Y.
      • Crauwels M.
      • De Pauw P.
      • Hassanzadeh G.G.
      • Devoogdt N.
      • Ackaert C.
      • Vincke C.
      • Muyldermans S.
      Identification of useful nanobodies by phage display of immune single domain libraries derived from camelid heavy chain antibodies.
      ). Phage display libraries with a diversity of 107 to 108 clones are common. Display-based approaches can also be applied using model single-cell organisms, such as Escherichia coli (
      • Salema V.
      • Fernández L.Á.
      Escherichia coli surface display for the selection of nanobodies.
      ), Staphylococcus sp. (
      • Cavallari M.
      Rapid and direct VHH and target identification by staphylococcal surface display libraries.
      ,
      • Fleetwood F.
      • Devoogdt N.
      • Pellis M.
      • Wernery U.
      • Muyldermans S.
      • Ståhl S.
      • Löfblom J.
      Surface display of a single-domain antibody library on Gram-positive bacteria.
      ), and yeast (
      • Uchański T.
      • Pardon E.
      • Steyaert J.
      Nanobodies to study protein conformational states.
      ,
      • McMahon C.
      • Baier A.S.
      • Pascolutti R.
      • Wegrecki M.
      • Zheng S.
      • Ong J.X.
      • Erlandson S.C.
      • Hilger D.
      • Rasmussen S.G.F.
      • Ring A.M.
      • Manglik A.
      • Kruse A.C.
      Yeast surface display platform for rapid discovery of conformationally selective nanobodies.
      ).
      Yeast display platforms have succeeded in the identification of nanobodies that bind to specific conformations of cell surface proteins, such as G protein–coupled receptors (GPCRs) (
      • Uchański T.
      • Pardon E.
      • Steyaert J.
      Nanobodies to study protein conformational states.
      ,
      • McMahon C.
      • Baier A.S.
      • Pascolutti R.
      • Wegrecki M.
      • Zheng S.
      • Ong J.X.
      • Erlandson S.C.
      • Hilger D.
      • Rasmussen S.G.F.
      • Ring A.M.
      • Manglik A.
      • Kruse A.C.
      Yeast surface display platform for rapid discovery of conformationally selective nanobodies.
      ). Bacterial and yeast display platforms of a complexity comparable with that of phage libraries have the advantage that antigen-binding clones can be detected and enriched by flow cytometry (
      • Salema V.
      • Fernández L.Á.
      Escherichia coli surface display for the selection of nanobodies.
      ).
      Ribosome display relies on a covalent bond between the nanobody and the encoding RNA chain. Both the translated nanobody sequence and the RNA that encodes it remain tethered to the ribosome when the mRNA lacks a stop codon. Nanobodies that bind to cell membrane proteins in specific conformations were thus obtained (
      • Hutter C.A.J.
      • Timachi M.H.
      • Hürlimann L.M.
      • Zimmermann I.
      • Egloff P.
      • Göddeke H.
      • Kucher S.
      • Štefanić S.
      • Karttunen M.
      • Schäfer L.V.
      • Bordignon E.
      • Seeger M.A.
      The extracellular gate shapes the energy profile of an ABC exporter.
      ,
      • Zimmermann I.
      • Egloff P.
      • Hutter C.A.
      • Arnold F.M.
      • Stohler P.
      • Bocquet N.
      • Hug M.N.
      • Huber S.
      • Siegrist M.
      • Hetemann L.
      • Gera J.
      • Gmür S.
      • Spies P.
      • Gygax D.
      • Geertsma E.R.
      • Dawson R.J.
      • Seeger M.A.
      Synthetic single domain antibodies for the conformational trapping of membrane proteins.
      ,
      • Ferrari D.
      • Garrapa V.
      • Locatelli M.
      • Bolchi A.
      A novel nanobody scaffold optimized for bacterial expression and suitable for the construction of ribosome display libraries.
      ). An approach called RNA display or cDNA display relies on the antibiotic puromycin applied in vitro to enter the ribosomal active site and form a cross-linking covalent bond with the nascent nanobody polypeptide and the encoding RNA sequence to enable selection (
      • Suzuki T.
      • Mochizuki Y.
      • Kimura S.
      • Akazawa-Ogawa Y.
      • Hagihara Y.
      • Nemoto N.
      Anti-survivin single-domain antibodies derived from an artificial library including three synthetic random regions by in vitro selection using cDNA display.
      ,
      • Takahashi K.
      • Sunohara M.
      • Terai T.
      • Kumachi S.
      • Nemoto N.
      Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions.
      ). Library complexity (up to 1012 unique clones) used in in vitro selection techniques can exceed by far those used in phage, bacterial, and yeast display, but screening then requires multiple rounds of selection to arrive at individual high-affinity binders (
      • Zimmermann I.
      • Egloff P.
      • Hutter C.A.
      • Arnold F.M.
      • Stohler P.
      • Bocquet N.
      • Hug M.N.
      • Huber S.
      • Siegrist M.
      • Hetemann L.
      • Gera J.
      • Gmür S.
      • Spies P.
      • Gygax D.
      • Geertsma E.R.
      • Dawson R.J.
      • Seeger M.A.
      Synthetic single domain antibodies for the conformational trapping of membrane proteins.
      ).
      Alternative methods of screening have been developed to identify nanobodies that function in their intended environment. In one such method, nanobody-coding sequences (minus the signal peptide) were inserted into lentiviral vectors for expression in the cytoplasm of mammalian cells. Nanobodies that protected cells from a lytic infection with influenza A virus or vesicular stomatitis virus were then identified through enrichment of surviving cells and recovery by PCR of the protective nanobody sequences (
      • Schmidt F.I.
      • Hanke L.
      • Morin B.
      • Brewer R.
      • Brusic V.
      • Whelan S.P.J.
      • Ploegh H.L.
      Phenotypic lentivirus screens to identify functional single domain antibodies.
      ). A similar approach was used to identify a nanobody that protected against porcine reproductive and respiratory syndrome virus (
      • Liu Z.-H.
      • Lei K.-X.
      • Han G.-W.
      • Xu H.-L.
      • He F.
      Novel lentivirus-based method for rapid selection of inhibitory nanobody against PRRSV.
      ). The use of a functional readout, like cell survival, ensured that the nanobodies were functional in the cytosol. The size of the library tested in the lentivirus-based approach is similar to that used in phage display (∼107 clones). Another method to identify nanobodies that are functional in the cytosol involves a yeast two-hybrid system, in which propagation of the yeast is contingent on the interaction of a nanobody clone with a target antigen of interest (
      • Visintin M.
      • Tse E.
      • Axelson H.
      • Rabbitts T.H.
      • Cattaneo A.
      Selection of antibodies for intracellular function using a two-hybrid in vivo system.
      ). This approach yielded nanobodies that bind HIV VPR and capsid proteins and the hemagglutinin-neuraminidase protein of the Newcastle disease virus (
      • Matz J.
      • Hérate C.
      • Bouchet J.
      • Dusetti N.
      • Gayet O.
      • Baty D.
      • Benichou S.
      • Chames P.
      Selection of intracellular single-domain antibodies targeting the HIV-1 Vpr protein by cytoplasmic yeast two-hybrid system.
      ,
      • Gao X.
      • Hu X.
      • Tong L.
      • Liu D.
      • Chang X.
      • Wang H.
      • Dang R.
      • Wang X.
      • Xiao S.
      • Du E.
      • Yang Z.
      Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus.
      ).

      Chemical and enzymatic functionalization

      Conventional recombinant expression in bacteria produces nanobodies in high yields, providing ample material for chemical functionalization. Conjugation of nanobodies with fluorescent dyes, small-molecule drugs, oligonucleotides, and other moieties allows complex yet controlled functionalization of nanobodies to extend their application to a wide range of areas, including imaging, therapeutics, and detection, and as delivery agents. Early examples of nanobody functionalization mostly relied on reactivity of cysteine and lysine residues using maleimide (
      • Massa S.
      • Xavier C.
      • De Vos J.
      • Caveliers V.
      • Lahoutte T.
      • Muyldermans S.
      • Devoogdt N.
      Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.
      ) and N-hydroxysuccinimide ester–based chemistry (
      • Ries J.
      • Kaplan C.
      • Platonova E.
      • Eghlidi H.
      • Ewers H.
      A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.
      ,
      • Nevoltris D.
      • Lombard B.
      • Dupuis E.
      • Mathis G.
      • Chames P.
      • Baty D.
      Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers.
      ). Nanobodies typically require the introduction of an unpaired Cys and disulfide reduction prior to labeling. N-Hydroxysuccinimide ester–based labeling lacks selectivity, resulting in heterogeneous mixtures of labeled proteins. Excessive labeling of nanobodies can cause loss of antigen recognition and specificity and result in altered pharmacokinetic properties (
      • Pleiner T.
      • Bates M.
      • Trakhanov S.
      • Lee C.-T.
      • Schliep J.E.
      • Chug H.
      • Böhning M.
      • Stark H.
      • Urlaub H.
      • Görlich D.
      Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.
      ,
      • Agarwal P.
      • Bertozzi C.R.
      Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.
      ,
      • Massa S.
      • Xavier C.
      • Muyldermans S.
      • Devoogdt N.
      Emerging site-specific bioconjugation strategies for radioimmunotracer development.
      ). Chemoenzymatic labeling methods, incorporation of unnatural amino acids, and expressed protein ligation are therefore attractive alternatives for the bioconjugation of nanobodies, as will be summarized below (Fig. 2). These methods enable the conjugation of nanobodies with a virtually unlimited selection of chemical cargoes. Even with these advances, it remains difficult to use nanobody conjugates prepared in vitro to address biology inside of live cells because of their membrane impermeability. The development of robust methods for delivery of nanobodies across the cell membrane (
      • Herce H.D.
      • Schumacher D.
      • Schneider A.F.L.
      • Ludwig A.K.
      • Mann F.A.
      • Fillies M.
      • Kasper M.-A.
      • Reinke S.
      • Krause E.
      • Leonhardt H.
      • Cardoso M.C.
      • Hackenberger C.P.R.
      Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.
      ,
      • Klein A.
      • Hank S.
      • Raulf A.
      • Joest E.F.
      • Tissen F.
      • Heilemann M.
      • Wieneke R.
      • Tampé R.
      Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies.
      ,
      • Teng K.W.
      • Ishitsuka Y.
      • Ren P.
      • Youn Y.
      • Deng X.
      • Ge P.
      • Lee S.H.
      • Belmont A.S.
      • Selvin P.R.
      Labeling proteins inside living cells using external fluorophores for microscopy.
      ) or labeling in cells with minimal background will empower new and powerful applications with conjugates.
      Figure thumbnail gr2
      Figure 2Recent examples of nanobody bioconjugation. A, enzymatic approaches, including OaAEP1 (a), formylglycine-generating enzyme (b), tubulin Butelase and tubulin tyrosine ligase need to be switched in the figure legend (Butelase is panel c, Tubulin tyrosine ligase is panel d) and sortase A (e). B, incorporation of unnatural amino acids by stop codon suppression. C, expressed protein ligation. See the Chemical and Enzymatic Functionalization section for a discussion of strengths and drawbacks of these approaches and associated references.

      Oldenlandia affinis asparaginyl endopeptidase (OaAEP1)

      Asparaginyl endopeptidases (AEPs) are an increasingly attractive class of enzymes for protein modification. AEPs are nominally Cys proteases that recognize a tripeptide motif, Asn/Asp-Xaa-Yaa, and generate a thioester intermediate C-terminally of the Asn or Asp residue. The thioester can be then attacked by a suitable nucleophile: a dipeptide Gly/Ala-Zaa, where Zaa is a hydrophobic amino acid residue. AEPs of plant origin, such as OaAEP1 from O. affinis, catalyze head-to-tail cyclization of peptides and have been used to prepare cyclic peptides (
      • Mylne J.S.
      • Chan L.Y.
      • Chanson A.H.
      • Daly N.L.
      • Schaefer H.
      • Bailey T.L.
      • Nguyencong P.
      • Cascales L.
      • Craik D.J.
      Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis.
      ,
      • Jackson M.A.
      • Gilding E.K.
      • Shafee T.
      • Harris K.S.
      • Kaas Q.
      • Poon S.
      • Yap K.
      • Jia H.
      • Guarino R.
      • Chan L.Y.
      • Durek T.
      • Anderson M.A.
      • Craik D.J.
      Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases.
      ,
      • Craik D.J.
      • Malik U.
      Cyclotide biosynthesis.
      ). The slow kinetics of OaAEP1 limited its application for protein labeling. Substitution of cysteine residue 247 by an alanine residue enhances OaAEP1's catalytic efficiency, making it an efficient tool for protein modification (
      • Yang R.
      • Wong Y.H.
      • Nguyen G.K.T.
      • Tam J.P.
      • Lescar J.
      • Wu B.
      Engineering a catalytically efficient recombinant protein ligase.
      ). The mutant OaAEP1 has been applied to the modification of nanobodies. By screening different nucleophiles, a Gly-Val dipeptide was identified that readily served as a nucleophile in the ligation reaction, but the product of that reaction (Asn-Gly-Val) was poorly recognized by the enzyme. This yields a ligation product resistant to the reverse reaction, a common shortcoming of enzymatic labeling methods such as sortagging (see “Sortase A”) (
      • Rehm F.B.H.
      • Harmand T.J.
      • Yap K.
      • Durek T.
      • Craik D.J.
      • Ploegh H.L.
      Site-specific sequential protein labeling catalyzed by a single recombinant ligase.
      ). The use of OaAEP1 with Asn-Gly-Leu–based modified tripeptides allowed efficient modification of the N terminus of a nanobody with the Gly-Val sequence at the N terminus. The use of OaAEP1 thus enabled conjugation of nanobodies with a broad range of different molecules: dyes, lipids, biotin, tetrazine, azide, cyclooctene, small-molecule drugs, PEG oligomers, d-amino acids, and β amino acids (
      • Rehm F.B.H.
      • Harmand T.J.
      • Yap K.
      • Durek T.
      • Craik D.J.
      • Ploegh H.L.
      Site-specific sequential protein labeling catalyzed by a single recombinant ligase.
      ).
      The resistance of the Asn-Gly-Val sequence—the ligation product—to attack by OaAEP1 allowed efficient site-specific modification of a nanobody both at the C and N terminus using the same enzyme, making possible the preparation of doubly functionalized nanobodies (
      • Rehm F.B.H.
      • Harmand T.J.
      • Yap K.
      • Durek T.
      • Craik D.J.
      • Ploegh H.L.
      Site-specific sequential protein labeling catalyzed by a single recombinant ligase.
      ), as follows. A C-terminal NGL sequence is enzymatically modified first with a Gly-Val modified peptide, yielding a cleavage-resistant product. The N-terminal modification required transient protection of the future N-terminal Gly-Val nucleophile by a TEV protease recognition sequence. Its removal freed up the N terminus for a second OaAEP reaction. Obviously, this method is not limited to modification of nanobodies and can be applied to other proteins of interest, and it might be particularly useful for single-molecule studies. It is also worth noting that, although not observed during nanobody modification, unwanted cleavage within the protein of interest is a side reaction that can occur while using enzyme from the AEP family.

