Skip to main content

Advertisement

SpringerLink
Log in

Nanobody: outstanding features for diagnostic and therapeutic applications

  • Trends
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Nanobodies (Nbs) have arisen as an alternative to conventional antibodies (Abs) and show great potential when used as tools in different biotechnology fields such as diagnostics and therapy. Different approaches have been described for the production of Nbs and these methods face new challenges focused on improving yield, affinity, and reducing production costs. This review summarizes these challenges, and also the latest advances in the detection of different kinds of molecules, such as proteins and small molecules, and describes their potential use for noninvasive in vivo imaging and for in vitro assays. Moreover, the unique properties of Nbs are outlined like internalization, size, thermal and chemical stability, affinity, blood clearance, and labeling procedures. Concerning therapeutic applications, we highlight some already reported examples about Nbs being used for the treatment of several diseases such as cancer, neurodegenerative or infectious diseases among others. Finally, future trends, opportunities, and disadvantages are also discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Ruigrok Vincent JB, Levisson M, Eppink Michel HM, Smidt H, van der Oost J. Alternative affinity tools: more attractive than antibodies? Biochem J. 2011;436(1):1–13. https://doi.org/10.1042/bj20101860.

    Article  CAS  PubMed  Google Scholar 

  2. Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013;82(1):775–97. https://doi.org/10.1146/annurev-biochem-063011-092449.

    Article  CAS  PubMed  Google Scholar 

  3. Ingram JR, Schmidt FI, Ploegh HL. Exploiting nanobodies’ singular traits. In: Littman DR, Yokoyama WM, editors. Annual review of immunology, vol 36. Annu rev Immunol. 2018; 695–715. https://doi.org/10.1146/annurev-immunol-042617-053327.

  4. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, et al. Naturally occurring antibodies devoid of light chains. Nature. 1993;363(6428):446–8. https://doi.org/10.1038/363446a0.

    Article  CAS  Google Scholar 

  5. Muyldermans S, Baral TN, Retamozzo VC, De Baetselier P, De Genst E, Kinne J, Leonhardt H, Magez S, Nguyen VK, Revets H, Rothbauer U, Stijlemans B, Tillib S, Wernery U, Wyns L, Hassanzadeh-Ghassabeh G, Saerens D. Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol. 2009;128:178–183. https://doi.org/10.1016/j.vetimm.2008.10.299.

  6. Steeland S, Vandenbroucke RE, Libert C. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today. 2016;21(7):1076–113. https://doi.org/10.1016/j.drudis.2016.04.003.

    Article  CAS  PubMed  Google Scholar 

  7. Liu W, Song H, Chen Q, Yu J, Xian M, Nian R, et al. Recent advances in the selection and identification of antigen-specific nanobodies. Mol Immunol. 2018;96:37–47. https://doi.org/10.1016/j.molimm.2018.02.012.

    Article  CAS  PubMed  Google Scholar 

  8. Kubala MH, Kovtun O, Alexandrov K, Collins BM. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 2010;19(12):2389–401. https://doi.org/10.1002/pro.519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kunz P, Zinner K, Mücke N, Bartoschik T, Muyldermans S, Hoheisel JD. The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep. 2018;8(1):7934. https://doi.org/10.1038/s41598-018-26338-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Siontorou CG. Nanobodies as novel agents for disease diagnosis and therapy. Int J Nanomed. 2013;8:4215–27. https://doi.org/10.2147/ijn.s39428.

    Article  Google Scholar 

  11. Crivianu-Gaita V, Thompson M. Aptamers, antibody scFv, and antibody Fab’ fragments: an overview and comparison of three of the most versatile biosensor biorecognition elements. Biosens Bioelectron. 2016;85:32–45. https://doi.org/10.1016/j.bios.2016.04.091.

    Article  CAS  PubMed  Google Scholar 

  12. Wang Y, Fan Z, Shao L, Kong X, Hou X, Tian D, et al. Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications. Int J Nanomed. 2016;11:3287–302. https://doi.org/10.2147/ijn.s107194.

    Article  CAS  Google Scholar 

  13. Iezzi ME, Policastro L, Werbajh S, Podhajcer O, Canziani GA. Single-domain antibodies and the promise of modular targeting in cancer imaging and treatment. Front Immunol. 2018;9:273. https://doi.org/10.3389/fimmu.2018.00273.

  14. Pardon E, Laeremans T, Triest S, Rasmussen SGF, Wohlkönig A, Ruf A, et al. A general protocol for the generation of nanobodies for structural biology. Nat Protocols. 2014;9(3):674–93. https://doi.org/10.1038/nprot.2014.039.

    Article  CAS  PubMed  Google Scholar 

  15. Liu Y, Huang H. Expression of single-domain antibody in different systems. Appl Microbiol Biotechnol. 2018;102(2):539–51. https://doi.org/10.1007/s00253-017-8644-3.

