Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells

BBAPAP-39348; No. of pages: 10; 4C: 3, 4, 7 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells☆ Philipp D. Kaiser, Julia Maier, Bjoern Traenkle, Felix Emele, Ulrich Rothbauer ⁎ Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany Department of Pharmaceutical Biotechnology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

a r t i c l e

i n f o

Article history: Received 22 February 2014 Received in revised form 16 April 2014 Accepted 21 April 2014 Available online xxxx Keywords: Antibody-scaffold Intrabody Protein transduction Functional assay Live cell imaging Chromobody

a b s t r a c t In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. © 2014 Published by Elsevier B.V.

1. Introduction Molecules with the ability to recognize, bind and modulate intracellular targets play an important role for basic research in cell biology as well as in the development of therapeutic and diagnostic agents. For real time visualization of cellular components, intracellular target identification and validation in living cells, such binding molecules essentially have to fulfill three criteria: (i) stable folding, (ii) compactness to enter subcellular compartments, and (iii) an affinity that is sufficient for specific antigen binding. During the last decade extensive research on recombinant antigen binding proteins, the generation of combinatorial libraries, screening methodologies and cellular targeting technologies led to the identification and generation of numerous intracellular functional binding molecules (intrabodies). Selected intrabody formats were shown to function for different applications including target inhibition/modulation, to study protein–protein interactions or to ☆ This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. ⁎ Corresponding author. Tel.: +49 7121 515 30 415; fax: +49 7212 515 30 16. E-mail address: [email protected] (U. Rothbauer).

visualize dynamic changes of cellular components in living cells. Here we give an update on the current state of selection and application of intrabodies focusing on cell-based analyses including live cell imaging. 2. Scaffolds for intrabodies 2.1. Non-antibody based scaffolds There is a constantly growing list of scaffold proteins, peptides or nucleic acids that can be designed as intracellular affinity reagents. We limit our discussion to emerging scaffold proteins which were previously shown to function within living cells. One prominent member of non-antibody like proteins is fibronectin. The tenth domain of type III fibronectin (also named FN3 or monobody) is one of the best characterized scaffolds of this type of protein. Its functional conformation does not rely on the formation of disulfide bonds, which is supposed to facilitate a correct folding in the reducing environment of the cytoplasm. It comprises ligand binding loops that are suitable to probe intracellular targets. Desired binders can be selected from randomized generic libraries via different selection processes including phage display or mRNA display [1–3]. In an early study it has been reported that such

http://dx.doi.org/10.1016/j.bbapap.2014.04.019 1570-9639/© 2014 Published by Elsevier B.V.