      Tub-tag

      Tubulin tyrosine ligase (TTL) modifies the C terminus of a protein through conjugation of an unnatural tyrosine residue. This modified tyrosine can be equipped with a wide range of chemical substituents and thus serves to introduce into the newly modified protein important functionalities, such as azides, aldehydes, iodides, alkynes, and dyes. The TTL enzyme recognizes a glutamic acid–rich 14-amino acid sequence, also called Tub-tag (VDSVEGEGEEEGEE), which must be placed at the C terminus of the protein of interest. Labeling with TTL and Tub-tag has been used to modify nanobodies (
      • Schumacher D.
      • Lemke O.
      • Helma J.
      • Gerszonowicz L.
      • Waller V.
      • Stoschek T.
      • Durkin P.M.
      • Budisa N.
      • Leonhardt H.
      • Keller B.G.
      • Hackenberger C.P.R.
      Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.
      ) with fluorescent coumarin and biotin derivatives. Recombinantly produced nanobodies equipped with a Tub-tag sequence were likewise used to prepare nanobody-based immunoprecipitation tools and superresolution probes (
      • Schumacher D.
      • Lemke O.
      • Helma J.
      • Gerszonowicz L.
      • Waller V.
      • Stoschek T.
      • Durkin P.M.
      • Budisa N.
      • Leonhardt H.
      • Keller B.G.
      • Hackenberger C.P.R.
      Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.
      ,
      • Schumacher D.
      • Helma J.
      • Mann F.A.
      • Pichler G.
      • Natale F.
      • Krause E.
      • Cardoso M.C.
      • Hackenberger C.P.R.
      • Leonhardt H.
      Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase.
      ).

      Formylglycine-generating enzyme

      Formylglycine-generating enzymes allow the post-translational modification of cysteine or serine residues within distinct consensus motifs ((C/S)XPXR) to produce formylglycine. Such aldehydes are of particular interest as they can selectively react with hydrazides and amino-oxy moieties for site-specific modification of proteins (
      • Frese M.-A.
      • Dierks T.
      Formylglycine aldehyde tag—protein engineering through a novel post-translational modification.
      ,
      • Krüger T.
      • Dierks T.
      • Sewald N.
      Formylglycine-generating enzymes for site-specific bioconjugation.
      ). This approach was used to install an aldehyde motif on two nanobodies that recognize different epitopes on human β2-microglobulin (
      • Zang B.
      • Ren J.
      • Li D.
      • Huang C.
      • Ma H.
      • Peng Q.
      • Ji F.
      • Han L.
      • Jia L.
      Freezing-assisted synthesis of covalent C-C linked bivalent and bispecific nanobodies.
      ). This method allowed the preparation of C-to-C–linked homodimer nanobodies using an unusual method: aqueous solutions of formylglycine-containing nanobodies and bivalent hydrazide or aminoxy linkers were frozen at −20 °C. This reduction in temperature and freezing drastically increased the rate of dimer formation. Using a sequential approach in which one nanobody was reacted with an excess of the bivalent linker, followed by the addition of the second nanobody, yielded heterodimeric bivalent C-to-C–linked conjugates that were superior in antigen binding relative to C-to-N–linked dimers provided by simple genetic fusion.

      Butelase 1

      Butelase 1 is also a cysteine protease of the AEP family found in the seed pods of Clitoria ternatea. It recognizes a C-terminal Asn/Asp-containing tripeptide motif, Asn/Asp-His-Val, to form an Asn/Asp-Xaa-Yaa peptide bond, where Xaa can be any amino acid and Yaa is a hydrophobic residue. Butelase 1 is more than 10,000 times faster than other known ligases, with catalytic efficiencies of up to 1,340,000 m−1 s−1 (
      • Nguyen G.K.T.
      • Wang S.
      • Qiu Y.
      • Hemu X.
      • Lian Y.
      • Tam J.P.
      Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.
      ,
      • Nguyen G.K.T.
      • Qiu Y.
      • Cao Y.
      • Hemu X.
      • Liu C.-F.
      • Tam J.P.
      Butelase-mediated cyclization and ligation of peptides and proteins.
      ). This unique characteristic has made butelase 1 a powerful tool for the preparation of cyclic peptides and proteins or for the direct labeling of a protein (
      • Nguyen G.K.T.
      • Wang S.
      • Qiu Y.
      • Hemu X.
      • Lian Y.
      • Tam J.P.
      Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.
      ,
      • Hemu X.
      • Zhang X.
      • Bi X.
      • Liu C.-F.
      • Tam J.P.
      Butelase 1-mediated ligation of peptides and proteins.
      ). Our group used this enzyme together with sortase A to prepare homodimeric and heterodimeric nanobody conjugates connected via DNA linkers (
      • Harmand T.J.
      • Bousbaine D.
      • Chan A.
      • Zhang X.
      • Liu D.R.
      • Tam J.P.
      • Ploegh H.L.
      One-pot dual labeling of IgG 1 and preparation of C-to-C fusion proteins through a combination of sortase A and butelase 1.
      ). The use of dsDNA as linker between two nanobodies imparts rigidity on the linkage and is a straightforward method to control the length of spacing. Such control can be precious for biophysical studies (
      • Galimidi R.P.
      • Klein J.S.
      • Politzer M.S.
      • Bai S.
      • Seaman M.S.
      • Nussenzweig M.C.
      • West A.P.
      • Bjorkman P.J.
      Intra-spike crosslinking overcomes antibody evasion by HIV-1.
      ). Although butelase 1 is an attractive enzyme for protein engineering, a major drawback remains its availability. Despite new approaches to produce it in bacteria (
      • James A.M.
      • Haywood J.
      • Leroux J.
      • Ignasiak K.
      • Elliott A.G.
      • Schmidberger J.W.
      • Fisher M.F.
      • Nonis S.G.
      • Fenske R.
      • Bond C.S.
      • Mylne J.S.
      The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity.
      ,
      • Pi N.
      • Gao M.
      • Cheng X.
      • Liu H.
      • Kuang Z.
      • Yang Z.
      • Yang J.
      • Zhang B.
      • Chen Y.
      • Liu S.
      • Huang Y.
      • Su Z.
      Recombinant butelase-mediated cyclization of the p53-binding domain of the oncoprotein MdmX-stabilized protein conformation as a promising model for structural investigation.
      ), the main source of butelase 1 remains its extraction from seed pods of Clitoria ternatea (
      • Nguyen G.K.T.
      • Qiu Y.
      • Cao Y.
      • Hemu X.
      • Liu C.-F.
      • Tam J.P.
      Butelase-mediated cyclization and ligation of peptides and proteins.
      ).

      Sortase A

      Sortase A is an enzyme from Staphylococcus aureus that recognizes the amino acid sequence LPXTGG with high specificity (
      • Mao H.
      • Hart S.A.
      • Schink A.
      • Pollok B.A.
      Sortase-mediated protein ligation: a new method for protein engineering.
      ). This tag can be placed at the C terminus of the protein of interest but also internal to its sequence, as long as the recognition tag remains accessible (
      • Guimaraes C.P.
      • Witte M.D.
      • Theile C.S.
      • Bozkurt G.
      • Kundrat L.
      • Blom A.E.M.
      • Ploegh H.L.
      Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions.
      ). After recognition, sortase A cleaves between the threonine and glycine residue to form a thioacyl intermediate. An N-terminal polyglycine equipped with a payload of choice can attack this intermediate and form a new peptide bond (
      • Popp M.W.-L.
      • Ploegh H.L.
      Making and breaking peptide bonds: protein engineering using sortase.
      ). We have used this approach to prepare diverse nanobody conjugates: nanobody dimers through C-to-C fusion (
      • Witte M.D.
      • Cragnolini J.J.
      • Dougan S.K.
      • Yoder N.C.
      • Popp M.W.
      • Ploegh H.L.
      Preparation of unnatural N-to-N and C-to-C protein fusions.
      ), bispecific nanobodies against GFP and mouse class II major histocompatibility complex (
      • Witte M.D.
      • Cragnolini J.J.
      • Dougan S.K.
      • Yoder N.C.
      • Popp M.W.
      • Ploegh H.L.
      Preparation of unnatural N-to-N and C-to-C protein fusions.
      ), nanobodies labeled with radionuclides for PET imaging in vivo (
      • Rashidian M.
      • Keliher E.J.
      • Bilate A.M.
      • Duarte J.N.
      • Wojtkiewicz G.R.
      • Jacobsen J.T.
      • Cragnolini J.
      • Swee L.K.
      • Victora G.D.
      • Weissleder R.
      • Ploegh H.L.
      Noninvasive imaging of immune responses.
      ,
      • Rashidian M.
      • Wang L.
      • Edens J.G.
      • Jacobsen J.T.
      • Hossain I.
      • Wang Q.
      • Victora G.D.
      • Vasdev N.
      • Ploegh H.
      • Liang S.H.
      Enzyme-mediated modification of single-domain antibodies for imaging modalities with different characteristics.
      ), nanobody-drug conjugates against B-cell lymphoma (
      • Fang T.
      • Van Elssen C.H.M.J.
      • Duarte J.N.
      • Guzman J.S.
      • Chahal J.S.
      • Ling J.
      • Ploegh H.L.
      Targeted antigen delivery by an anti-class II MHC VHH elicits focused αMUC1(Tn) immunity.
      ), and many fluorescently labeled versions (
      • Rashidian M.
      • Wang L.
      • Edens J.G.
      • Jacobsen J.T.
      • Hossain I.
      • Wang Q.
      • Victora G.D.
      • Vasdev N.
      • Ploegh H.
      • Liang S.H.
      Enzyme-mediated modification of single-domain antibodies for imaging modalities with different characteristics.
      ). The orthogonality of sortase A and butelase 1 allowed the preparation, in a one-pot reaction, of C-C fusion nanobody dimers linked together with PEG and oligonucleotide linkers (
      • Harmand T.J.
      • Bousbaine D.
      • Chan A.
      • Zhang X.
      • Liu D.R.
      • Tam J.P.
      • Ploegh H.L.
      One-pot dual labeling of IgG 1 and preparation of C-to-C fusion proteins through a combination of sortase A and butelase 1.
      ). Proteins equipped with a suitably exposed stretch of Gly residues at the N terminus can be labeled with LPXTG-based peptides in a similar way (
      • Theile C.S.
      • Witte M.D.
      • Blom A.E.M.
      • Kundrat L.
      • Ploegh H.L.
      • Guimaraes C.P.
      Site-specific N-terminal labeling of proteins using sortase-mediated reactions.
      ).

      Native chemical and expressed protein ligation

      Native chemical ligation links unprotected polypeptides through an amide bond that relies on the reaction of a C-terminal thioester with an N-terminal Cys. Expressed protein ligation is based on the naturally occurring splicing of proteins, which proceeds via formation of a thioester intermediate (
      • Muir T.W.
      • Sondhi D.
      • Cole P.A.
      Expressed protein ligation: a general method for protein engineering.
      ). The protein of interest is expressed as a fusion with a mutant version of an intein. Activation with a thiol-containing small molecule, such as 2-mercaptoethanol, generates a C-terminal thioester on the protein of interest, such as a nanobody. Using this thioester, native chemical ligation can be used to attach the desired Cys-containing moiety to the nanobody. The expressed protein ligation approach was used to install on nanobodies two distinct arginine-rich cell-penetrating peptides (CPPs) for comparison. These synthetic CPPs contained d-amino acids, are cyclized, and are therefore impossible to introduce by standard genetic means (
      • Herce H.D.
      • Schumacher D.
      • Schneider A.F.L.
      • Ludwig A.K.
      • Mann F.A.
      • Fillies M.
      • Kasper M.-A.
      • Reinke S.
      • Krause E.
      • Leonhardt H.
      • Cardoso M.C.
      • Hackenberger C.P.R.
      Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.
      ). After attachment of the CPPs to an anti-GFP nanobody, these conjugates were delivered to the interior of the cell with an efficiency of up to 95% of cells, in different cell lines, and at relatively low concentrations (10 μm). Nucleolar localization caused by CPP-nucleic acid interactions could be avoided by attaching the CPP via a disulfide linkage.