    Article  CAS  PubMed  Google Scholar 

  16. Sockolosky JT, Dougan M, Ingram JR, Ho CCM, Kauke MJ, Almo SC, et al. Durable antitumor responses to CD47 blockade require adaptive immune stimulation. Proc Natl Acad Sci. 2016;113(19):E2646–54. https://doi.org/10.1073/pnas.1604268113.

    Article  CAS  PubMed  Google Scholar 

  17. Vuchelen A, O’Day E, De Genst E, Pardon E, Wyns L, Dumoulin M, et al. (1)H, (13)C and (15)N assignments of a camelid nanobody directed against human alpha-synuclein. Biomol NMR Assign. 2009;3(2):231–3. https://doi.org/10.1007/s12104-009-9182-4.

    Article  CAS  PubMed  Google Scholar 

  18. Kumar H, Finer-Moore JS, Jiang X, Smirnova I, Kasho V, Pardon E, et al. Crystal structure of a ligand-bound LacY-Nanobody complex. Proc Natl Acad Sci U S A. 2018;115(35):8769–74. https://doi.org/10.1073/pnas.1801774115.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Robert B, Dorvillius M, Buchegger F, Garambois V, Mani JC, Pugnieres M, et al. Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA). Int J Cancer. 1999;81(2):285–91.

    Article  CAS  Google Scholar 

  20. Els Conrath K, Lauwereys M, Wyns L, Muyldermans S. Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem. 2001;276(10):7346–50. https://doi.org/10.1074/jbc.M007734200.

    Article  CAS  PubMed  Google Scholar 

  21. Sedykh SE, Prinz VV, Buneva VN, Nevinsky GA. Bispecific antibodies: design, therapy, perspectives. Drug Design Dev Ther. 2018;12:195–208. https://doi.org/10.2147/DDDT.S151282.

    Article  CAS  Google Scholar 

  22. Davies J, Riechmann L. Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. Protein Eng. 1996;9(6):531–7.

    Article  CAS  Google Scholar 

  23. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997;414(3):521–6.

    Article  CAS  Google Scholar 

  24. Perez JM, Renisio JG, Prompers JJ, van Platerink CJ, Cambillau C, Darbon H, et al. Thermal unfolding of a llama antibody fragment: a two-state reversible process. Biochemistry. 2001;40(1):74–83.

    Article  CAS  Google Scholar 

  25. Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, et al. Single-domain antibody fragments with high conformational stability. Protein Sci. 2002;11(3):500–15. https://doi.org/10.1110/ps.34602.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wolfgang WJ, Miller TW, Webster JM, Huston JS, Thompson LM, Marsh JL, et al. Suppression of Huntington’s disease pathology in Drosophila by human single-chain Fv antibodies. Proc Natl Acad Sci U S A. 2005;102(32):11563–8. https://doi.org/10.1073/pnas.0505321102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wang J, Mukhtar H, Ma L, Pang Q, Wang X. VHH antibodies: reagents for mycotoxin detection in food products. Sensors. 2018;18(2):485.

    Article  CAS  Google Scholar 

  28. McMurphy T, Xiao R, Magee D, Slater A, Zabeau L, Tavernier J, et al. The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma. PLoS One. 2014;9(2):e89895. https://doi.org/10.1371/journal.pone.0089895.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Blick SK, Curran MP. Certolizumab pegol: in Crohn’s disease. BioDrugs. 2007;21(3):195–201; discussion 202-193. https://doi.org/10.2165/00063030-200721030-00006.

    Article  CAS  PubMed  Google Scholar 

  30. Padlan EA. X-ray crystallography of antibodies. Adv Protein Chem. 1996;49:57–133.

    Article  CAS  Google Scholar 

  31. Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, Wyns L, Muyldermans S. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother. 2001;45(10):2807–2812. https://doi.org/10.1128/aac.45.10.2807-2812.2001.

  32. Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Holzer W, De Genst E, et al. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J. 1998;17(13):3512–20. https://doi.org/10.1093/emboj/17.13.3512.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009;284(5):3273–84. https://doi.org/10.1074/jbc.M806889200.

    Article  CAS  PubMed  Google Scholar 

  34. Saerens D, Huang L, Bonroy K, Muyldermans S. Antibody fragments as probe in biosensor development. Sensors (Basel). 2008;8(8):4669–86. https://doi.org/10.3390/s8084669.

    Article  CAS  Google Scholar 

  35. Pinto Torres JE, Goossens J, Ding J, Li Z, Lu S, Vertommen D, et al. Development of a nanobody-based lateral flow assay to detect active Trypanosoma congolense infections. Sci Rep. 2018;8(1):9019. https://doi.org/10.1038/s41598-018-26732-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Qiu Y, Li P, Dong S, Zhang X, Yang Q, Wang Y, et al. Phage-mediated competitive chemiluminescent immunoassay for detecting Cry1Ab toxin by using an anti-idiotypic camel nanobody. J Agric Food Chem. 2018;66(4):950–6. https://doi.org/10.1021/acs.jafc.7b04923.

