**3. Camelid intrabodies: a versatile research tool**

Over the years, nanobodies have earned their mark as a research tool. A variety of extracellular and intracellular applications using nanobodies exist, and the latter will be discussed here. Intrabodies are often used to unravel protein functions and to gain insight into their dynamics. The versatility of nanobodies and the ease by which they can be engineered allow researchers to use different lines of approach (**Figure 2**). Chromobodies, consisting of a nanobody fused with a fluorescent protein, allow researchers to recognize and trace endogenous proteins in living cells [70]. Since they are already well known, they will not be discussed in detail here.

and thus to confirm the in vivo functionality of nanobodies. Moreover, in this way, one can also disturb protein function by restricting free diffusion of the protein and limiting its availability at places where it is needed [71]. Considering that the paratope of the nanobody is located at its N-terminal end, it is safer to fuse the tag at the C-terminal end of the nanobody. Otherwise, a substantial risk at disturbing antigen binding exists [2], although there are examples where a long tag is added to the nanobody N-terminus without disturbing its functionality [72]. Beghein et al. elegantly demonstrated how effectively nanobodies can delocalize their target protein to a variety of subcellular organelles. A survivin Nb (Kd ~ 1 nM) was capable of guiding endogenous survivin in or out the nucleus (*nuclear localization sequence* tag and *nuclear export sequence* tag), capturing survivin on the outer membrane of mitochondria (*mitochondrial outer membrane* tag) and even at the intermembrane area (*mitofilin* tag) which probably required (partial) unfolding of the nanobody and possibly chaperone-assisted entry into mitochondria. This had not yet been investigated. Also, transport of survivin in the peroxisomes (PST-1 tag) was demonstrated [72]. Since interaction between the nanobody and survivin apparently did not perturb survivin functionality, the tagged nanobody is a perfect research tool for further elucidating survivin biology. This strategy also provided evidence that only actin-free gelsolin is able to migrate to the nucleus (in contrast to the actin-gelsolin complex) to potentially act as a nuclear cofactor for

specific protein-protein interaction, the co-localization is absent.

**Figure 2.** Schematic overview of nanobody-based applications in research. Upper panel. *Left*: Nanobodies can be equipped with a delocalization signal sequence, which allows the enrichment of a protein of interest (POI) at a specific subcellular localization, such as mitochondria. *Middle*: Nanobodies can exert a direct inhibitory effect which induces a protein knockout. *Right*: Nanobodies can be used to target a POI for proteasomal degradation. For this purpose, the nanobody is fused with a cullin-RING E3 ubiquitin ligase. Lower panel. *Left*: A chromobody, consisting of a fusion between a nanobody and a fluorescent protein (FP), allows the visualization and tracing of endogenous proteins in a background of contaminating proteins. *Right*: This nanobody-based application allows one to determine whether or not two proteins of interest interact. Both proteins are labeled with a different fluorophore. One protein, labeled with GFP, is recruited to a specific location via a GFP Nb coupled with a targeting signal. If the second protein, in this example labeled with RFP, is an interaction partner, both fluorescent signals will co-localize. When a compound interferes with a

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#### **3.1. Pinpointing protein functions**

Nanobodies are an attractive tool for the determination of endogenous protein function. They not only complement well-known RNAi and CRISPR/Cas9 techniques but also allow a more detailed insight by pinpointing specific functions with "surgical precision" by targeting individual protein domains (rather than eliminating the entire protein altogether) and protein conformations, which cannot be achieved by expression modulation. In other words, nanobodies can be of inestimable value to deepen our knowledge of several biological pathways. Researchers have employed several strategies for assessing the functionality of proteins or protein domains, and the different options will be discussed here.