      Unnatural amino acid incorporation using stop codon suppression

      Introduction of an unnatural bio-orthogonally functionalized amino acid can be achieved by using the cellular translational machinery and reassignment of a stop codon (
      • Neumann-Staubitz P.
      • Neumann H.
      The use of unnatural amino acids to study and engineer protein function.
      ,
      • Lee K.J.
      • Kang D.
      • Park H.-S.
      Site-specific labeling of proteins using unnatural amino acids.
      ). This new functional group then enables conjugation of a payload of choice in a site-selective manner. For example, a bifunctional unnatural amino acid derivative was introduced into an anti EGFR nanobody. The modified amino acid (AmAzZLys) contains an aryl amine and an azido group, which allows the conjugation of two different probes in orthogonal and selective fashion. Moreover, the azide moiety was used to perform a photoinduced cross-linking reaction to EGFR upon antigen binding (
      • Yamaguchi A.
      • Matsuda T.
      • Ohtake K.
      • Yanagisawa T.
      • Yokoyama S.
      • Fujiwara Y.
      • Watanabe T.
      • Hohsaka T.
      • Sakamoto K.
      Incorporation of a doubly functionalized synthetic amino acid into proteins for creating chemical and light-induced conjugates.
      ). Although attractive in principle, codon reassignment remains technically demanding and often suffers from reduced yields compared with conventional production methods.

      Targeting membrane proteins

      The high stability, propensity to bind and stabilize specific receptor conformations, and ease of production by recombinant expression make nanobodies well-suited for studying membrane proteins. These characteristics have inspired investigators from a variety of disciplines to use nanobodies. Immense efforts have gone into the identification of membrane protein–binding nanobodies. These efforts have positioned nanobodies as useful reagents for structural studies of membrane proteins using cryo-EM (
      • Mukherjee S.
      • Erramilli S.K.
      • Ammirati M.
      • Alvarez F.J.D.
      • Fennell K.F.
      • Purdy M.D.
      • Skrobek B.M.
      • Radziwon K.
      • Coukos J.
      • Kang Y.
      • Dutka P.
      • Gao X.
      • Qiu X.
      • Yeager M.
      • Eric Xu H.
      • et al.
      Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins.
      ,
      • Uchański T.
      • Masiulis S.
      • Fischer B.
      • Kalichuk V.
      • Wohlkönig A.
      • Zögg T.
      • Remaut H.
      • Vranken W.
      • Aricescu A.R.
      • Pardon E.
      • Steyaert J.
      Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.
      ) in a way analogous to past work with X-ray crystallography (
      • Pardon E.
      • Laeremans T.
      • Triest S.
      • Rasmussen S.G.F.
      • Wohlkönig A.
      • Ruf A.
      • Muyldermans S.
      • Hol W.G.J.
      • Kobilka B.K.
      • Steyaert J.
      A general protocol for the generation of Nanobodies for structural biology.
      ) (see also Fig. 3).
      Figure thumbnail gr3
      Figure 3Three examples of nanobodies used as crystallization chaperones. A, in the structure of nucleoporin Nup133 from S. cerevisiae, three nanobodies (shades of orange) generated the critical packing interface necessary to build up the crystal lattice (PDB code 6X04). B, in the structure of the nucleoporin complex of Nup107 and Nup133 from H. sapiens two different nanobodies that bind the Nup107 moiety in separate locations were co-crystallized (PDB code 6X03). C, in the TorsinA-LULL1 complex structure, the nanobody recognizes both binding partners and binds neither TorsinA (white) nor LULL1 (gray) individually (PDB codes 5J1S and 5J1T).
      Visualization of the trafficking of membrane proteins using monovalent nanobodies avoids cross-linking–induced artifacts that can arise from the use of bivalent antibodies (
      • Cheloha R.W.
      • Li Z.
      • Bousbaine D.
      • Woodham A.W.
      • Perrin P.
      • Volarić J.
      • Ploegh H.L.
      Internalization of influenza virus and cell surface proteins monitored by site-specific conjugation of protease-sensitive probes.
      ,
      • Buser D.P.
      • Schleicher K.D.
      • Prescianotto-Baschong C.
      • Spiess M.
      A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface.
      ). Nanobodies that bind Igs from mice (
      • Pleiner T.
      • Bates M.
      • Görlich D.
      A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies.
      ), rabbits (
      • Pleiner T.
      • Bates M.
      • Görlich D.
      A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies.
      ), pigs (
      • Harmsen M.M.
      • Van Solt C.B.
      • Fijten H.P.D.
      • Van Setten M.C.
      Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins.
      ), and humans (
      • Ereño-Orbea J.
      • Sicard T.
      • Cui H.
      • Carson J.
      • Hermans P.
      • Julien J.-P.
      Structural basis of enhanced crystallizability induced by a molecular chaperone for antibody antigen-binding fragments.
      ) can facilitate their use in place of conventional secondary antibodies (
      • Sograte-Idrissi S.
      • Schlichthaerle T.
      • Duque-Afonso C.J.
      • Alevra M.
      • Strauss S.
      • Moser T.
      • Jungmann R.
      • Rizzoli S.O.
      • Opazo F.
      Circumvention of common labelling artefacts using secondary nanobodies.
      ). Nanobodies have also proven useful for affinity purification of delicate membrane protein complexes (
      • Götzke H.
      • Kilisch M.
      • Martínez-Carranza M.
      • Sograte-Idrissi S.
      • Rajavel A.
      • Schlichthaerle T.
      • Engels N.
      • Jungmann R.
      • Stenmark P.
      • Opazo F.
      • Frey S.
      The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications.
      ,
      • Thomsen A.R.B.
      • Plouffe B.
      • Cahill T.J.
      • Shukla A.K.
      • Tarrasch J.T.
      • Dosey A.M.
      • Kahsai A.W.
      • Strachan R.T.
      • Pani B.
      • Mahoney J.P.
      • Huang L.
      • Breton B.
      • Heydenreich F.M.
      • Sunahara R.K.
      • Skiniotis G.
      • et al.
      GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling.
      ), but these are properties they share with conventional antibodies of similar specificity.
      Structural biologists are well-aware of the ability of nanobodies to facilitate crystallization of otherwise difficult-to-crystallize proteins. Nanobodies frequently contribute to crystal-packing contacts that facilitate structure determination (Fig. 3A). Structures where even two nanobodies bind to a single polypeptide have been produced (Fig. 3B). The binding of nanobodies to discontinuous epitopes that span more than one protein can also facilitate crystallization of the nanobody-bound complex (Fig. 3C). The single-domain nature of nanobodies implies that the universe of epitopes they can sample overlaps with, but is distinct from, that of conventional Igs. These unconventional modes of nanobody-antigen interaction should inspire new modes of application in biological settings, such as nanobody-induced target heterodimerization and target-induced nanobody dimerization. Moreover, such nanobodies can stabilize a receptor in a particular, functionally relevant conformation, as shown for various GPCRs and bacterial proteins. Structural studies have benefited from the use of nanobodies as chaperones, most notably for membrane proteins such as GPCRs (
      • Rasmussen S.G.F.
      • Choi H.-J.
      • Fung J.J.
      • Pardon E.
      • Casarosa P.
      • Chae P.S.
      • Devree B.T.
      • Rosenbaum D.M.
      • Thian F.S.
      • Kobilka T.S.
      • Schnapp A.
      • Konetzki I.
      • Sunahara R.K.
      • Gellman S.H.
      • Pautsch A.
      • et al.
      Structure of a nanobody-stabilized active state of the β2 adrenoceptor.
      ,
      • Shukla A.K.
      • Gupta C.
      • Srivastava A.
      • Jaiman D.
      Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.
      ,
      • De Groof T.W.M.
      • Bobkov V.
      • Heukers R.
      • Smit M.J.
      Nanobodies: new avenues for imaging, stabilizing and modulating GPCRs.
      ). In most cases, GPCR-binding nanobodies generated for structural studies bind the cytoplasmic face of the protein (
      • Rasmussen S.G.F.
      • Choi H.-J.
      • Fung J.J.
      • Pardon E.
      • Casarosa P.
      • Chae P.S.
      • Devree B.T.
      • Rosenbaum D.M.
      • Thian F.S.
      • Kobilka T.S.
      • Schnapp A.
      • Konetzki I.
      • Sunahara R.K.
      • Gellman S.H.
      • Pautsch A.
      • et al.
      Structure of a nanobody-stabilized active state of the β2 adrenoceptor.
      ,
      • Kruse A.C.
      • Ring A.M.
      • Manglik A.
      • Hu J.
      • Hu K.
      • Eitel K.
      • Hübner H.
      • Pardon E.
      • Valant C.
      • Sexton P.M.
      • Christopoulos A.
      • Felder C.C.
      • Gmeiner P.
      • Steyaert J.
      • Weis W.I.
      • et al.
      Activation and allosteric modulation of a muscarinic acetylcholine receptor.
      ,
      • Burg J.S.
      • Ingram J.R.
      • Venkatakrishnan A.J.
      • Jude K.M.
      • Dukkipati A.
      • Feinberg E.N.
      • Angelini A.
      • Waghray D.
      • Dror R.O.
      • Ploegh H.L.
      • Garcia K.C.
      Structural biology: structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.
      ) and have visualized different conformers of the GPCRs to which they bind. These features have been exploited by intracellular expression of GPCR-binding nanobodies, as will be discussed below. Nanobodies that bind to the extracellular domain of GPCRs facilitated crystallization of metabolic glutamate receptor-2 (mGluR2) (
      • Koehl A.
      • Hu H.
      • Feng D.
      • Sun B.
      • Zhang Y.
      • Robertson M.J.
      • Chu M.
      • Kobilka T.S.
      • Laeremans T.
      • Steyaert J.
      • Tarrasch J.
      • Dutta S.
      • Fonseca R.
      • Weis W.I.
      • Mathiesen J.M.
      • et al.
      Structural insights into the activation of metabotropic glutamate receptors.
      ) and of the apelin receptor (
      • Ma Y.
      • Ding Y.
      • Song X.
      • Ma X.
      • Li X.
      • Zhang N.
      • Song Y.
      • Sun Y.
      • Shen Y.
      • Zhong W.
      • Hu L.A.
      • Ma Y.
      • Zhang M.-Y.
      Structure-guided discovery of a single-domain antibody agonist against human apelin receptor.
      ). The latter enabled the first identification of nanobodies that activate a GPCR (
      • Ren H.
      • Li J.
      • Zhang N.
      • Hu L.A.
      • Ma Y.
      • Tagari P.
      • Xu J.
      • Zhang M.-Y.
      Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors.
      ,
      • Ma Y.
      • Ding Y.
      • Song X.
      • Ma X.
      • Li X.
      • Zhang N.
      • Song Y.
      • Sun Y.
      • Shen Y.
      • Zhong W.
      • Hu L.A.
      • Ma Y.
      • Zhang M.-Y.
      Structure-guided discovery of a single-domain antibody agonist against human apelin receptor.
      ). Bacterial membrane proteins have also been trapped by conformation-specific nanobodies, both for structural characterization (
      • Smirnova I.
      • Kasho V.
      • Jiang X.
      • Pardon E.
      • Steyaert J.
      • Kaback H.R.
      Transient conformers of LacY are trapped by nanobodies.
      ,
      • Kumar H.
      • Finer-Moore J.S.
      • Jiang X.
      • Smirnova I.
      • Kasho V.
      • Pardon E.
      • Steyaert J.
      • Kaback H.R.
      • Stroud R.M.
      Crystal structure of a ligand-bound LacY-nanobody complex.
      ) and for inhibition of their activity (
      • Perez C.
      • Köhler M.
      • Janser D.
      • Pardon E.
      • Steyaert J.
      • Zenobi R.
      • Locher K.P.
      Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody.
      ). Several accounts and reviews cover the development of nanobodies as reagents for structural studies (
      • Pardon E.
      • Laeremans T.
      • Triest S.
      • Rasmussen S.G.F.
      • Wohlkönig A.
      • Ruf A.
      • Muyldermans S.
      • Hol W.G.J.
      • Kobilka B.K.
      • Steyaert J.
      A general protocol for the generation of Nanobodies for structural biology.
      ) and their use as chaperones (
      • Manglik A.
      • Kobilka B.K.
      • Steyaert J.
      Nanobodies to study G protein-coupled receptor structure and function.
      ,
      • Shukla A.K.
      • Gupta C.
      • Srivastava A.
      • Jaiman D.
      Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.
      ). A comprehensive overview of structures containing nanobodies was published (
      • Zavrtanik U.
      • Lukan J.
      • Loris R.
      • Lah J.
      • Hadži S.
      Structural basis of epitope recognition by heavy-chain camelid antibodies.
      ), and a regularly updated database is available online (
      • Dunbar J.
      • Krawczyk K.
      • Leem J.
      • Baker T.
      • Fuchs A.
      • Georges G.
      • Shi J.
      • Deane C.M.
      SAbDab: the structural antibody database.
      ).
      Conformation-specific nanobodies that bind the cytoplasmic face of GPCRs have been valuable for structural studies of GPCRs in distinct conformations (
      • Staus D.P.
      • Strachan R.T.
      • Manglik A.
      • Pani B.
      • Kahsai A.W.
      • Kim T.H.
      • Wingler L.M.
      • Ahn S.
      • Chatterjee A.
      • Masoudi A.
      • Kruse A.C.
      • Pardon E.
      • Steyaert J.
      • Weis W.I.
      • Prosser R.S.
      • et al.
      Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.
      ,
      • Ring A.M.
      • Manglik A.
      • Kruse A.C.
      • Enos M.D.
      • Weis W.I.
      • Garcia K.C.
      • Kobilka B.K.
      Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody.
      ,
      • Che T.
      • English J.
      • Krumm B.E.
      • Kim K.
      • Pardon E.
      • Olsen R.H.J.
      • Wang S.
      • Zhang S.
      • Diberto J.F.
      • Sciaky N.
      • Carroll F.I.
      • Steyaert J.
      • Wacker D.
      • Roth B.L.
      Nanobody-enabled monitoring of κ opioid receptor states.
      ,
      • Wingler L.M.
      • McMahon C.
      • Staus D.P.
      • Lefkowitz R.J.
      • Kruse A.C.
      Distinctive activation mechanism for angiotensin receptor revealed by a synthetic nanobody.
      ,
      • Che T.
      • Majumdar S.
      • Zaidi S.A.
      • Ondachi P.
      • McCorvy J.D.
      • Wang S.
      • Mosier P.D.
      • Uprety R.
      • Vardy E.
      • Krumm B.E.
      • Han G.W.
      • Lee M.-Y.
      • Pardon E.
      • Steyaert J.
      • Huang X.-P.
      • et al.
      Structure of the nanobody-stabilized active state of the κ opioid receptor.
      ). When expressed in cells, they can serve as sensors to report on the localization of active receptors. Several mGluR2-binding nanobodies that bind the receptor ectodomain act as positive allosteric modulators and sensitize the receptors to respond to subthreshold levels of glutamate (
      • Koehl A.
      • Hu H.
      • Feng D.
      • Sun B.
      • Zhang Y.
      • Robertson M.J.
      • Chu M.
      • Kobilka T.S.
      • Laeremans T.
      • Steyaert J.
      • Tarrasch J.
      • Dutta S.
      • Fonseca R.
      • Weis W.I.
      • Mathiesen J.M.
      • et al.
      Structural insights into the activation of metabotropic glutamate receptors.
      ,
      • Scholler P.
      • Nevoltris D.
      • de Bundel D.
      • Bossi S.
      • Moreno-Delgado D.
      • Rovira X.
      • Møller T.C.
      • El Moustaine D.
      • Mathieu M.
      • Blanc E.
      • McLean H.
      • Dupuis E.
      • Mathis G.
      • Trinquet E.
      • Daniel H.
      • et al.
      Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation.
      ). A nanobody that binds the extracellular face of CXCR4 reports on conformational changes induced by small-molecule allosteric modulators (
      • Soave M.
      • Heukers R.
      • Kellam B.
      • Woolard J.
      • Smit M.J.
      • Briddon S.J.
      • Hill S.J.
      Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies.
      ). The use of nanobodies that lock β2-adrenergic receptor (β2AR) into active or inactive receptor conformations allowed identification of small-molecule agonists, antagonists, and inverse agonists using binding assays (
      • Pardon E.
      • Betti C.
      • Laeremans T.
      • Chevillard F.
      • Guillemyn K.
      • Kolb P.
      • Ballet S.
      • Steyaert J.
      Nanobody-enabled reverse pharmacology on G-protein-coupled receptors.
      ). The high yield of nanobodies from recombinant expression systems and the variety of chemical functionalization methods available have enabled the synthesis of conjugates between nanobodies and other complex molecules. A small-molecule ligand for mGlu2R was tethered to the receptor through a GFP-specific nanobody that recognized GFP grafted onto the receptor(
      • Farrants H.
      • Gutzeit V.A.
      • Acosta-Ruiz A.
      • Trauner D.
      • Johnsson K.
      • Levitz J.
      • Broichhagen J.
      SNAP-tagged nanobodies enable reversible optical control of a G protein-coupled receptor via a remotely tethered photoswitchable ligand.
      ). The use of a photoactivatable ligand provided reversible photocontrol of receptor activation on a time scale of seconds. In another approach, using a combination of enzymatic labeling and click chemistry, a truncated peptide ligand (PTH1-11) for the parathyroid hormone receptor (PTHR) was conjugated to a PTHR-specific nanobody (Fig. 4) (
      • Cheloha R.W.
      • Fischer F.A.
      • Woodham A.W.
      • Daley E.
      • Suminski N.
      • Gardella T.J.
      • Ploegh H.L.
      Improved GPCR ligands from nanobody tethering.
      ). Conjugation of a suboptimal peptide ligand to the nanobody enhanced the potency of the peptide by >100-fold in some cases and improved selectivity for one PTHR subtype over another. In a separate set of studies, the nanobodies that recognize surface proteins on antigen-presenting cells were conjugated to weakly immunogenic peptides, including those with nonnatural amino acids. These conjugates showed dramatically enhanced immunogenicity relative to free peptides in vitro and in vivo (
      • Duarte J.N.
      • Cragnolini J.J.
      • Swee L.K.
      • Bilate A.M.
      • Bader J.
      • Ingram J.R.
      • Rashidfarrokhi A.
      • Fang T.
      • Schiepers A.
      • Hanke L.
      • Ploegh H.L.
      Generation of immunity against pathogens via single-domain antibody-antigen constructs.
      ,
      • Cheloha R.W.
      • Woodham A.W.
      • Bousbaine D.
      • Wang T.
      • Liu S.
      • Sidney J.
      • Sette A.
      • Gellman S.H.
      • Ploegh H.L.
      Recognition of class II MHC peptide ligands that contain β-amino acids.
      ). It was thus possible to generate antibodies against the otherwise poorly immunogenic cyclotides, heavily disulfide-bonded circular peptides found in plants (
      • Kwon S.
      • Duarte J.N.
      • Li Z.
      • Ling J.J.
      • Cheneval O.
      • Durek T.
      • Schroeder C.I.
      • Craik D.J.
      • Ploegh H.L.
      Targeted delivery of cyclotides via conjugation to a nanobody.
      ).
      Figure thumbnail gr4
      Figure 4Nanobody-ligand conjugates to target a G protein–coupled receptor. Synthetic fragments of parathyroid hormone were site-specifically linked to nanobodies to provide conjugates (bottom right) with biological activity (EC50) superior to the free ligand (bottom left). Structures are based on human parathyroid hormone receptor (gray) in complex with PTH (orange) (PDB code 6FJ3) and a generic VHH (blue) with complementarity-determining regions highlighted (red) (PDB code 3K1K). The binding of the nanobody to PTHR1 (bottom right) is shown in two possible orientations as the actual site of binding is unknown.