    Article  CAS  PubMed  Google Scholar 

  37. Tu Z, Chen Q, Li Y, Xiong Y, Xu Y, Hu N, et al. Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk. Anal Biochem. 2016;493:1–7. https://doi.org/10.1016/j.ab.2015.09.023.

    Article  CAS  PubMed  Google Scholar 

  38. Zafra O, Fraile S, Gutiérrez C, Haro A, Páez-Espino AD, Jiménez JI, et al. Monitoring biodegradative enzymes with nanobodies raised in Camelus dromedarius with mixtures of catabolic proteins. Environ Microbiol. 2011;13(4):960–74. https://doi.org/10.1111/j.1462-2920.2010.02401.x.

    Article  CAS  PubMed  Google Scholar 

  39. Campuzano S, Salema V, Moreno-Guzmán M, Gamella M, Yáñez-Sedeño P, Fernández LA, et al. Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element. Determination of fibrinogen in plasma. Biosens Bioelectron. 2014;52:255–60. https://doi.org/10.1016/j.bios.2013.08.055.

    Article  CAS  PubMed  Google Scholar 

  40. Marco M-P, Gee S, Hammock BD. Immunochemical techniques for environmental analysis II. Antibody production and immunoassay development. TrAC Trends Anal Chem. 1995;14(8):415–25. https://doi.org/10.1016/0165-9936(95)90920-I.

    Article  CAS  Google Scholar 

  41. Fodey T, Leonard P, O’Mahony J, O’Kennedy R, Danaher M. Developments in the production of biological and synthetic binders for immunoassay and sensor-based detection of small molecules. TrAC Trends Anal Chem. 2011;30(2):254–69. https://doi.org/10.1016/j.trac.2010.10.011.

    Article  CAS  Google Scholar 

  42. Bever CS, Dong J-X, Vasylieva N, Barnych B, Cui Y, Xu Z-L, et al. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016;408(22):5985–6002. https://doi.org/10.1007/s00216-016-9585-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Alvarez-Rueda N, Behar G, Ferré V, Pugnière M, Roquet F, Gastinel L, et al. Generation of llama single-domain antibodies against methotrexate, a prototypical hapten. Mol Immunol. 2007;44(7):1680–90. https://doi.org/10.1016/j.molimm.2006.08.007.

    Article  CAS  PubMed  Google Scholar 

  44. Makvandi-Nejad S, Fjällman T, Arbabi-Ghahroudi M, MacKenzie CR, Hall JC. Selection and expression of recombinant single domain antibodies from a hyper-immunized library against the hapten azoxystrobin. J Immunol Methods. 2011;373(1):8–18. https://doi.org/10.1016/j.jim.2011.07.006.

    Article  CAS  PubMed  Google Scholar 

  45. Wang J, Bever CRS, Majkova Z, Dechant JE, Yang J, Gee SJ, et al. Heterologous antigen selection of camelid heavy chain single domain antibodies against tetrabromobisphenol A. Anal Chem. 2014;86(16):8296–302. https://doi.org/10.1021/ac5017437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kim H-J, McCoy MR, Majkova Z, Dechant JE, Gee SJ, Tabares-da Rosa S, et al. Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal Chem. 2012;84(2):1165–71. https://doi.org/10.1021/ac2030255.

    Article  CAS  PubMed  Google Scholar 

  47. Pan D, Li G, Hu H, Xue H, Zhang M, Zhu M, et al. Direct immunoassay for facile and sensitive detection of small molecule aflatoxin B1 based on nanobody. Chemistry. 2018;24(39):9869–76. https://doi.org/10.1002/chem.201801202.

    Article  CAS  PubMed  Google Scholar 

  48. Liu X, Tang Z, Duan Z, He Z, Shu M, Wang X, et al. Nanobody-based enzyme immunoassay for ochratoxin a in cereal with high resistance to matrix interference. Talanta. 2017;164:154–8. https://doi.org/10.1016/j.talanta.2016.11.039.

    Article  CAS  PubMed  Google Scholar 

  49. Wang J, Majkova Z, Bever CRS, Yang J, Gee SJ, Li J, et al. One-step immunoassay for tetrabromobisphenol A using a camelid single domain antibody–alkaline phosphatase fusion protein. Anal Chem. 2015;87(9):4741–8. https://doi.org/10.1021/ac504735p.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Tang X, Li P, Zhang Q, Zhang Z, Zhang W, Jiang J. Time-resolved fluorescence immunochromatographic assay developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin and zearalenone in maize and its products. Anal Chem. 2017;89(21):11520–8. https://doi.org/10.1021/acs.analchem.7b02794.