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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binding molecules detect conformational changes of intracellular targets like the estrogen receptor alpha ligand binding domain (ERαLBD) in the nucleus of living cells [4]. Another important class of binding molecules reported to be functional in living cells are the so called “repeat proteins”. These proteins are characterized by repetitive structural units of 30–50 amino acids (aa) forming elongated domains as binding interfaces which were shown to recognize a variety of cellular target structures. The most prominent members of this protein family are the tetratricopeptide (TPR), HEAT and designed ankyrin repeat proteins (DARPins) [5,6]. Ankyrin repeats (AR) are common motifs in many binding proteins of different species. Each AR consists of 33 amino acids (aa) forming a β-loop followed by two antiparallel α-helices and each DARPin is built of two to four repeats flanked by a N- and C-terminal capping repeat, giving rise to molecular weights between 14 and 21 kDa. Like fibronectins, DARPins neither contain disulfide bonds nor free cysteines and they are reported to be functional in living cells. Intracellular expressed DARPins have mainly been reported for targeted modulation of particular enzymes. Previous studies include DARPins selected against the NIa(pro) proteinase of tobacco etch virus (TEV) which decrease proteinase activity upon expression in bacteria. Furthermore DARPin-mediated inhibition of c-Jun N-terminal kinases (JNKs) in human cells was shown [7,8]. Recently, two DARPins were described recognizing distinct conformational changes of ERK2 either in its non-phosphorylated or phosphorylated form. GFP-fusions of these binding molecules specifically recognize the modification status of the kinase in COS7 cells and were shown to block the phosphorylation of ERK2 in living cells [9]. In addition to protein scaffolds, peptides or short single stranded DNA or RNA oligomers (aptamers) can also serve as affinity reagents in living cells. In 2006 Kunz et al. reported new peptide aptamers, which specifically interact with defined domains of the intracellular part of the ErbB2 receptor. The peptides were either expressed intracellularly or introduced as recombinant aptamer proteins. Upon binding to the ErbB2 receptor the peptides were shown to inhibit the induction of AKT kinase in breast cancer (MCF7) cells, which accordingly became sensitized to Taxol [10]. Besides peptides, the intracellular expression of target specific RNA aptamers (so called “intramers”) was demonstrated to interfere with protein function in living cells. This includes the blockade of specific DNA recognition sites of transcription factors or the specific binding to a guanine nucleotide exchange factor (Cytohesin-2) resulting in a knockdown of kinase activities in HeLa cells [11–13]. Such alternative intracellular binding formats become attractive for targeted functional studies e.g. for target validation. However, the applicability of these molecules for intracellular imaging has so far been limited. 2.2. Antibody scaffolds/IgG derived intrabodies Besides their steadily growing impact as therapeutic molecules, antibodies are the most powerful tools for biomedical research and represent a well-established class of clinical diagnostics. Conventional antibodies (IgGs) of vertebrates consist of two identical heavy (H) and two light (L) chains, each comprising a variable domain at the N-terminus (abbreviated as VH and VL, respectively). Antigen binding is mediated by three complementarity determining regions (CDRs) located in the VH and VL [14]. Antibodies per se are large and complex molecules (~ 150 kDa) and for a correct heterotetrameric structure four covalent disulfide bonds have to be formed [15] (Fig. 1). Antibodies are naturally secreted or presented at the cellular surface as an essential part of the host response towards extracellular pathogens. During the last two decades recombinant antibody technology has rapidly progressed and led to the development of smaller antigen binding molecules. Starting from conventional IgGs (150 kDa), the antigen recognition part can be reduced to a monovalent antibody fragment (Fab, 50 kDa) or a single-chain variable fragment (scFv, 25 kDa) built of an isolated VH and VL domain, connected by a flexible peptide linker,

usually comprising glycine/serine (Gly4Ser)2–4 repeats [16,17]. Originally, scFvs were derived by linking the isolated VH and VL domains of a specific monoclonal antibody [16,18,19] (Fig. 1). In contrast to full length antibodies, scFvs were proposed to be suitable for intracellular applications because of their reduced size, ease of expression from different vector systems and absence of inter-chain disulfide linkages. However, first attempts to express scFvs within living cells show only limited success due to aggregation, impaired intra-molecular disulfide bond formation, inefficient assembly of the epitope recognizing parts and hampered subcellular targeting [20–23]. Advances in the structural formulation of such recombinant antibody molecules, the generation of comprehensive libraries from either immunized or naïve sources and the implementation of new screening protocols (please refer to the review provided by Terence H. Rabbitts & Jing Zhang in this issue) have led to a substantial progress in the development of intracellular functional binding molecules [24–30]. Besides scFvs, in few examples stabilized singular VH and VL domains derived from conventional IgGs have been applied as intrabodies [31–34]. However, the loss of the corresponding variable light or heavy chain often reduces binding affinities and specificities. The discovery of naturally occurring heavy-chain only antibodies in Camelidae and cartilaginous fish (e.g. sharks) in the early nineties impressively demonstrated the superiority of nature in downsizing the most essential antigen-binding regions [35,36]. 2.3. Single domain antibodies Up to now, single domain antibodies (sdAbs) are the smallest engineered antibody fragments still capable of binding antigens. sdAbs consist of only the variable domain of a light chain or a heavy chain. The most prominent alternative to conventional antibodies are heavychain antibodies (HCAbs) of the Camelidae that do not have light chains [35]. HCAbs recognize and bind their antigens via a single variable domain (referred to as VHH or nanobody, Fig. 1), which represents the smallest intact antigen binding fragment (~13 kDa) occurring in nature [37,38]. Compared to other small antibody fragments like Fab or scFv, nanobodies have numerous technological advantages. First, only one domain has to be cloned and expressed to generate an in vivo matured binding molecule. Second, specific nanobodies can easily be selected with standard screening technologies (e.g. phage display). Third, nanobodies are highly soluble and stable and can be efficiently expressed in heterologous systems [39]. The affinities found for nanobodies so far are in the nanomolar range and comparable with those of scFvs [40–42]. Similar to the VH domain of conventional IgGs the singular VHH domain of HCAbs consists of four conserved framework (FR) regions, that are connected by loops representing the three CDRs. The framework regions of VHH and VH are highly conserved, including the canonical disulfide bond between FR1 and FR3 (Cys23–Cys94) [43]. However, there are minor but characteristic differences. In FR2 of a conventional VH domain the four highly conserved hydrophobic amino acids Val37, Gly44, Leu45 and Trp47 are crucial for the interaction between the variable heavy chain and the variable light chain [44,45]. Those so called “hallmark amino acids” are replaced by the more hydrophilic amino acids Phe37, Glu44, Arg45 and Gly47 in the VHH domain leading to a higher solubility of the entire single domain binding molecule [43,44, 46]. Another difference between VH and VHHs is observed in the CDRs. Compared to a conventional antibody the VHH domain of HCAbs recognizes and binds its antigen with only three CDRs instead of six. To provide an appropriate antigen interaction surface (600–800 Å2) the CDRs of a VHH domain are slightly extended. Especially VHH domains isolated from camels often have an extended CDR3 that can form convex structures to recognize cavity epitopes of the antigen [47]. Owing to the small size (2 nm × 4 nm), increased solubility and singular structure, it can be proposed that VHHs have a higher probability to be functional upon intracellular expression [42,48].