      Targeting extracellular proteins

      Many nanobodies were developed to target soluble extracellular proteins. The list of such targets continues to expand at a rapid pace. The only nanobody currently approved for clinical use targets the secreted protein von Willebrand factor to treat a blood-clotting disorder (
      • Scully M.
      • Cataland S.R.
      • Peyvandi F.
      • Coppo P.
      • Knöbl P.
      • Kremer Hovinga J.A.
      • Metjian A.
      • de la Rubia J.
      • Pavenski K.
      • Callewaert F.
      • Biswas D.
      • De Winter H.
      • Zeldin R.K.
      Caplacizumab treatment for acquired thrombotic thrombocytopenic purpura.
      ). More nanobodies will find application as therapeutics and diagnostics, as evidenced by a wealth of preclinical data, some of which are summarized below. Nanobodies' small size endows them with a short circulatory t½ and superior tissue penetration. These two features in combination distinguish nanobodies from full-size conventional Igs and will determine the investigative and therapeutic areas to which they can be applied. One ongoing challenge is to identify targets, either extracellular or otherwise, for which nanobody application has advantages over conventional antibodies.
      Nanobodies can be used to block a variety of biological processes, such as placental growth factor-induced angiogenesis in cancer (
      • Arezumand R.
      • Mahdian R.
      • Zeinali S.
      • Hassanzadeh-Ghassabeh G.
      • Mansouri K.
      • Khanahmad H.
      • Namvar-Asl N.
      • Rahimi H.
      • Behdani M.
      • Cohan R.A.
      • Eavazalipour M.
      • Ramazani A.
      • Muyldermans S.
      Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis.
      ); the action of inflammatory proteins, such as tumor necrosis factor, interleukin-23, granulocyte colony–stimulating factor, and macrophage migration inhibitory factor (
      • Desmyter A.
      • Spinelli S.
      • Boutton C.
      • Saunders M.
      • Blachetot C.
      • de Haard H.
      • Denecker G.
      • Van Roy M.
      • Cambillau C.
      • Rommelaere H.
      Neutralization of human interleukin 23 by multivalent nanobodies explained by the structure of cytokine-nanobody complex.
      ,
      • Gorshkova E.N.
      • Efimov G.A.
      • Ermakova K.D.
      • Vasilenko E.A.
      • Yuzhakova D.V.
      • Shirmanova M.V.
      • Mokhonov V.V.
      • Tillib S.V.
      • Nedospasov S.A.
      • Astrakhantseva I.V.
      Properties of fluorescent far-red anti-TNF nanobodies.
      ,
      • Nosenko M.A.
      • Atretkhany K.-S.N.
      • Mokhonov V.V.
      • Efimov G.A.
      • Kruglov A.A.
      • Tillib S.V.
      • Drutskaya M.S.
      • Nedospasov S.A.
      VHH-based bispecific antibodies targeting cytokine production.
      ,
      • Bakherad H.
      • Gargari S.L.M.
      • Sepehrizadeh Z.
      • Aghamollaei H.
      • Taheri R.A.
      • Torshabi M.
      • Yazdi M.T.
      • Ebrahimizadeh W.
      • Setayesh N.
      Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.
      ,
      • Sparkes A.
      • De Baetselier P.
      • Brys L.
      • Cabrito I.
      • Sterckx Y.G.-J.
      • Schoonooghe S.
      • Muyldermans S.
      • Raes G.
      • Bucala R.
      • Vanlandschoot P.
      • Van Ginderachter J.A.
      • Stijlemans B.
      Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.
      ); and the action of various toxins and venoms (
      • Kaur M.
      • Dubey A.
      • Khatri M.
      • Sehrawat S.
      Secretory PLA2 specific single domain antibody neutralizes Russell viper venom induced cellular and organismal toxicity.
      ,
      • Vasylieva N.
      • Kitamura S.
      • Dong J.
      • Barnych B.
      • Hvorecny K.L.
      • Madden D.R.
      • Gee S.J.
      • Wolan D.W.
      • Morisseau C.
      • Hammock B.D.
      Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif).
      ,
      • Alirahimi E.
      • Kazemi-Lomedasht F.
      • Shahbazzadeh D.
      • Habibi-Anbouhi M.
      • Hosseininejad Chafi M.
      • Sotoudeh N.
      • Ghaderi H.
      • Muyldermans S.
      • Behdani M.
      Nanobodies as novel therapeutic agents in envenomation.
      ). A chimeric heavy chain–only antibody consisting of a proprotein convertase subtilisin/kexin type 9–binding nanobody and a portion of the human immunoglobulin heavy chain lowers low-density lipoprotein levels when administered in transgenic rats (
      • Li X.
      • Wang M.
      • Zhang X.
      • Liu C.
      • Xiang H.
      • Huang M.
      • Ma Y.
      • Gao X.
      • Jiang L.
      • Liu X.
      • Li B.
      • Hou Y.
      • Zhang X.
      • Yang S.
      • Yang N.
      The novel llama-human chimeric antibody has potent effect in lowering LDL-c levels in hPCSK9 transgenic rats.
      ). Nanobodies against neuronal tau (
      • Dupré E.
      • Danis C.
      • Arrial A.
      • Hanoulle X.
      • Homa M.
      • Cantrelle F.-X.
      • Merzougui H.
      • Colin M.
      • Rain J.-C.
      • Buée L.
      • Landrieu I.
      Single domain antibody fragments as new tools for the detection of neuronal tau protein in cells and in mice studies.
      ), human prion protein (
      • Abskharon R.N.N.
      • Giachin G.
      • Wohlkonig A.
      • Soror S.H.
      • Pardon E.
      • Legname G.
      • Steyaert J.
      Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.
      ), and α-synuclein (
      • Guilliams T.
      • El-Turk F.
      • Buell A.K.
      • O'Day E.M.
      • Aprile F.A.
      • Esbjörner E.K.
      • Vendruscolo M.
      • Cremades N.
      • Pardon E.
      • Wyns L.
      • Welland M.E.
      • Steyaert J.
      • Christodoulou J.
      • Dobson C.M.
      • De Genst E.
      Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages.
      ) provided insight into structural transitions that lead to amyloid formation (
      • Abskharon R.N.N.
      • Giachin G.
      • Wohlkonig A.
      • Soror S.H.
      • Pardon E.
      • Legname G.
      • Steyaert J.
      Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.
      ) and served as sensors to differentiate between fibrils at characteristically different stages (
      • Guilliams T.
      • El-Turk F.
      • Buell A.K.
      • O'Day E.M.
      • Aprile F.A.
      • Esbjörner E.K.
      • Vendruscolo M.
      • Cremades N.
      • Pardon E.
      • Wyns L.
      • Welland M.E.
      • Steyaert J.
      • Christodoulou J.
      • Dobson C.M.
      • De Genst E.
      Nanobodies raised against monomeric α-synuclein distinguish between fibrils at different maturation stages.
      ). Nanobodies raised against β2-microglobulin, a protein for which mutations frequently lead to amyloidosis, have illuminated structural features of aggregation intermediates in mutant versions of β2-microglobulin (
      • Domanska K.
      • Vanderhaegen S.
      • Srinivasan V.
      • Pardon E.
      • Dupeux F.
      • Marquez J.A.
      • Giorgetti S.
      • Stoppini M.
      • Wyns L.
      • Bellotti V.
      • Steyaert J.
      Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant.
      ,
      • Vanderhaegen S.
      • Fislage M.
      • Domanska K.
      • Versées W.
      • Pardon E.
      • Bellotti V.
      • Steyaert J.
      Structure of an early native-like intermediate of β2-microglobulin amyloidogenesis.
      ). They can prevent amyloid formation (
      • Raimondi S.
      • Porcari R.
      • Mangione P.P.
      • Verona G.
      • Marcoux J.
      • Giorgetti S.
      • Taylor G.W.
      • Ellmerich S.
      • Ballico M.
      • Zanini S.
      • Pardon E.
      • Al-Shawi R.
      • Simons J.P.
      • Corazza A.
      • Fogolari F.
      • et al.
      A specific nanobody prevents amyloidogenesis of D76N β2-microglobulin in vitro and modifies its tissue distribution in vivo.
      ) and remove β2-microglobulin from blood to treat dialysis-related amyloidosis (
      • Zhang L.
      • Zang B.
      • Huang C.
      • Ren J.
      • Jia L.
      One-step preparation of a VHH-based immunoadsorbent for the extracorporeal removal of β2-microglobulin.
      ). Gelsolin, a protein for which mutations lead to aberrant proteolytic processing and the formation of amyloidogenic fragments, has likewise been targeted with nanobodies. Nanobodies that bind gelsolin prevent proteolysis, either extracellularly or in the secretory pathway and reduce amyloidosis (
      • Van Overbeke W.
      • Wongsantichon J.
      • Everaert I.
      • Verhelle A.
      • Zwaenepoel O.
      • Loonchanta A.
      • Burtnick L.D.
      • De Ganck A.
      • Hochepied T.
      • Haigh J.
      • Cuvelier C.
      • Derave W.
      • Robinson R.C.
      • Gettemans J.
      An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model.
      ,
      • Van Overbeke W.
      • Verhelle A.
      • Everaert I.
      • Zwaenepoel O.
      • Vandekerckhove J.
      • Cuvelier C.
      • Derave W.
      • Gettemans J.
      Chaperone nanobodies protect gelsolin against MT1-MMP degradation and alleviate amyloid burden in the gelsolin amyloidosis mouse model.
      ). In vivo delivery of gelsolin-binding nanobodies using a viral vector reduces the amyloid burden in a mouse model (
      • Verhelle A.
      • Nair N.
      • Everaert I.
      • Van Overbeke W.
      • Supply L.
      • Zwaenepoel O.
      • Peleman C.
      • Van Dorpe J.
      • Lahoutte T.
      • Devoogdt N.
      • Derave W.
      • Chuah M.K.
      • VandenDriessche T.
      • Gettemans J.
      AAV9 delivered bispecific nanobody attenuates amyloid burden in the gelsolin amyloidosis mouse model.
      ). Anti-gelsolin nanobodies have also been used to visualize gelsolin amyloid deposits by SPECT/CT (
      • Verhelle A.
      • Van Overbeke W.
      • Peleman C.
      • De Smet R.
      • Zwaenepoel O.
      • Lahoutte T.
      • Van Dorpe J.
      • Devoogdt N.
      • Gettemans J.
      Non-invasive imaging of amyloid deposits in a mouse model of AGel using 99mTc-modified nanobodies and SPECT/CT.
      ).
      Early evaluation of nanobodies raised against carbonic anhydrase and amylase demonstrated inhibitory activity for several of them (
      • Lauwereys M.
      • Arbabi Ghahroudi M.
      • Desmyter A.
      • Kinne J.
      • Hölzer W.
      • De Genst E.
      • Wyns L.
      • Muyldermans S.
      Potent enzyme inhibitors derived from dromedary heavy-chain antibodies.
      ), encouraging further experiments to deploy nanobodies to modulate enzyme activity. Nanobodies that bind to and inhibit the protease urokinase-type plasminogen activator (uPA) (
      • Kromann-Hansen T.
      • Oldenburg E.
      • Yung K.W.Y.
      • Ghassabeh G.H.
      • Muyldermans S.
      • Declerck P.J.
      • Huang M.
      • Andreasen P.A.
      • Ngo J.C.K.
      A camelid-derived antibody fragment targeting the active site of a serine protease balances between inhibitor and substrate behavior.
      ,
      • Kaczmarek J.Z.
      • Skottrup P.D.
      Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator.
      ,
      • Kromann-Hansen T.
      • Louise Lange E.
      • Peter Sørensen H.
      • Hassanzadeh-Ghassabeh G.
      • Huang M.
      • Jensen J.K.
      • Muyldermans S.
      • Declerck P.J.
      • Komives E.A.
      • Andreasen P.A.
      Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator.
      ), which can contribute to cancer metastasis, may find clinical application. Crystallization of complexes between uPA and nanobodies shows how substrate binds and reveals the conformational equilibria that contribute (
      • Kromann-Hansen T.
      • Oldenburg E.
      • Yung K.W.Y.
      • Ghassabeh G.H.
      • Muyldermans S.
      • Declerck P.J.
      • Huang M.
      • Andreasen P.A.
      • Ngo J.C.K.
      A camelid-derived antibody fragment targeting the active site of a serine protease balances between inhibitor and substrate behavior.
      ,
      • Kromann-Hansen T.
      • Louise Lange E.
      • Peter Sørensen H.
      • Hassanzadeh-Ghassabeh G.
      • Huang M.
      • Jensen J.K.
      • Muyldermans S.
      • Declerck P.J.
      • Komives E.A.
      • Andreasen P.A.
      Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator.
      ). An inhibitory nanobody against matrix metalloprotease-8, one of ∼25 matrix metalloprotease family members in mammals that contribute to inflammatory responses, provides protection against pathological inflammation induced by lipopolysaccharide (
      • Demeestere D.
      • Dejonckheere E.
      • Steeland S.
      • Hulpiau P.
      • Haustraete J.
      • Devoogdt N.
      • Wichert R.
      • Becker-Pauly C.
      • Van Wonterghem E.
      • Dewaele S.
      • Van Imschoot G.
      • Aerts J.
      • Arckens L.
      • Saeys Y.
      • Libert C.
      • et al.
      Development and validation of a small single-domain antibody that effectively inhibits matrix metalloproteinase 8.
      ). Nanobodies that bind β-secretase affect enzyme function, with two nanobodies increasing and one inhibiting activity (
      • Dorresteijn B.
      • Rotman M.
      • Faber D.
      • Schravesande R.
      • Suidgeest E.
      • van der Weerd L.
      • van der Maarel S.M.
      • Verrips C.T.
      • El Khattabi M.
      Camelid heavy chain only antibody fragment domain against β-site of amyloid precursor protein cleaving enzyme 1 inhibits β-secretase activity in vitro in vivo.
      ). Injection of the inhibitory nanobody directly into the cerebrospinal fluid decreased deposition of β-amyloid as a result of β-secretase inhibition in a mouse model of Alzheimer's disease (
      • Dorresteijn B.
      • Rotman M.
      • Faber D.
      • Schravesande R.
      • Suidgeest E.
      • van der Weerd L.
      • van der Maarel S.M.
      • Verrips C.T.
      • El Khattabi M.
      Camelid heavy chain only antibody fragment domain against β-site of amyloid precursor protein cleaving enzyme 1 inhibits β-secretase activity in vitro in vivo.
      ). γ-Secretase, also relevant for Alzheimer's disease, has likewise been targeted for inhibition by nanobodies (
      • Veugelen S.
      • Dewilde M.
      • De Strooper B.
      • Chávez-Gutiérrez L.
      Screening and characterization strategies for nanobodies targeting membrane proteins.
      ). Nanobodies raised against plasminogen activator inhibitor-1 induced a profibrinolytic effect through stimulation of protease activity via neutralization of the protease inhibitor (
      • Zhou X.
      • Hendrickx M.L.V.
      • Hassanzadeh-Ghassabeh G.
      • Muyldermans S.
      • Declerck P.J.
      Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1.
      ,
      • Sillen M.
      • Weeks S.D.
      • Zhou X.
      • Komissarov A.A.
      • Florova G.
      • Idell S.
      • Strelkov S.V.
      • Declerck P.J.
      Molecular mechanism of two nanobodies that inhibit PAI-1 activity reveals a modulation at distinct stages of the PAI-1/plasminogen activator interaction.
      ). Nanobodies that bind thrombin-activatable fibrinolysis inhibitor (procarboxypeptidase U) block protease activation and thereby promote fibrinolysis (
      • Zhou X.
      • Weeks S.D.
      • Ameloot P.
      • Callewaert N.
      • Strelkov S.V.
      • Declerck P.J.
      Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin-activatable fibrinolysis inhibitor activation and activated thrombin-activatable fibrinolysis inhibitor activity.
      ). Combined, these examples demonstrate the versatility of the various nanobody platforms in their application to extracellular space.

