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Exploring cellular biochemistry with nanobodies

2020; Elsevier BV; Volume: 295; Issue: 45 Linguagem: Inglês

10.1074/jbc.rev120.012960

ISSN

1083-351X

Autores

Ross W. Cheloha, Thibault J. Harmand, Charlotte Wijne, Thomas Schwartz, Hidde L. Ploegh,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

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. 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. 1Chanier T. Chames P. Nanobody engineering: toward next generation immunotherapies and immunoimaging of cancer.Antibodies (Basel). 2019; 8 (31544819): 1310.3390/antib8010013Crossref Google Scholar and 2Manglik A. Kobilka B.K. Steyaert J. Nanobodies to study G protein-coupled receptor structure and function.Annu. Rev. Pharmacol. Toxicol. 2017; 57 (27959623): 19-3710.1146/annurev-pharmtox-010716-104710Crossref PubMed Scopus (100) Google Scholar). Similar to full-sized antibodies, nanobodies are suitable for flow cytometry, immunoprecipitation, affinity purification, and microscopy (3Beghein E. Gettemans J. Nanobody technology: a versatile toolkit for microscopic imaging, protein-protein interaction analysis, and protein function exploration.Front. Immunol. 2017; 8 (28725224): 77110.3389/fimmu.2017.00771Crossref PubMed Scopus (73) Google Scholar, 4Braun 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.Sci. Rep. 2016; 6 (26791954): 1921110.1038/srep19211Crossref PubMed Scopus (0) Google Scholar, 5Traenkle B. Emele F. Anton R. Poetz O. Haeussler R.S. Maier J. Kaiser P.D. Scholz A.M. Nueske S. Buchfellner A. Romer T. Rothbauer U. Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.Mol. Cell. Proteomics. 2015; 14 (25595278): 707-72310.1074/mcp.M114.044016Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 6Götzke 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.Nat. Commun. 2019; 10 (31562305): 440310.1038/s41467-019-12301-7Crossref PubMed Scopus (35) Google Scholar, 7Bruce V.J. McNaughton B.R. Evaluation of nanobody conjugates and protein fusions as bioanalytical reagents.Anal. Chem. 2017; 89: 3819-382310.1021/acs.analchem.7b00470Crossref PubMed Scopus (12) Google Scholar). 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. 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. Methods to identify nanobodies that bind to targets of interest are essential for their effective deployment (8Liu W. Song H. Chen Q. Yu J. Xian M. Nian R. Feng D. Recent advances in the selection and identification of antigen-specific nanobodies.Mol. Immunol. 2018; 96 (29477934): 37-4710.1016/j.molimm.2018.02.012Crossref PubMed Scopus (24) Google Scholar). 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 (9Comor L. Dolinska S. Bhide K. Pulzova L. Jiménez-Munguía I. Bencurova E. Flachbartova Z. 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.Microb. Cell Fact. 2017; 16 (28114943): 1310.1186/s12934-017-0630-zCrossref PubMed Scopus (6) Google Scholar). Purified proteins (10Pardon 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.Nat. Protoc. 2014; 9 (24577359): 674-69310.1038/nprot.2014.039Crossref PubMed Scopus (273) Google Scholar), cells or cell lysates containing antigens of interest (11Jähnichen 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.Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (21059953): 20565-2057010.1073/pnas.1012865107Crossref PubMed Scopus (155) Google Scholar, 12Itoh K. Reis A.H. Hayhurst A. Sokol S.Y. 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DNA-based immunization has been particularly valuable for the generation of nanobodies against properly folded membrane proteins (15Eden 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.Front. Immunol. 2017; 8 (29410663): 198910.3389/fimmu.2017.01989Crossref PubMed Scopus (10) Google Scholar, 16Peyrassol 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.J. Immunol. 2016; 196 (26864035): 2893-290110.4049/jimmunol.1500888Crossref PubMed Scopus (29) Google Scholar). 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 (17Tiller 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.Front. Immunol. 2017; 8 (28928732): 98610.3389/fimmu.2017.00986Crossref PubMed Scopus (24) Google Scholar), and virus-mediated directed evolution (18English 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.Cell. 2019; 178 (31280962): 748-761.e1710.1016/j.cell.2019.05.051Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) 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) (8Liu W. Song H. Chen Q. Yu J. Xian M. Nian R. Feng D. Recent advances in the selection and identification of antigen-specific nanobodies.Mol. Immunol. 2018; 96 (29477934): 37-4710.1016/j.molimm.2018.02.012Crossref PubMed Scopus (24) Google Scholar, 19Moutel 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.Elife. 2016; 5 (27434673): e1622810.7554/elife.16228Crossref PubMed Scopus (99) Google Scholar), 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) (20Salema V. Fernández L.Á. Escherichia coli surface display for the selection of nanobodies.Microb Biotechnol. 2017; 10 (28772027): 1468-148410.1111/1751-7915.12819Crossref PubMed Scopus (17) Google Scholar), 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) (21Kavousipour 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.Protein Pept. Lett. 2018; 25 (29848261): 580-58810.2174/0929866525666180530122159Crossref PubMed Scopus (3) Google Scholar). 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 (22Veugelen S. Dewilde M. De Strooper B. Chávez-Gutiérrez L. Screening and characterization strategies for nanobodies targeting membrane proteins.Methods Enzymol. 