    Article  CAS  PubMed  Google Scholar 

  51. Traenkle B, Rothbauer U. Under the microscope: single-domain antibodies for live-cell imaging and super-resolution microscopy. Front Immunol. 2017;8:1030. https://doi.org/10.3389/fimmu.2017.01030.

  52. Roeder R, Helma J, Prei T, Raedler JO, Leonhardt H, Wagner E. Intracellular delivery of nanobodies for imaging of target proteins in live cells. Pharm Res. 2017;34(1):161–74. https://doi.org/10.1007/s11095-016-2052-8.

    Article  CAS  Google Scholar 

  53. Schumacher D, Helma J, Schneider AFL, Leonhardt H, Hackenberger CPR. Nanobodies: chemical functionalization strategies and intracellular applications. Angew Chem Int Ed. 2018;57(9):2314–33. https://doi.org/10.1002/anie.201708459.

    Article  CAS  Google Scholar 

  54. Gainkam LOT, Huang L, Caveliers V, Keyaerts M, Hernot S, Vaneycken I, et al. Comparison of the biodistribution and tumor targeting of two Tc-99m-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CT. J Nucl Med. 2008;49(5):788–95. https://doi.org/10.2967/jnumed.107.048538.

    Article  CAS  PubMed  Google Scholar 

  55. Debie P, Van Quathem J, Hansen I, Bala G, Massa S, Devoogdt N, et al. Effect of dye and conjugation chemistry on the biodistribution profile of near-infrared-labeled nanobodies as tracers for image-guided surgery. Mol Pharm. 2017;14(4):1145–53. https://doi.org/10.1021/acs.molpharmaceut.6b01053.

    Article  CAS  PubMed  Google Scholar 

  56. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–7.

    Article  CAS  Google Scholar 

  57. Fraser G, Smith CA, Imrie K, Meyer R, Hematology Disease Site Group of Cancer Care Ontario’s Program in Evidence-Based Care. Alemtuzumab in chronic lymphocytic leukemia. Curr Oncol. 2007;14(3):96–109.

    Article  CAS  Google Scholar 

  58. Casadevall A. The case for pathogen-specific therapy. Expert Opin Pharmacother. 2009;10(11):1699–703. https://doi.org/10.1517/14656560903066837.

    Article  CAS  PubMed  Google Scholar 

  59. Pankhurst T, Adu D. Antibodies in the prevention of renal allograft rejection. Expert Opin Biol Ther. 2004;4(2):243–52. https://doi.org/10.1517/14712598.4.2.243.

    Article  CAS  PubMed  Google Scholar 

  60. Ibanez LI, De Filette M, Hultberg A, Verrips T, Temperton N, Weiss RA, et al. Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus infection. J Infect Dis. 2011;203(8):1063–72. https://doi.org/10.1093/infdis/jiq168.

    Article  CAS  Google Scholar 

  61. Unger M, Eichhoff AM, Schumacher L, Strysio M, Menzel S, Schwan C, et al. Selection of nanobodies that block the enzymatic and cytotoxic activities of the binary Clostridium difficile toxin CDT. Sci Rep. 2015;5:7850. https://doi.org/10.1038/srep07850.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Lafaye P, Achour I, England P, Duyckaerts C, Rougeon F. Single-domain antibodies recognize selectively small oligomeric forms of amyloid beta, prevent Abeta-induced neurotoxicity and inhibit fibril formation. Mol Immunol. 2009;46(4):695–704. https://doi.org/10.1016/j.molimm.2008.09.008.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work has been funded by the ImmunoQS project funded by MINECO, Programa Estatal de Investigación Desarrollo e Innovación Orientada a los Retos de la Sociedad (SAF2015-67476-R). The Nb4D group is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient: 2017 SGR 1441). CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation 2013-2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

Author information

Author notes

  1. J.-Pablo Salvador and Lluïsa Vilaplana contributed equally to this work.

Authors and Affiliations

  1. Nanobiotechnology for Diagnostics Group (Nb4D), Department of Chemical and Biomolecular Nanotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) of the Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034, Barcelona, Spain

    J.-Pablo Salvador, Lluïsa Vilaplana & M.-Pilar Marco

  2. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Jordi Girona 18-26, 08034, Barcelona, Spain

    J.-Pablo Salvador, Lluïsa Vilaplana & M.-Pilar Marco

Authors
  1. J.-Pablo Salvador
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lluïsa Vilaplana
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M.-Pilar Marco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J.-Pablo Salvador.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Salvador, JP., Vilaplana, L. & Marco, MP. Nanobody: outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem 411, 1703–1713 (2019). https://doi.org/10.1007/s00216-019-01633-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-01633-4

Keywords

Search

Navigation