      Targeting intracellular proteins

      Many nanobodies require neither glycosylation nor disulfide bond formation to retain their antigen-binding properties. They can thus be expressed as targeting reagents in the reducing environment of the cytosol. Conventional antibodies and their fragments mostly rely on their entry into the endoplasmic reticulum for assembly and glycosylation. The cytosol precludes association of immunoglobulin heavy and light chains, thus compromising their intracellular assembly into a functional unit. Intracellular nanobodies are typically introduced through transfection of DNA. This allows expression of nanobodies in either a constitutive or an inducible manner. Nanobodies can be expressed as monomeric units to modulate the activity of their targets upon binding or as fusions with fluorescent proteins or taggable protein domains for visualization of targets. Nanobodies, their variants, and fusions have also been used to redirect protein localization, induce protein degradation, and serve as biological sensors of protein conformation, abundance, and localization. Nanobodies that target nuclear proteins are often equipped with a nuclear localization sequence, although this is not always required (
      • Ju Shin Y.
      • Kyun Park S.
      • Jung Jung Y.
      • Na Kim Y.
      • Sung Kim K.
      • Kyu Park O.
      • Kwon S.-H.
      • Ho Jeon S.
      • Trinh L.A.
      • Fraser S.E.
      • Kee Y.
      • Joon Hwang B.
      Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.
      ). One of the main bottlenecks restricting the deployment of nanobodies in cells is the paucity of intracellular target-specific nanobodies. We provide summary of many relevant examples below without making claims as to completeness of the list provided (Table 1).
      Table 1Summary of nanobodies used to target intracellular proteins
      Intracellular target (function)Species
      Species of the antigen bound by the referenced nanobody. H, human; M, murine; B, bacterial; S, Salmonella; Y, yeast; R, rat; G, guinea pig.
      Application and biological impactReference
      Cytoplasmic proteins
      STAT3 (transcription factor)HSlows breast cancer growth in vitro and in vivo
      • Singh S.
      • Murillo G.
      • Chen D.
      • Parihar A.S.
      • Mehta R.G.
      Suppression of breast cancer cell proliferation by selective single-domain antibody for intracellular STAT3.
      TUT4 (uridytransferase)H, MBlocks microRNA uridylation and degradation
      • Yu C.
      • Wang L.
      • Rowe R.G.
      • Han A.
      • Ji W.
      • McMahon C.
      • Baier A.S.
      • Huang Y.-C.
      • Marion W.
      • Pearson D.S.
      • Kruse A.C.
      • Daley G.Q.
      • Wu H.
      • Sliz P.
      A nanobody targeting the LIN28:let-7 interaction fragment of TUT4 blocks uridylation of let-7.
      Calcium-dependent kinaseTInhibits kinase activity, crystallization chaperone
      • Ingram J.R.
      • Knockenhauer K.E.
      • Markus B.M.
      • Mandelbaum J.
      • Ramek A.
      • Shan Y.
      • Shaw D.E.
      • Schwartz T.U.
      • Ploegh H.L.
      • Lourido S.
      Allosteric activation of apicomplexan calcium-dependent protein kinases.
      CapG (actin-capping enzyme)HBlocks actin binding, inhibits cancer metastasis
      • Van Impe K.
      • Bethuyne J.
      • Cool S.
      • Impens F.
      • Ruano-Gallego D.
      • De Wever O.
      • Vanloo B.
      • Van Troys M.
      • Lambein K.
      • Boucherie C.
      • Martens E.
      • Zwaenepoel O.
      • Hassanzadeh-Ghassabeh G.
      • Vandekerckhove J.
      • Gevaert K.
      • et al.
      A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis.
      HypE (AMPylation)HInhibits or activates enzyme, cellular imaging
      • Truttmann M.C.
      • Wu Q.
      • Stiegeler S.
      • Duarte J.N.
      • Ingram J.
      • Ploegh H.L.
      HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation.
      SpvB (ADP-ribosylation)SInhibits enzyme, blocks cytoskeletal changes
      • Alzogaray V.
      • Danquah W.
      • Aguirre A.
      • Urrutia M.
      • Berguer P.
      • García Véscovi E.
      • Haag F.
      • Koch-Nolte F.
      • Goldbaum F.A.
      Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium.
      ASC (inflammasome adaptor)HInterrupts assembly, cellular imaging
      • Schmidt F.I.
      • Lu A.
      • Chen J.W.
      • Ruan J.
      • Tang C.
      • Wu H.
      • Ploegh H.L.
      A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.
      Roco (GTPase)BDestabilizes dimer, enhances GTP hydrolysis
      • Leemans M.
      • Galicia C.
      • Deyaert E.
      • Daems E.
      • Krause L.
      • Paesmans J.
      • Pardon E.
      • Steyaert J.
      • Kortholt A.
      • Sobott F.
      • Klostermeier D.
      • Versées W.
      Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies.
      RhoA (GTPase)HEither inhibits RhoA or tracks localization
      • Moutel S.
      • Bery N.
      • Bernard V.
      • Keller L.
      • Lemesre E.
      • de Marco A.
      • Ligat L.
      • Rain J.-C.
      • Favre G.
      • Olichon A.
      • Perez F.
      NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.
      ,
      • Keller L.
      • Bery N.
      • Tardy C.
      • Ligat L.
      • Favre G.
      • Rabbitts T.H.
      • Olichon A.
      Selection and characterization of a nanobody biosensor of GTP-bound RHO activities.
      RhoB (GTPase)HTargeted degradation of GTP-bound RhoB
      • Bery N.
      • Keller L.
      • Soulié M.
      • Gence R.
      • Iscache A.-L.
      • Cherier J.
      • Cabantous S.
      • Sordet O.
      • Lajoie-Mazenc I.
      • Pedelacq J.-D.
      • Favre G.
      • Olichon A.
      A targeted protein degradation cell-based screening for nanobodies selective toward the cellular RHOB GTP-bound conformation.
      Gβ/γ (GTPase subunit)HBlockade of signaling following GPCR activation
      • Gulati S.
      • Jin H.
      • Masuho I.
      • Orban T.
      • Cai Y.
      • Pardon E.
      • Martemyanov K.A.
      • Kiser P.D.
      • Stewart P.L.
      • Ford C.P.
      • Steyaert J.
      • Palczewski K.
      Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor.
      L-plastin (actin-bundling protein)HInhibits enzyme function, defective immune synapse formation
      • De Clercq S.
      • Zwaenepoel O.
      • Martens E.
      • Vandekerckhove J.
      • Guillabert A.
      • Gettemans J.
      Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation.
      ,
      • Delanote V.
      • Vanloo B.
      • Catillon M.
      • Friederich E.
      • Vandekerckhove J.
      • Gettemans J.
      An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling.
      p53 (tumor suppressor protein)HRelocalization to mitochondria or protection from proteasomal degradation
      • Steels A.
      • Verhelle A.
      • Zwaenepoel O.
      • Gettemans J.
      Intracellular displacement of p53 using transactivation domain (p53 TAD) specific nanobodies.
      ,
      • Steels A.
      • Vannevel L.
      • Zwaenepoel O.
      • Gettemans J.
      Nb-induced stabilisation of p53 in HPV-infected cells.
      H2A/H2B (histone)Y, M, HDirects ubiquitination to induce DNA damage signaling, imaging
      • Jullien D.
      • Vignard J.
      • Fedor Y.
      • Béry N.
      • Olichon A.
      • Crozatier M.
      • Erard M.
      • Cassard H.
      • Ducommun B.
      • Salles B.
      • Mirey G.
      Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells.
      UBC6e (E2 enzyme)M, HEnhances enzyme function in vitro
      • Ling J.
      • Cheloha R.W.
      • McCaul N.
      • Sun Z.-Y.J.
      • Wagner G.
      • Ploegh H.L.
      A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.
      Dynamin (GTPase)HBinds GTP-bound enzyme, visualization of localization
      • Galli V.
      • Sebastian R.
      • Moutel S.
      • Ecard J.
      • Perez F.
      • Roux A.
      Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab.
      Intracellular face of plasma membrane protein
      VGLUT (Glu transporter)RInhibits glutamate transport, visualization
      • Schenck S.
      • Kunz L.
      • Sahlender D.
      • Pardon E.
      • Geertsma E.R.
      • Savtchouk I.
      • Suzuki T.
      • Neldner Y.
      • Štefanić S.
      • Steyaert J.
      • Volterra A.
      • Dutzler R.
      Generation and characterization of anti-VGLUT nanobodies acting as inhibitors of transport.
      P-glycoprotein (transporter)MInhibits function in vitro
      • Ward A.B.
      • Szewczyk P.
      • Grimard V.
      • Lee C.-W.
      • Martinez L.
      • Doshi R.
      • Caya A.
      • Villaluz M.
      • Pardon E.
      • Cregger C.
      • Swartz D.J.
      • Falson P.G.
      • Urbatsch I.L.
      • Govaerts C.
      • Steyaert J.
      • et al.
      Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.
      β2AR (GPCR)HBinds and stabilizes the receptor active or inactive states for structural studies and visualization
      • Irannejad R.
      • Tomshine J.C.
      • Tomshine J.R.
      • Chevalier M.
      • Mahoney J.P.
      • Steyaert J.
      • Rasmussen S.G.F.
      • Sunahara R.K.
      • El-Samad H.
      • Huang B.
      • von Zastrow M.
      Conformational biosensors reveal GPCR signalling from endosomes.
      ,
      • Staus D.P.
      • Wingler L.M.
      • Strachan R.T.
      • Rasmussen S.G.F.
      • Pardon E.
      • Ahn S.
      • Steyaert J.
      • Kobilka B.K.
      • Lefkowitz R.J.
      Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.
      Muscarinic acetylcholine receptor (GPCR)HBinds and stabilizes the receptor active state for structural studies
      • Kruse A.C.
      • Ring A.M.
      • Manglik A.
      • Hu J.
      • Hu K.
      • Eitel K.
      • Hübner H.
      • Pardon E.
      • Valant C.
      • Sexton P.M.
      • Christopoulos A.
      • Felder C.C.
      • Gmeiner P.
      • Steyaert J.
      • Weis W.I.
      • et al.
      Activation and allosteric modulation of a muscarinic acetylcholine receptor.
      κ- and μ-opioid receptors (GPCR)HBinds and stabilizes the receptor active or inactive states for structural studies and visualization
      • Che T.
      • English J.
      • Krumm B.E.
      • Kim K.
      • Pardon E.
      • Olsen R.H.J.
      • Wang S.
      • Zhang S.
      • Diberto J.F.
      • Sciaky N.
      • Carroll F.I.
      • Steyaert J.
      • Wacker D.
      • Roth B.L.
      Nanobody-enabled monitoring of κ opioid receptor states.
      ,
      • Stoeber M.
      • Jullié D.
      • Lobingier B.T.
      • Laeremans T.
      • Steyaert J.
      • Schiller P.W.
      • Manglik A.
      • von Zastrow M.
      A genetically encoded biosensor reveals location bias of opioid drug action.
      ,
      • Livingston K.E.
      • Mahoney J.P.
      • Manglik A.
      • Sunahara R.K.
      • Traynor J.R.
      Measuring ligand efficacy at the μ-opioid receptor using a conformational biosensor.
      CaV1/CaV2 (high voltage–activated calcium channel)H, M, GExpressed as E3 fusion to ubiquitinate, redirect localization, inhibit function
      • Morgenstern T.J.
      • Park J.
      • Fan Q.R.
      • Colecraft H.M.
      A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits.
      a Species of the antigen bound by the referenced nanobody. H, human; M, murine; B, bacterial; S, Salmonella; Y, yeast; R, rat; G, guinea pig.
      Transfection-based approaches do not allow direct installation on nanobodies of bright organic fluorophores, which mostly requires chemical methods applied to purified nanobodies. Delivery of labeled nanobodies and other antibody derivatives across the plasma membrane (
      • Slastnikova T.A.
      • Ulasov A.V.
      • Rosenkranz A.A.
      • Sobolev A.S.
      Targeted intracellular delivery of antibodies: the state of the art.
      ) has relied on appending cell-penetrating peptides (
      • Herce H.D.
      • Schumacher D.
      • Schneider A.F.L.
      • Ludwig A.K.
      • Mann F.A.
      • Fillies M.
      • Kasper M.-A.
      • Reinke S.
      • Krause E.
      • Leonhardt H.
      • Cardoso M.C.
      • Hackenberger C.P.R.
      Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.
      ,
      • Tabtimmai L.
      • Suphakun P.
      • Srisook P.
      • Kiriwan D.
      • Phanthong S.
      • Kiatwuthinon P.
      • Chaicumpa W.
      • Choowongkomon K.
      Cell-penetrable nanobodies (transbodies) that inhibit the tyrosine kinase activity of EGFR leading to the impediment of human lung adenocarcinoma cell motility and survival.
      ,
      • van Lith S.A.M.
      • van den Brand D.
      • Wallbrecher R.
      • Wübbeke L.
      • van Duijnhoven S.M.J.
      • Mäkinen P.I.
      • Hoogstad-van Evert J.S.
      • Massuger L.
      • Ylä-Herttuala S.
      • Brock R.
      • Leenders W.P.J.
      The effect of subcellular localization on the efficiency of EGFR-targeted VHH photosensitizer conjugates.
      ), mutagenesis of nanobody surface residues to increase their positive charge (
      • Bruce V.J.
      • Lopez-Islas M.
      • McNaughton B.R.
      Resurfaced cell-penetrating nanobodies: a potentially general scaffold for intracellularly targeted protein discovery.
      ), complexation with cell-penetrating mesoporous silica nanoparticles (
      • Chiu H.-Y.
      • Deng W.
      • Engelke H.
      • Helma J.
      • Leonhardt H.
      • Bein T.
      Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time.
      ), and the use of a microfluidics-based cell permeabilization platform (
      • Klein A.
      • Hank S.
      • Raulf A.