2017; 584 (28065273): 59-9710.1016/bs.mie.2016.10.029Crossref PubMed Scopus (9) Google Scholar, 23Doshi R. Chen B.R. Vibat C.R.T. Huang N. Lee C.-W. Chang G. In vitro nanobody discovery for integral membrane protein targets.Sci. Rep. 2014; 4 (25342225): 676010.1038/srep06760Crossref PubMed Scopus (0) Google Scholar, 24Crepin 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.Biochem. Biophys. Res. Commun. 2017; 493 (29017919): 1567-157210.1016/j.bbrc.2017.10.036Crossref PubMed Scopus (9) Google Scholar) and that either block or induce activation (25Ren 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.Commun. Biol. 2020; 3 (32218528): 14610.1038/s42003-020-0867-7Crossref PubMed Scopus (2) Google Scholar). 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 (26Reader R.H. Workman R.G. Maddison B.C. Gough K.C. Advances in the production and batch reformatting of phage antibody libraries.Mol. Biotechnol. 2019; 61 (31468301): 801-81510.1007/s12033-019-00207-0Crossref PubMed Scopus (3) Google Scholar, 27Romao 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.Curr. Pharm. Des. 2016; 22 (27669966): 6500-651810.2174/1381612822666160923114417Crossref PubMed Scopus (16) Google Scholar). 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 (20Salema V. Fernández L.Á. Escherichia coli surface display for the selection of nanobodies.Microb Biotechnol. 2017; 10 (28772027): 1468-148410.1111/1751-7915.12819Crossref PubMed Scopus (17) Google Scholar), Staphylococcus sp. (28Cavallari M. Rapid and direct VHH and target identification by staphylococcal surface display libraries.Int. J. Mol. Sci. 2017; 18 (28704956): 150710.3390/ijms18071507Crossref Scopus (9) Google Scholar, 29Fleetwood 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.Cell. Mol. Life Sci. 2013; 70 (23064703): 1081-109310.1007/s00018-012-1179-yCrossref PubMed Scopus (41) Google Scholar), and yeast (30Uchański T. Pardon E. Steyaert J. Nanobodies to study protein conformational states.Curr. Opin. Struct. Biol. 2020; 60 (32036243): 117-12310.1016/j.sbi.2020.01.003Crossref PubMed Scopus (0) Google Scholar, 31McMahon 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.Nat. Struct. Mol. Biol. 2018; 25 (29434346): 289-29610.1038/s41594-018-0028-6Crossref PubMed Scopus (103) Google Scholar). 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) (30Uchański T. Pardon E. Steyaert J. Nanobodies to study protein conformational states.Curr. Opin. Struct. Biol. 2020; 60 (32036243): 117-12310.1016/j.sbi.2020.01.003Crossref PubMed Scopus (0) Google Scholar, 31McMahon 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.Nat. Struct. Mol. Biol. 2018; 25 (29434346): 289-29610.1038/s41594-018-0028-6Crossref PubMed Scopus (103) Google Scholar). 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 (20Salema V. Fernández L.Á. Escherichia coli surface display for the selection of nanobodies.Microb Biotechnol. 2017; 10 (28772027): 1468-148410.1111/1751-7915.12819Crossref PubMed Scopus (17) Google Scholar). 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 (32Hutter 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.Nat. Commun. 2019; 10 (31113958): 226010.1038/s41467-019-09892-6Crossref PubMed Scopus (21) Google Scholar, 33Zimmermann 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.Elife. 2018; 7 (29792401): e3431710.7554/elife.34317Crossref PubMed Scopus (33) Google Scholar, 34Ferrari D. Garrapa V. Locatelli M. Bolchi A. A novel nanobody scaffold optimized for bacterial expression and suitable for the construction of ribosome display libraries.Mol. Biotechnol. 2020; 62 (31720928): 43-5510.1007/s12033-019-00224-zCrossref PubMed Scopus (4) Google Scholar). 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 (35Suzuki 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.Biochem. 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Seeger M.A. Synthetic single domain antibodies for the conformational trapping of membrane proteins.Elife. 2018; 7 (29792401): e3431710.7554/elife.34317Crossref PubMed Scopus (33) Google Scholar). 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 (37Schmidt 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.Nat. Microbiol. 2016; 1 (27573105): 1608010.1038/nmicrobiol.2016.80Crossref PubMed Scopus (27) Google Scholar). 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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 (42Massa 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.Bioconjug. Chem. 2014; 25 (24815083): 979-98810.1021/bc500111tCrossref PubMed Scopus (79) Google Scholar) and N-hydroxysuccinimide ester–based chemistry (43Ries J. Kaplan C. Platonova E. Eghlidi H. Ewers H. 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Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.Elife. 2015; 4 (26633879): e1134910.7554/eLife.11349Crossref PubMed Scopus (94) Google Scholar, 46Agarwal P. Bertozzi C.R. Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.Bioconjug. Chem. 2015; 26 (25494884): 176-19210.1021/bc5004982Crossref PubMed Scopus (305) Google Scholar, 47Massa S. Xavier C. Muyldermans S. Devoogdt N. Emerging site-specific bioconjugation strategies for radioimmunotracer development.Expert Opin. Drug Deliv. 2016; 13 (27116898): 1149-116310.1080/17425247.2016.1178235Crossref PubMed Scopus (17) Google Scholar). 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 (48Herce 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.Nat. Chem. 2017; 9 (28754949): 762-77110.1038/nchem.2811Crossref PubMed Google Scholar, 49Klein 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.Chem. Sci. 2018; 9 (30429993): 7835-784210.1039/c8sc02910eCrossref PubMed Google Scholar, 50Teng K.W. Ishitsuka Y. 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