As stated earlier, nanobody cDNAs are available, and these are easily engineered. This implies that the addition of a delocalization tag is fairly straightforward. A variety of targeting sequences are available and can be used to induce the enrichment of both nanobody and its target at specific (ectopic) subcellular compartments. This strategy allows researchers to assess the interaction between the nanobody and its target in the strongly reducing intracellular environment Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs http://dx.doi.org/10.5772/intechopen.70495 217

[24]. Using adeno-associated virus as a vehicle, a bispecific nanobody was introduced in these mice that protects against both furin and MT1-MMP, yielding similar effects on muscle contraction speed [64, 65]. Inhibiting the enzymatic activity of furin could be an alternative strategy, and noncompetitive furin-inhibiting nanobodies have been identified although they have not been tested for treatment of gelsolin amyloidosis [66]. However, despite the involvement of furin in several pathological processes, some considerations have to be made regarding its use as a therapeutic target. Although a complete/partial cleavage redundancy of furin toward several substrates was observed in the liver of an interferon-inducible Mx-Cre/loxP, furin knockout mouse model and obvious adverse effect were absent; a complete knockout of furin in a mouse model resulted in embryonic lethality at day 11 [67, 68]. This observation probably precludes their use in chronic treatments because it is rather unlikely that the long-term inhibition of furin does not go hand in hand with severe adverse effects. Therefore, shielding mutant PG from aberrant cleavage seems to be the better strategy. Moreover, this approach is already successfully implemented in the treatment of early-stage familial amyloid polyneuropathy caused by amyloidogenic variants of transthyretin, thus highlighting its feasibility [69].

Over the years, nanobodies have earned their mark as a research tool. A variety of extracellular and intracellular applications using nanobodies exist, and the latter will be discussed here. Intrabodies are often used to unravel protein functions and to gain insight into their dynamics. The versatility of nanobodies and the ease by which they can be engineered allow researchers to use different lines of approach (**Figure 2**). Chromobodies, consisting of a nanobody fused with a fluorescent protein, allow researchers to recognize and trace endogenous proteins in living cells [70]. Since they are already well known, they will not be discussed in

Nanobodies are an attractive tool for the determination of endogenous protein function. They not only complement well-known RNAi and CRISPR/Cas9 techniques but also allow a more detailed insight by pinpointing specific functions with "surgical precision" by targeting individual protein domains (rather than eliminating the entire protein altogether) and protein conformations, which cannot be achieved by expression modulation. In other words, nanobodies can be of inestimable value to deepen our knowledge of several biological pathways. Researchers have employed several strategies for assessing the functionality of proteins or

As stated earlier, nanobody cDNAs are available, and these are easily engineered. This implies that the addition of a delocalization tag is fairly straightforward. A variety of targeting sequences are available and can be used to induce the enrichment of both nanobody and its target at specific (ectopic) subcellular compartments. This strategy allows researchers to assess the interaction between the nanobody and its target in the strongly reducing intracellular environment

**3. Camelid intrabodies: a versatile research tool**

protein domains, and the different options will be discussed here.

detail here.

216 Antibody Engineering

**3.1. Pinpointing protein functions**

**Figure 2.** Schematic overview of nanobody-based applications in research. Upper panel. *Left*: Nanobodies can be equipped with a delocalization signal sequence, which allows the enrichment of a protein of interest (POI) at a specific subcellular localization, such as mitochondria. *Middle*: Nanobodies can exert a direct inhibitory effect which induces a protein knockout. *Right*: Nanobodies can be used to target a POI for proteasomal degradation. For this purpose, the nanobody is fused with a cullin-RING E3 ubiquitin ligase. Lower panel. *Left*: A chromobody, consisting of a fusion between a nanobody and a fluorescent protein (FP), allows the visualization and tracing of endogenous proteins in a background of contaminating proteins. *Right*: This nanobody-based application allows one to determine whether or not two proteins of interest interact. Both proteins are labeled with a different fluorophore. One protein, labeled with GFP, is recruited to a specific location via a GFP Nb coupled with a targeting signal. If the second protein, in this example labeled with RFP, is an interaction partner, both fluorescent signals will co-localize. When a compound interferes with a specific protein-protein interaction, the co-localization is absent.