      • Joest E.F.
      • Tissen F.
      • Heilemann M.
      • Wieneke R.
      • Tampé R.
      Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies.
      ). Cell-penetrating nanobodies have been used for imaging (
      • Herce H.D.
      • Schumacher D.
      • Schneider A.F.L.
      • Ludwig A.K.
      • Mann F.A.
      • Fillies M.
      • Kasper M.-A.
      • Reinke S.
      • Krause E.
      • Leonhardt H.
      • Cardoso M.C.
      • Hackenberger C.P.R.
      Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.
      ,
      • Klein A.
      • Hank S.
      • Raulf A.
      • Joest E.F.
      • Tissen F.
      • Heilemann M.
      • Wieneke R.
      • Tampé R.
      Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies.
      ), to inhibit EGFR function from the cytoplasmic side of the membrane in lung cancer cells (
      • Tabtimmai L.
      • Suphakun P.
      • Srisook P.
      • Kiriwan D.
      • Phanthong S.
      • Kiatwuthinon P.
      • Chaicumpa W.
      • Choowongkomon K.
      Cell-penetrable nanobodies (transbodies) that inhibit the tyrosine kinase activity of EGFR leading to the impediment of human lung adenocarcinoma cell motility and survival.
      ), and to evaluate the importance of subcellular localization in photosensitizer-induced cell killing (
      • van Lith S.A.M.
      • van den Brand D.
      • Wallbrecher R.
      • Wübbeke L.
      • van Duijnhoven S.M.J.
      • Mäkinen P.I.
      • Hoogstad-van Evert J.S.
      • Massuger L.
      • Ylä-Herttuala S.
      • Brock R.
      • Leenders W.P.J.
      The effect of subcellular localization on the efficiency of EGFR-targeted VHH photosensitizer conjugates.
      ). The design of methods to deliver proteins such as nanobodies across the plasma membrane is an area of active research, but accumulation of most such exogenously added proteins in the endocytic compartment is difficult to avoid and remains a major confounding factor.
      Nanobodies that bind intracellular proteins enable characterization and modulation of proteins of interest and can avoid the need for genetic modification of the target protein. A summary of several nanobodies that target intracellular proteins is shown in Table 1. Targets include mammalian and bacterial proteins; soluble cytoplasmic proteins and those embedded in membranes; and proteins in the nucleus, endoplasmic reticulum membrane, and inner leaflet of the plasma membrane. Nanobodies can inhibit intracellular signaling proteins, such as the transcription factor STAT3 (
      • Singh S.
      • Murillo G.
      • Chen D.
      • Parihar A.S.
      • Mehta R.G.
      Suppression of breast cancer cell proliferation by selective single-domain antibody for intracellular STAT3.
      ), the uridyltransferase TUT4 (
      • Yu C.
      • Wang L.
      • Rowe R.G.
      • Han A.
      • Ji W.
      • McMahon C.
      • Baier A.S.
      • Huang Y.-C.
      • Marion W.
      • Pearson D.S.
      • Kruse A.C.
      • Daley G.Q.
      • Wu H.
      • Sliz P.
      A nanobody targeting the LIN28:let-7 interaction fragment of TUT4 blocks uridylation of let-7.
      ), apicomplexan calcium-dependent kinase (
      • Ingram J.R.
      • Knockenhauer K.E.
      • Markus B.M.
      • Mandelbaum J.
      • Ramek A.
      • Shan Y.
      • Shaw D.E.
      • Schwartz T.U.
      • Ploegh H.L.
      • Lourido S.
      Allosteric activation of apicomplexan calcium-dependent protein kinases.
      ), the actin-capping protein CapG (
      • Van Impe K.
      • Bethuyne J.
      • Cool S.
      • Impens F.
      • Ruano-Gallego D.
      • De Wever O.
      • Vanloo B.
      • Van Troys M.
      • Lambein K.
      • Boucherie C.
      • Martens E.
      • Zwaenepoel O.
      • Hassanzadeh-Ghassabeh G.
      • Vandekerckhove J.
      • Gevaert K.
      • et al.
      A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis.
      ), the AMPylation enzyme HypeE (
      • Truttmann M.C.
      • Wu Q.
      • Stiegeler S.
      • Duarte J.N.
      • Ingram J.
      • Ploegh H.L.
      HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation.
      ) and the Salmonella ADP-ribosylation enzyme SpvB (
      • Alzogaray V.
      • Danquah W.
      • Aguirre A.
      • Urrutia M.
      • Berguer P.
      • García Véscovi E.
      • Haag F.
      • Koch-Nolte F.
      • Goldbaum F.A.
      Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium.
      ). A nanobody that targets an allosteric site on a bacterial LRRK2 GTPase homologue modulates oligomerization and enzymatic activity (
      • Leemans M.
      • Galicia C.
      • Deyaert E.
      • Daems E.
      • Krause L.
      • Paesmans J.
      • Pardon E.
      • Steyaert J.
      • Kortholt A.
      • Sobott F.
      • Klostermeier D.
      • Versées W.
      Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies.
      ). The ER-localized E2 enzyme UBC6e is targeted by a nanobody that augments enzymatic activity without obvious biological consequences (
      • Ling J.
      • Cheloha R.W.
      • McCaul N.
      • Sun Z.-Y.J.
      • Wagner G.
      • Ploegh H.L.
      A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.
      ). A biological sensing platform that relies on the expression of two nanobodies that bind to different epitopes on GFP, each linked to one part of a transcription factor, converts the production of intracellular GFP to a transcriptional output (
      • Tang J.C.Y.
      • Szikra T.
      • Kozorovitskiy Y.
      • Teixiera M.
      • Sabatini B.L.
      • Roska B.
      • Cepko C.L.
      A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation.
      ).
      Intracellular proteins that require oligomerization and those trafficked through the endoplasmic reticulum have also been probed using nanobodies. Co-translational delivery of a nanobody to the endoplasmic reticulum prevents aberrant processing and aggregation of a variant of the secreted form of gelsolin (
      • Van Overbeke W.
      • Wongsantichon J.
      • Everaert I.
      • Verhelle A.
      • Zwaenepoel O.
      • Loonchanta A.
      • Burtnick L.D.
      • De Ganck A.
      • Hochepied T.
      • Haigh J.
      • Cuvelier C.
      • Derave W.
      • Robinson R.C.
      • Gettemans J.
      An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model.
      ). Expression of a nanobody that recognizes ASC, an adaptor protein important in inflammasome assembly, enabled visualization of inflammasome assembly in cells and altered the morphology of assembled inflammasomes (
      • Schmidt F.I.
      • Lu A.
      • Chen J.W.
      • Ruan J.
      • Tang C.
      • Wu H.
      • Ploegh H.L.
      A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.
      ). Nanobodies raised against the active (GTP-bound) forms of the GTPases RhoA and dynamin enabled tracking of active enzymes in living cells (
      • Keller L.
      • Bery N.
      • Tardy C.
      • Ligat L.
      • Favre G.
      • Rabbitts T.H.
      • Olichon A.
      Selection and characterization of a nanobody biosensor of GTP-bound RHO activities.
      ,
      • Galli V.
      • Sebastian R.
      • Moutel S.
      • Ecard J.
      • Perez F.
      • Roux A.
      Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab.
      ). Using these tools, active RhoA was detected at the inner plasma membrane upon overt activation; active dynamin was formed in stochastic bursts associated with membrane fission. A nanobody that binds and inhibits the function of β/γ-subunit complex of the heterotrimeric G proteins showed that blockade of β/γ-subunit function has minimal impact on Gα function (
      • Gulati S.
      • Jin H.
      • Masuho I.
      • Orban T.
      • Cai Y.
      • Pardon E.
      • Martemyanov K.A.
      • Kiser P.D.
      • Stewart P.L.
      • Ford C.P.
      • Steyaert J.
      • Palczewski K.
      Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor.
      ). The expression of nanobodies that inhibit the actin-bundling protein L-plastin uncovered a role for this protein and the T cell integrin LFA-1 in facilitating the formation of the immune synapse (
      • De Clercq S.
      • Zwaenepoel O.
      • Martens E.
      • Vandekerckhove J.
      • Guillabert A.
      • Gettemans J.
      Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation.
      ,
      • Delanote V.
      • Vanloo B.
      • Catillon M.
      • Friederich E.
      • Vandekerckhove J.
      • Gettemans J.
      An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling.
      ). Two proteins found in the plasma membrane, VGLUT and P-glycoprotein, are targeted at their cytoplasmic faces by nanobodies that inhibit their function (
      • Schenck S.
      • Kunz L.
      • Sahlender D.
      • Pardon E.
      • Geertsma E.R.
      • Savtchouk I.
      • Suzuki T.
      • Neldner Y.
      • Štefanić S.
      • Steyaert J.
      • Volterra A.
      • Dutzler R.
      Generation and characterization of anti-VGLUT nanobodies acting as inhibitors of transport.
      ,
      • Ward A.B.
      • Szewczyk P.
      • Grimard V.
      • Lee C.-W.
      • Martinez L.
      • Doshi R.
      • Caya A.
      • Villaluz M.
      • Pardon E.
      • Cregger C.
      • Swartz D.J.
      • Falson P.G.
      • Urbatsch I.L.
      • Govaerts C.
      • Steyaert J.
      • et al.
      Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.
      ).
      Intracellular nanobody constructs can be adapted to yield fusion proteins that enable the relocalization, destruction, or enzymatic modification of nanobody-bound targets. The fusion of a nanobody to O-GlcNAc transferase enabled directed glycosylation of proteins targeted by the nanobody fusion (
      • Ramirez D.H.
      • Aonbangkhen C.
      • Wu H.-Y.
      • Naftaly J.A.
      • Tang S.
      • O'Meara T.R.
      • Woo C.M.
      Engineering a proximity-directed O-GlcNAc transferase for selective protein O-GlcNAcylation in cells.
      ), a post-translational modification that is otherwise widespread. Nanobodies, expressed as fusions with tags that dictate a particular subcellular localization, can redirect the localization of proteins of interest and serve to control protein diffusion. GFP-binding nanobodies routed to subcellular sites have been used to assess the impact of redirecting GFP-tagged targets in living multicellular organisms (Fig. 5A) (
      • Harmansa S.
      • Alborelli I.
      • Bieli D.
      • Caussinus E.
      • Affolter M.
      A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila.
      ,
      • Roubinet C.
      • Tsankova A.
      • Pham T.T.
      • Monnard A.
      • Caussinus E.
      • Affolter M.
      • Cabernard C.
      Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells.
      ,
      • Rodriguez J.
      • Peglion F.
      • Martin J.
      • Hubatsch L.
      • Reich J.
      • Hirani N.
      • Gubieda A.G.
      • Roffey J.
      • Fernandes A.R.
      • St Johnston D.
      • Ahringer J.
      • Goehring N.W.
      aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity.
      ). Forced mislocalization of the drosophila regulatory myosin light chain, tagged with GFP, to either the basolateral or apical membrane, caused an alteration in the shape of wing cells or aberrant sibling cell asymmetry in neural cells (
      • Harmansa S.
      • Alborelli I.
      • Bieli D.
      • Caussinus E.
      • Affolter M.
      A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila.
      ,
      • Roubinet C.
      • Tsankova A.
      • Pham T.T.
      • Monnard A.
      • Caussinus E.
      • Affolter M.
      • Cabernard C.
      Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells.
      ). Membrane anchoring of a protein essential for the development of cellular polarity in Caenorhabditis elegans revealed the importance of clustering for asymmetry (
      • Rodriguez J.
      • Peglion F.
      • Martin J.
      • Hubatsch L.
      • Reich J.
      • Hirani N.
      • Gubieda A.G.
      • Roffey J.
      • Fernandes A.R.
      • St Johnston D.
      • Ahringer J.
      • Goehring N.W.
      aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity.
      ). This approach has been used to redirect mRNAs engineered to contain the GFP-binding binding sequence MS2, which showed that forced mRNA relocalization also caused protein relocalization (
      • Ramat A.
      • Hannaford M.
      • Januschke J.
      Maintenance of Miranda localization in Drosophila neuroblasts involves interaction with the cognate mRNA.
      ). In a variation on this approach, a secreted protein was tethered to the cell surface, with rates of diffusion controlled by the strength of the nanobody-epitope interaction employed, to assess the importance of local and distal action of secreted proteins (
      • Harmansa S.
      • Hamaratoglu F.
      • Affolter M.
      • Caussinus E.
      Dpp spreading is required for medial but not for lateral wing disc growth.
      ,
      • Mörsdorf D.
      • Müller P.
      Tuning protein diffusivity with membrane tethers.
      ). This approach showed that diffusion of a secreted morphogenic protein in Drosophila is essential for proper wing patterning (
      • Harmansa S.
      • Hamaratoglu F.
      • Affolter M.
      • Caussinus E.
      Dpp spreading is required for medial but not for lateral wing disc growth.
      ). A p53-binding nanobody with a mitochondrial localization tag showed that mitochondrial mislocalization led to loss of cell viability in some cases (Fig. 5B) (
      • Steels A.
      • Verhelle A.
      • Zwaenepoel O.
      • Gettemans J.
