The future of
single-domain antibodies



Intracellular VHH or Intrabody

Nanobodies of high quality that work inside living cell: learn more about Intrabody (Intracellular Antibody)

Since the mid 20th century, antibodies have become major components of the scientific toolkit to analyze fundamental biological mechanisms or develop diagnostics. In the last 25 years, antibodies have even turned into an inexhaustible source of new drugs. An antibody is a large molecule secreted by B lymphocytes. This large molecule (Figure 1)  is difficult to use and to monitor. To be able to modify intracellular targets, antibodies need to be injected inside the cells which is not an easy task [1]

Molecular manipulations of antibodies now allow the expression of different parts of antibodies inside the cells from a cDNA clone[2]. The most classical one is the scFv (fig1), a molecule in which the VH and the VL parts of an antibody have been fused by a small peptidic linker. A new family of antibodies, discovered in the camelid in 1993 [3], displays the unique feature to specifically recognize the antigen with a single VH domain. They are called VHH or Nanobodies. 
 

IgG-scFv-VHH

Figure 1: Comparison of the size and structure of the different types of antibodies.

 

VHH and Yeast two-Hybrid: keys to obtain powerful tools for your experiments

Thanks to their small size, the fragments derived from this new type of antibodies (VHH, fig 1) are very good candidates for intrabody expression.

However, since the folding in the secretion pathway and in the intracellular context could be different, the intrabody capacity of a fragment has to be tested by screening inside mammalian cells or using Yeast Two-Hybrid technology [4-6]

In fundamental research, intrabodies have been successfully used to visualize and understand molecular dynamics of biological processes. For example, uncoupling of dynamin polymerization and GTPase activity have been recently revealed by a conformation-specific nanobody selected from our synthetic library [7] (see also [4, 8, 9])

You can read the paper relating this discovery in eLIFE.

E12-1 Anti-Tau intrabody fused to mCherry protein expressed in Hela cell recognized over expressed GFP-Tau

Figure 2: E12-1 Anti-Tau intrabody fused to mCherry, expressed in Hela cell, recognizes over expressed GFP-Tau

 

Intrabodies could also be:

- Blocking molecule that are incredible tools for target engagement studies [10, 11]
- Fused to an F-box or a PEST signal they could mediate specific target degradation in cell [11-13]
- Block virus replication [14, 15]
- Inhibitors of protein/protein interactions

 

Video: mcherry-Rab6 expressed in HeLa cells with an anti-mCherry VHH fused to GFP, imaged using a spinning disk confocal microscope.[11]

Intrabodies may be the tools you need to improve your experiments.

Contact our scientist about your project and join us in the fascinating word of intrabodies.

 

References

1. Kreis, T.E., Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J, 1986. 5(5): p. 931-41.
2. Biocca, S., M.S. Neuberger, and A. Cattaneo,Expression and targeting of intracellular antibodies in mammalian cells.EMBO J, 1990. 9(1): p. 101-8.
3. Hamers-Casterman, C., et al., Naturally occurring antibodies devoid of light chains. Nature, 1993. 363(6428): p. 446-8.
4. Nizak, C., et al., Recombinant antibodies to the small GTPase Rab6 as conformation sensors. Science, 2003. 300(5621): p. 984-7.
5. Tanaka, T. and T.H. Rabbitts, Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J, 2003. 22(5): p. 1025-35.
6. Rothbauer, U., et al., Targeting and tracing antigens in live cells with fluorescent nanobodies.Nat Methods, 2006. 3(11): p. 887-9.
7. Galli, V., et al., Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab. Elife, 2017. 6.
8. Fukata, Y., et al., Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J Cell Biol, 2013. 202(1): p. 145-61.
9. Dimitrov, A., et al.,Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues. Science, 2008. 322(5906): p. 1353-6.
10. Tanaka, T., R.L. Williams, and T.H. Rabbitts, Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J, 2007. 26(13): p. 3250-9.
11. Moutel, S., et al., NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife, 2016. 5.
12. Butler, D.C. and A. Messer, Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments. PLoS One, 2011. 6(12): p. e29199.
13. Caussinus, E. and M. Affolter, deGradFP: A System to Knockdown GFP-Tagged Proteins. Methods Mol Biol, 2016. 1478: p. 177-187.
14. Gal-Tanamy, M., et al., Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies. Antiviral Res, 2010. 88(1): p. 95-106.
15. Kaku, Y., et al., Inhibition of rabies virus propagation in mouse neuroblastoma cells by an intrabody against the viral phosphoprotein. Antiviral Res, 2011. 91(1): p. 64-71.

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