and thus to confirm the in vivo functionality of nanobodies. Moreover, in this way, one can also disturb protein function by restricting free diffusion of the protein and limiting its availability at places where it is needed [71]. Considering that the paratope of the nanobody is located at its N-terminal end, it is safer to fuse the tag at the C-terminal end of the nanobody. Otherwise, a substantial risk at disturbing antigen binding exists [2], although there are examples where a long tag is added to the nanobody N-terminus without disturbing its functionality [72]. Beghein et al. elegantly demonstrated how effectively nanobodies can delocalize their target protein to a variety of subcellular organelles. A survivin Nb (Kd ~ 1 nM) was capable of guiding endogenous survivin in or out the nucleus (*nuclear localization sequence* tag and *nuclear export sequence* tag), capturing survivin on the outer membrane of mitochondria (*mitochondrial outer membrane* tag) and even at the intermembrane area (*mitofilin* tag) which probably required (partial) unfolding of the nanobody and possibly chaperone-assisted entry into mitochondria. This had not yet been investigated. Also, transport of survivin in the peroxisomes (PST-1 tag) was demonstrated [72]. Since interaction between the nanobody and survivin apparently did not perturb survivin functionality, the tagged nanobody is a perfect research tool for further elucidating survivin biology. This strategy also provided evidence that only actin-free gelsolin is able to migrate to the nucleus (in contrast to the actin-gelsolin complex) to potentially act as a nuclear cofactor for the androgen receptor [73], that fascin plays a role in the formation of filopodia/cell spreading and is also involved in MMP9 secretion, and that the SH3 domain of cortactin directly regulates MMP secretion [74].

called DeGradFP, was expressed in mammalian cells or *D. melanogaster* embryos, certain GFPtagged proteins were depleted. DeGradFP was also capable of phenocopying specific loss of function mutations. In spite of these successful results, treatment with DeGradFP was not always followed by the degradation of the targeted protein (e.g., GFP) [81]. In addition to that,

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219

Just like DeGradFP, the cullin-RING E3 ubiquitin ligases were used as the framework for synthetic E3 ligase design. In an attempt to enhance the E3 activity, however, the GFP Nb was fused directly to a truncated adaptor protein instead of the substrate recognition protein. The best results were obtained with Ab-SPOP, a synthetic version of the CLR3 E3 ligase complex, displaying a 10-fold stronger signal reduction of a GFP-tagged protein compared to DeGradFP (50-fold vs. 5-fold). Importantly, the construct degrades only nuclear proteins, and possibly in the future, similar constructs may become available that degrade cytoplasmic proteins. The in vivo effectiveness of Ab-SPOP was confirmed in zebra fish embryos. Ab-SPOP-induced depletion of Hmg2a-citrine, a protein responsible for the modulation of nucleosome and chromatin structure, resulted in various early developmental defects [83]. Fulcher et al. tailored the von Hippel-Lindau (VHL) protein as an affinity-directed protein missile, called AdPROM. Under normoxic conditions, this substrate recognition protein recruits the hypoxia-inducible factor (HIF1α) to the CLR2 E3 ligase. AdPROM is composed of a fusion between the VHL protein and a GFP Nb. It was of crucial importance that the GFP Nb was positioned at the C-terminus of the VHL protein in order to obtain a proper orientation of the target proteins to the CLR2 E3 ligase complex. Since the paratope of a nanobody is localized at the N-terminal end, one should definitely check for potential detrimental effects of this fusion on the binding capacity of the nanobody itself. However, the affinity-directed protein missile was competent in inducing the specific degradation of GFP-tagged VPS34 and PAWS1 proteins in human cell lines, which was further substantiated by the observation of functional effects. Interestingly, during these experiments the researchers observed the co-degradation of UVRAG which is a regulatory component of the VPS34 kinase complex. This suggests that AdPROM has the potential of destroying protein complexes although only individual proteins are targeted [82]. Targeted degradation of proteins of interest by the use of nanobodies holds great potential and might be the perfect complement to CRISPR/Cas systems or RNAi in the elucidation of protein function. The tunability of this system is a huge benefit. Future experiments should point out whether the GFP Nb can be replaced by highly selective nanobodies targeted against specific proteins. In this way, one could investigate the functions of the protein of interest in a more direct manner, without the requirement of protein tags.