      Intracellular displacement of p53 using transactivation domain (p53 TAD) specific nanobodies.
      ). A p53-binding nanobody that blocked the degradation of p53 mediated by human papillomavirus E6 protein failed to promote apoptosis (
      • Steels A.
      • Vannevel L.
      • Zwaenepoel O.
      • Gettemans J.
      Nb-induced stabilisation of p53 in HPV-infected cells.
      ).
      Figure thumbnail gr5
      Figure 5Nanobodies as redirecting and sensing agents in live cells. A, use of GFP-binding nanobodies to redirect tagged proteins to subcellular locations or for degradation. B, use of a p53-binding nanobody to block HPV E6-mediated ubiquitination and degradation. C, use of orthogonal anti-NP nanobodies as biosensors coupled to a transcriptional output (
      • Cao J.
      • Zhong N.
      • Wang G.
      • Wang M.
      • Zhang B.
      • Fu B.
      • Wang Y.
      • Zhang T.
      • Zhang Y.
      • Yang K.
      • Chen Y.
      • Yuan Q.
      • Xia N.
      Nanobody-based sandwich reporter system for living cell sensing influenza A virus infection.
      ). The DNA-binding domain (DBD) and VP64 activation domain are separately fused to anti-NP nanobodies (VHHs). UAS, upstream activator sequence that binds DBD. Transcription of the reporter gene produces GFP. D, use of nanobodies as biosensors to detect active and inactive states of GPCRs.
      The targeting function of nanobodies can be exploited to link particular substrates to the degradation machinery. The modular nature of nanobodies thus enabled their use in a variety of fusion proteins for targeted degradation of proteins of interest (
      • Böldicke T.
      Single domain antibodies for the knockdown of cytosolic and nuclear proteins.
      ). This approach typically involves genetic fusion of a nanobody with a (fragment of a) ubiquitin ligase (E3), which recruits the endogenous ubiquitination machinery to tag nanobody-targeted proteins for degradation by the proteasome. One widely deployed version of this approach relies on a fusion of a nanobody that binds GFP/YFP with an F-box protein domain from the Skp cullin F-box E3 complex, which can degrade GFP/YFP-tagged proteins (Fig. 5A) (
      • Caussinus E.
      • Kanca O.
      • Affolter M.
      Fluorescent fusion protein knockout mediated by anti-GFP nanobody.
      ,
      • Caussinus E.
      • Affolter M.
      deGradFP: a system to knockdown GFP-tagged proteins.
      ). An alternative version uses von Hippel–Lindau (VHL)-nanobody fusions to degrade GFP-tagged proteins (
      • Fulcher L.J.
      • Hutchinson L.D.
      • Macartney T.J.
      • Turnbull C.
      • Sapkota G.P.
      Targeting endogenous proteins for degradation through the affinity-directed protein missile system.
      ,
      • Fulcher L.J.
      • Macartney T.
      • Bozatzi P.
      • Hornberger A.
      • Rojas-Fernandez A.
      • Sapkota G.P.
      An affinity-directed protein missile system for targeted proteolysis.
      ). Targeted degradation using this approach, in combination with expression under tissue-specific promoters, has enabled evaluation of the role of targeted proteins in specific tissues in Drosophila development (
      • Ochoa-Espinosa A.
      • Harmansa S.
      • Caussinus E.
      • Affolter M.
      Myosin II is not required for Drosophila tracheal branch elongation and cell intercalation.
      ,
      • Pasakarnis L.
      • Frei E.
      • Caussinus E.
      • Affolter M.
      • Brunner D.
      Amnioserosa cell constriction but not epidermal actin cable tension autonomously drives dorsal closure.
      ). Tissue-specific degradation of myosin-II showed it is not essential for tracheal elongation or the closure of the dorsal opening during development of Drosophila. Alternative versions of nanobody-mediated target degradation, relying on other E3 fragments, have also been developed for application in zebrafish (
      • Yamaguchi N.
      • Colak-Champollion T.
      • Knaut H.
      zGrad is a nanobody-based degron system that inactivates proteins in zebrafish.
      ,
      • Daniel K.
      • Icha J.
      • Horenburg C.
      • Müller D.
      • Norden C.
      • Mansfeld J.
      Conditional control of fluorescent protein degradation by an auxin-dependent nanobody.
      ) and C. elegans (
      • Wang S.
      • Tang N.H.
      • Lara-Gonzalez P.
      • Zhao Z.
      • Cheerambathur D.K.
      • Prevo B.
      • Chisholm A.D.
      • Desai A.
      • Oegema K.
      A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans.
      ). The use of nanobody-E3 fragment fusions to target cell surface ion channels, either as a YFP fusion or as the WT protein, showed that ubiquitylation can have divergent consequences for the trafficking and function of cell surface proteins (
      • Kanner S.A.
      • Morgenstern T.
      • Colecraft H.M.
      Sculpting ion channel functional expression with engineered ubiquitin ligases.
      ,
      • Morgenstern T.J.
      • Park J.
      • Fan Q.R.
      • Colecraft H.M.
      A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits.
      ). An E3 fragment–nanobody fusion that bound directly to the histone H2A-H2B protein dimer enabled targeted ubiquitylation of histones and caused signaling associated with DNA damage (
      • Jullien D.
      • Vignard J.
      • Fedor Y.
      • Béry N.
      • Olichon A.
      • Crozatier M.
      • Erard M.
      • Cassard H.
      • Ducommun B.
      • Salles B.
      • Mirey G.
      Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells.
      ). A nanobody that selectively bound to the active (GTP-bound) form of RhoB GTPase was applied as an F-box fusion to knock down active RhoB. It showed that the GTP-bound fraction of RhoB mediates its role in cell invasion (
      • Bery N.
      • Keller L.
      • Soulié M.
      • Gence R.
      • Iscache A.-L.
      • Cherier J.
      • Cabantous S.
      • Sordet O.
      • Lajoie-Mazenc I.
      • Pedelacq J.-D.
      • Favre G.
      • Olichon A.
      A targeted protein degradation cell-based screening for nanobodies selective toward the cellular RHOB GTP-bound conformation.
      ). Control of the properties of the E3 fragment–nanobody produced in cells, such as expression level and degradation rate, allows quantitative control of cellular protein levels (
      • Zhao W.
      • Pferdehirt L.
      • Segatori L.
      Quantitatively predictable control of cellular protein levels through proteasomal degradation.
      ).
      Specialized, targeted approaches have produced nanobodies that bind to the target only when found in a specific conformation, with a particular emphasis on membrane proteins. These approaches rely on screening with antigens locked into the desired conformation: amyloidogenic protein variants at various stages of self-assembly, complexes formed by protein-protein interactions, and receptors bound to ligands constitute some of the targets of conformation-specific nanobodies (
      • Uchański T.
      • Pardon E.
      • Steyaert J.
      Nanobodies to study protein conformational states.
      ). They have served as biosensors to visualize the distribution of proteins in a specific conformation in living cells (Fig. 5D) (
      • Ortiz Zacarías N.V.
      • Lenselink E.B.
      • IJzerman A.P.
      • Handel T.M.
      • Heitman L.H.
      Intracellular receptor modulation: novel approach to target GPCRs.
      ,
      • Heukers R.
      • De Groof T.W.M.
      • Smit M.J.
      Nanobodies detecting and modulating GPCRs outside in and inside out.
      ), with a particular emphasis on GPCRs. A nanobody raised against the β2AR, used as a chaperone to facilitate its crystallization (
      • Rasmussen S.G.F.
      • Choi H.-J.
      • Fung J.J.
      • Pardon E.
      • Casarosa P.
      • Chae P.S.
      • Devree B.T.
      • Rosenbaum D.M.
      • Thian F.S.
      • Kobilka T.S.
      • Schnapp A.
      • Konetzki I.
      • Sunahara R.K.
      • Gellman S.H.
      • Pautsch A.
      • et al.
      Structure of a nanobody-stabilized active state of the β2 adrenoceptor.
      ), binds to the cytoplasmic face of β2AR and stabilizes its active state, much like a G protein would. This same nanobody, when expressed as a fluorescent fusion protein served as a biosensor to visualize ligand-bound β2AR in its active state (
      • Irannejad R.
      • Tomshine J.C.
      • Tomshine J.R.
      • Chevalier M.
      • Mahoney J.P.
      • Steyaert J.
      • Rasmussen S.G.F.
      • Sunahara R.K.
      • El-Samad H.
      • Huang B.
      • von Zastrow M.
      Conformational biosensors reveal GPCR signalling from endosomes.
      ). Surprisingly, activated β2AR was found both at the plasma membrane and in early endosomes. Further characterization of other β2AR-binding nanobodies identified one that bound to and stabilized the inactive form of β2AR (
      • Staus D.P.
      • Strachan R.T.
      • Manglik A.
      • Pani B.
      • Kahsai A.W.
      • Kim T.H.
      • Wingler L.M.
      • Ahn S.
      • Chatterjee A.
      • Masoudi A.
      • Kruse A.C.
      • Pardon E.
      • Steyaert J.
      • Weis W.I.
      • Prosser R.S.
      • et al.
      Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.
      ,
      • Staus D.P.
      • Wingler L.M.
      • Strachan R.T.
      • Rasmussen S.G.F.
      • Pardon E.
      • Ahn S.
      • Steyaert J.
      • Kobilka B.K.
      • Lefkowitz R.J.
      Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.
      ). This assembly of nanobodies enabled the classification of several β2AR ligands as agonists, antagonists, or inverse agonists (
      • Staus D.P.
      • Strachan R.T.
      • Manglik A.
      • Pani B.
      • Kahsai A.W.
      • Kim T.H.
      • Wingler L.M.
      • Ahn S.
      • Chatterjee A.
      • Masoudi A.
      • Kruse A.C.
      • Pardon E.
      • Steyaert J.
      • Weis W.I.
      • Prosser R.S.
      • et al.
      Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.
      ,
      • Pardon E.
      • Betti C.
      • Laeremans T.
      • Chevillard F.
      • Guillemyn K.
      • Kolb P.
      • Ballet S.
      • Steyaert J.
      Nanobody-enabled reverse pharmacology on G-protein-coupled receptors.
      ). Certain β-adrenergic receptor ligands affect the conformation of receptor molecules found in the Golgi, suggesting that receptors en route to the cell surface can be activated by cell-permeable ligands (
      • Irannejad R.
      • Pessino V.
      • Mika D.
      • Huang B.
      • Wedegaertner P.B.
      • Conti M.
      • von Zastrow M.
      Functional selectivity of GPCR-directed drug action through location bias.
      ). This possibility is of interest also in view of the exclusive Golgi localization of GPCRs such as GPR107 (
      • Tafesse F.G.
      • Guimaraes C.P.
      • Maruyama T.
      • Carette J.E.
      • Lory S.
      • Brummelkamp T.R.
      • Ploegh H.L.
      GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.
      ). Conformation-specific nanobodies have been applied for similar applications to the muscarinic acetylcholine receptor (
      • Kruse A.C.
      • Ring A.M.
      • Manglik A.
      • Hu J.
      • Hu K.
      • Eitel K.
      • Hübner H.
      • Pardon E.
      • Valant C.
      • Sexton P.M.
      • Christopoulos A.
      • Felder C.C.
      • Gmeiner P.
      • Steyaert J.
      • Weis W.I.
      • et al.
      Activation and allosteric modulation of a muscarinic acetylcholine receptor.
      ) and for the κ- and μ-opioid receptors (
      • Che T.
      • English J.
      • Krumm B.E.
      • Kim K.
      • Pardon E.
      • Olsen R.H.J.
      • Wang S.
      • Zhang S.
      • Diberto J.F.
      • Sciaky N.
      • Carroll F.I.
      • Steyaert J.
      • Wacker D.
      • Roth B.L.
      Nanobody-enabled monitoring of κ opioid receptor states.
      ,
      • Stoeber M.
      • Jullié D.
      • Lobingier B.T.
      • Laeremans T.
      • Steyaert J.
      • Schiller P.W.
      • Manglik A.
      • von Zastrow M.
      A genetically encoded biosensor reveals location bias of opioid drug action.
      ,
      • Livingston K.E.
      • Mahoney J.P.
      • Manglik A.
      • Sunahara R.K.
      • Traynor J.R.
      Measuring ligand efficacy at the μ-opioid receptor using a conformational biosensor.
      ). Although these conformation-specific nanobodies are restricted to the indicated receptors, study of a nanobody specific for the κ-opioid receptor in its active state showed that the intracellular loop fragment responsible for nanobody binding could be grafted onto other GPCRs with retention of binding, highlighting the exciting possibility of designing receptor chimeras bound by conformation-specific nanobodies (
      • Che T.
      • English J.
      • Krumm B.E.
      • Kim K.
      • Pardon E.
      • Olsen R.H.J.
      • Wang S.
      • Zhang S.
      • Diberto J.F.
      • Sciaky N.
      • Carroll F.I.
      • Steyaert J.
      • Wacker D.
      • Roth B.L.
      Nanobody-enabled monitoring of κ opioid receptor states.
      ). This select set of examples shows that intracellular expression of nanobodies directed against cytoplasmic targets clearly is a feasible approach to modulate cellular functions. However, considerable efforts must be expended to identify nanobodies with the desired properties. As methods for the production of large and completely synthetic nanobody libraries improve, the need for deliberate immunization of animals is reduced. Synthetic libraries may also make it possible to obtain nanobodies against proteins that are not immunogenic in camelids.