Nanobodies can be utilized for the detection of protein-protein interactions in cell-based assays. There is a large supply of in vitro methods which can be used for the detection of protein-protein interactions. These methods are widely used and highly efficient for highthroughput screenings but are limited by the fact that they don't operate in intact mammalian cells. Screening for interaction between proteins in their native environment guarantees their proper folding and the presence of necessary cofactors or regulatory proteins. Both

a broader application of DeGradFP is still to be demonstrated.

**3.3. Detection of protein-protein interactions**

Some nanobodies exert a direct inhibitory effect, resulting in a functional knockout of the protein. These nanobodies can help researchers to define the biochemical activities of proteins. For example, mechanistic insights in podosome formation were revealed by two inhibitory nanobodies targeted against L-plastin (LPL). LPL Nb5 is capable of blocking the actin-bundling activity of L-plastin, and LPL-Nb9 locks LPL in an inactive conformation. Experiments involving these nanobodies revealed the participation of L-plastin (LPL) in podosome formation and stability [75]. Furthermore, L-plastin is a component of cancer cell invadopodia and contributes to matrix degradation and cancer cell invasion. These effects are mediated by the actin-bundling activity of L-plastin and its bundling independent role in MMP9 secretion and activity, as revealed by the differential effects observed in the presence of LPL Nb5 and LPL Nb9 [76]. One can also interfere with signaling pathways by specific inhibition of the transcriptional activity of proteins, like beta-catenin and p53 [77, 78]. These nanobodies can be used to elucidate the impact of cofactors and post-translational modifications on the targeted protein and allow us to broaden our understanding of the respective signaling pathways. Insight into pathological mechanisms, which might result in the identification of druggable targets, can also be obtained. For example, nanobodies were used to investigate the role of two enzymatic domains of TcdB, a toxin produced by *Clostridium difficile*. Using specific inhibition of the effector glycosyltransferase activity or the cysteine protease, it was, among other things, established that the TcdB-cytopathic effects are mainly mediated by the glycosyltransferase activity [79].

Finally, nanobodies are known to stabilize certain protein conformations and are often used as an aid in crystallization experiments [2]. This property also comes in handy when one wants to study the mechanisms by which cellular receptors translate extracellular cues into intracellular responses. Depending on which conformation the receptor adopts after ligand binding, certain downstream signaling events can be either activated or inhibited. Staus et al. have identified nanobodies that preferentially recognize and stabilize the β2 adrenergic receptor in its active or inactive conformation resulting in a variety of functional effects [80]. These experiments indicate that nanobodies, by acting as an allosteric modulator of receptors, can help us to understand receptor biology.

#### **3.2. Depleting endogenous proteins through proteasomal targeting**

An alternative way to determine the function of a protein of interest (POI) in an in vivo setting is to selectively induce their degradation and study the resulting knockout phenotype. To achieve this goal, three different groups have exploited a combination of nanobodies and the endogenous ubiquitin proteasome pathway, a system that is responsible for selective protein degradation in eukaryotes [81–83]. Caussinus et al. were the first to use the ubiquitin pathway for targeted degradation by making adaptations of an E3 ubiquitin ligase, more specifically the cullin-RING 1 (CLR1) E3 ligase complex. For this purpose, a fusion between the F-box domain of *Slmb* and a GFP Nb (VHH GFP4) was made. *Slmb* is part of an F-box protein, responsible for substrate recognition that is expressed in *Drosophila melanogaster*. When this construct, called DeGradFP, was expressed in mammalian cells or *D. melanogaster* embryos, certain GFPtagged proteins were depleted. DeGradFP was also capable of phenocopying specific loss of function mutations. In spite of these successful results, treatment with DeGradFP was not always followed by the degradation of the targeted protein (e.g., GFP) [81]. In addition to that, a broader application of DeGradFP is still to be demonstrated.

Just like DeGradFP, the cullin-RING E3 ubiquitin ligases were used as the framework for synthetic E3 ligase design. In an attempt to enhance the E3 activity, however, the GFP Nb was fused directly to a truncated adaptor protein instead of the substrate recognition protein. The best results were obtained with Ab-SPOP, a synthetic version of the CLR3 E3 ligase complex, displaying a 10-fold stronger signal reduction of a GFP-tagged protein compared to DeGradFP (50-fold vs. 5-fold). Importantly, the construct degrades only nuclear proteins, and possibly in the future, similar constructs may become available that degrade cytoplasmic proteins. The in vivo effectiveness of Ab-SPOP was confirmed in zebra fish embryos. Ab-SPOP-induced depletion of Hmg2a-citrine, a protein responsible for the modulation of nucleosome and chromatin structure, resulted in various early developmental defects [83]. Fulcher et al. tailored the von Hippel-Lindau (VHL) protein as an affinity-directed protein missile, called AdPROM. Under normoxic conditions, this substrate recognition protein recruits the hypoxia-inducible factor (HIF1α) to the CLR2 E3 ligase. AdPROM is composed of a fusion between the VHL protein and a GFP Nb. It was of crucial importance that the GFP Nb was positioned at the C-terminus of the VHL protein in order to obtain a proper orientation of the target proteins to the CLR2 E3 ligase complex. Since the paratope of a nanobody is localized at the N-terminal end, one should definitely check for potential detrimental effects of this fusion on the binding capacity of the nanobody itself. However, the affinity-directed protein missile was competent in inducing the specific degradation of GFP-tagged VPS34 and PAWS1 proteins in human cell lines, which was further substantiated by the observation of functional effects. Interestingly, during these experiments the researchers observed the co-degradation of UVRAG which is a regulatory component of the VPS34 kinase complex. This suggests that AdPROM has the potential of destroying protein complexes although only individual proteins are targeted [82]. Targeted degradation of proteins of interest by the use of nanobodies holds great potential and might be the perfect complement to CRISPR/Cas systems or RNAi in the elucidation of protein function. The tunability of this system is a huge benefit. Future experiments should point out whether the GFP Nb can be replaced by highly selective nanobodies targeted against specific proteins. In this way, one could investigate the functions of the protein of interest in a more direct manner, without the requirement of protein tags.

#### **3.3. Detection of protein-protein interactions**

the androgen receptor [73], that fascin plays a role in the formation of filopodia/cell spreading and is also involved in MMP9 secretion, and that the SH3 domain of cortactin directly regulates

Some nanobodies exert a direct inhibitory effect, resulting in a functional knockout of the protein. These nanobodies can help researchers to define the biochemical activities of proteins. For example, mechanistic insights in podosome formation were revealed by two inhibitory nanobodies targeted against L-plastin (LPL). LPL Nb5 is capable of blocking the actin-bundling activity of L-plastin, and LPL-Nb9 locks LPL in an inactive conformation. Experiments involving these nanobodies revealed the participation of L-plastin (LPL) in podosome formation and stability [75]. Furthermore, L-plastin is a component of cancer cell invadopodia and contributes to matrix degradation and cancer cell invasion. These effects are mediated by the actin-bundling activity of L-plastin and its bundling independent role in MMP9 secretion and activity, as revealed by the differential effects observed in the presence of LPL Nb5 and LPL Nb9 [76]. One can also interfere with signaling pathways by specific inhibition of the transcriptional activity of proteins, like beta-catenin and p53 [77, 78]. These nanobodies can be used to elucidate the impact of cofactors and post-translational modifications on the targeted protein and allow us to broaden our understanding of the respective signaling pathways. Insight into pathological mechanisms, which might result in the identification of druggable targets, can also be obtained. For example, nanobodies were used to investigate the role of two enzymatic domains of TcdB, a toxin produced by *Clostridium difficile*. Using specific inhibition of the effector glycosyltransferase activity or the cysteine protease, it was, among other things, established that the TcdB-cytopathic effects are mainly mediated by the glycosyltransferase activity [79]. Finally, nanobodies are known to stabilize certain protein conformations and are often used as an aid in crystallization experiments [2]. This property also comes in handy when one wants to study the mechanisms by which cellular receptors translate extracellular cues into intracellular responses. Depending on which conformation the receptor adopts after ligand binding, certain downstream signaling events can be either activated or inhibited. Staus et al. have identified nanobodies that preferentially recognize and stabilize the β2 adrenergic receptor in its active or inactive conformation resulting in a variety of functional effects [80]. These experiments indicate that nanobodies, by acting as an allosteric modulator of receptors,

MMP secretion [74].