      Targeting viral proteins

      Nanobodies have been used to target viral proteins. Inhibition of viral entry by nanobodies is well-documented (
      • De Vlieger D.
      • Ballegeer M.
      • Rossey I.
      • Schepens B.
      • Saelens X.
      Single-domain antibodies and their formatting to combat viral infections.
      ,
      • Laursen N.S.
      • Friesen R.H.E.
      • Zhu X.
      • Jongeneelen M.
      • Blokland S.
      • Vermond J.
      • van Eijgen A.
      • Tang C.
      • van Diepen H.
      • Obmolova G.
      • van der Neut Kolfschoten M.
      • Zuijdgeest D.
      • Straetemans R.
      • Hoffman R.M.B.
      • Nieusma T.
      • et al.
      Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.
      ). The recent description of camelid-derived nanobodies capable of neutralizing SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, is one such example (
      • Wrapp D.
      • De Vlieger D.
      • Corbett K.S.
      • Torres G.M.
      • Wang N.
      • Van Breedam W.
      • Roose K.
      • van Schie L.
      • Response Team V.-C.C.-1.
      • Hoffmann M.
      • Pöhlmann S.
      • Graham B.S.
      • Callewaert N.
      • Schepens B.
      • Saelens X.
      • et al.
      Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies.
      ,
      • Hanke L.
      • Perez L.V.
      • Sheward D.J.
      • Das H.
      • Schulte T.
      • Morro A.M.
      • Corcoran M.
      • Achour A.
      • Hedestam G.K.
      • Hällberg B.M.
      • Murrell B.
      • McInerney G.M.
      An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction.
      ). There are fewer examples of nanobodies that target cytoplasmic viral proteins. Because infected cells produce cytoplasmic proteins required for proper virus replication, assembly, and release, nanobody-mediated interference with intracellular viral proteins requires cytoplasmic expression as well. Inhibition of viral polymerases by a cytoplasmically expressed nanobody inhibits influenza propagation (
      • Fan H.
      • Walker A.P.
      • Carrique L.
      • Keown J.R.
      • Serna Martin I.
      • Karia D.
      • Sharps J.
      • Hengrung N.
      • Pardon E.
      • Steyaert J.
      • Grimes J.M.
      • Fodor E.
      Structures of influenza A virus RNA polymerase offer insight into viral genome replication.
      ). A nanobody that blocks multimeri