218 Antibody Engineering

can help us to understand receptor biology.

**3.2. Depleting endogenous proteins through proteasomal targeting**

An alternative way to determine the function of a protein of interest (POI) in an in vivo setting is to selectively induce their degradation and study the resulting knockout phenotype. To achieve this goal, three different groups have exploited a combination of nanobodies and the endogenous ubiquitin proteasome pathway, a system that is responsible for selective protein degradation in eukaryotes [81–83]. Caussinus et al. were the first to use the ubiquitin pathway for targeted degradation by making adaptations of an E3 ubiquitin ligase, more specifically the cullin-RING 1 (CLR1) E3 ligase complex. For this purpose, a fusion between the F-box domain of *Slmb* and a GFP Nb (VHH GFP4) was made. *Slmb* is part of an F-box protein, responsible for substrate recognition that is expressed in *Drosophila melanogaster*. When this construct,

Nanobodies can be utilized for the detection of protein-protein interactions in cell-based assays. There is a large supply of in vitro methods which can be used for the detection of protein-protein interactions. These methods are widely used and highly efficient for highthroughput screenings but are limited by the fact that they don't operate in intact mammalian cells. Screening for interaction between proteins in their native environment guarantees their proper folding and the presence of necessary cofactors or regulatory proteins. Both nanobody-based methods rely on the interaction between a GFP Nb and a GFP-tagged protein. Herce et al. covalently linked a GFP Nb with a protein that accumulates at a specific subcellular location. In mammalian cells, this protein could be, for example, laminin B1 or centrin, which results in the delocalization of the GFP Nb to the nuclear lamina or the cytoplasmic centrioles, respectively. Subsequently, a GFP-tagged protein will be recruited to a specific location. If the second protein of interest, labeled with another fluorophore, interacts with the first protein of interest, the fluorophores will co-localize at a discrete spot. This interaction can be visualized by a single-fluorescence snapshot. Interestingly, this technique also allows screening for inhibitors of protein-protein interactions [84]. Another recently developed technique uses biocompatible engineered upconversion nanoparticles (UCNPs) conjugated with GFP Nbs. Visualization of the interaction between two proteins of interest is based on the lanthanide resonance energy transfer (LRET). As a proof of concept, they probed for the indirect interaction between the mitochondrial proteins TOM20 and TOM7. The latter was expressed as a fusion protein with EGFP and the former as a fusion protein with dsRed and a Halo tag. This Halo tag was subsequently labeled with tetramethylrhodamine (TMR), while the EGFP was recognized by the GFP Nb-labeled UCNPs. Co-localization of both proteins results in the detection of LRET-sensitized TMR emission. Remarkably, TOM7 and TOM20 are spatially separated by TOM40. The capacity of this technique for reporting indirect longdistance interactions might be of interest to unravel cellular protein complexes [85].

[86, 87]. Indeed, particularly polyclonal antibodies run out of stock at some point in the future, making experimental verification impossible. Because nanobody cDNAs are readily obtained and researchers all over the world can use exactly the same nanobody in their experiments, problems of reproducibility can be reduced. In the future, we hope to stimulate a closer consultation within the nanobody field and by doing so taking the research to

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This work was supported by grants from the Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen) and Ghent University (BOF13/GOA/010). AS and LB are supported by the Agency for Innovation by Science and Technology in Flanders

(IWT-Vlaanderen). We apologize to those researchers whose work could not be cited.

Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University,

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Anneleen Steels, Laurence Bertier and Jan Gettemans\*

\*Address all correspondence to: jan.gettemans@ugent.be

the next level.

**Acknowledgements**

**Author details**

Belgium

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