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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Fig. 1. Schematic representation of antibody formats shown to be functional within living cells.

3. Selection process of intracellular binding proteins The immune response system is a sophisticated machinery yielding antibodies with excellent functionality as they bind their antigen with high affinity and selectivity. This is achieved by the integration of large antibody diversities – practically capable of recognizing any antigen structure – with a multistep affinity maturation process. While the generation of complex in vitro antibody libraries is well established per se, the selection and in vitro maturation of antibodies are often laborious and cumbersome as their functionality has to be confirmed for each binding molecule individually. Although it is possible to obtain binding molecules from naïve or synthetic sources, immunization of animals (incl. mice, rats, goats, donkeys, camels) is frequently the first step in antibody development to make use of the above-mentioned natural selection. Using recombinant technologies, the whole antibody repertoire of an immunized animal can be easily transferred to expression libraries, which are subsequently screened for functional binding molecules in high-throughput methods such as phage-, ribosome- or mRNA-display [49–51]. While these test tube technologies are suitable to select high affinity binders, they are unable to predict whether those binders are functional within living cells. One of the first methods developed to identify intracellular functional scFvs was the intracellular antibody capture technology (IACT), a combination of phage display and a modified yeast two-hybrid (Y2H) assay [26,52,53]. After an initial phage display step, a library of in vitro identified antibodies is transferred to a plasmid expression system composed of a scFv fused to the VP16 transcriptional activation domain and the antigen linked to the LexA–DNA binding domain. Upon transfection of a yeast strain, that is unable to synthesize histidine, but carries the HIS3 gene under the control of a promotor with a LexA–DNA binding site, cells with positive antibody–antigen interaction can be identified

through growth on selective medium. However, in many cases selected scFvs, do not properly bind their antigen after expression in mammalian cells and therefore require additional validation [29]. Recent progress in this technology is reviewed in detail in the IACT section of this issue. Recently, a modified bacterial-two-hybrid selection system was developed enabling a one-step isolation of intracellular functional sdAbs (nanobodies) from large libraries (N107 potential binders). For high throughput selection in Escherichia coli reporter cells nanobodies were fused to a lambda repressor (λcI, binding to the phage λ operator) and cotransformed with an antigen fused to the α-subunit of RNApolymerase. A proper antigen–nanobody interaction recruits the transcription machinery to the λ operator and initiates expression of selective marker genes. Within fast growing bacteria this allows the survival of the transformed cells on a selective medium in medium to high throughput [54]. This method allows a rapid and efficient selection of nanobodies which are functional within bacteria. With respect to mammalian cells intrabodies require further validation regarding expression, localization, target specificity and/or interference in the cellular environment. One possibility to evaluate antigen binding and target specificity within living cells are colocalization assays. Therefore antigen and respective binding molecules are coexpressed as distinguishable fluorescent fusion proteins in living cells. Binding events become visible as an overlay of both fluorescent signals. Recently, this has been demonstrated in an assay where the antigen was redirected to the Golgi by fusing the antigen to a Golgi targeting sequence [55] and by the fluorescent-2-hybrid assay (F2H) [54,56]. The latter is performed in recombinant mammalian cell lines harboring a large copy number of the lac operator array stably integrated into the genome. Upon expression of a tripartite fusion protein comprising a fluorescent protein, the lac repressor (lacI) domain followed by a binding partner (antigen/antibody) a distinct fluorescent spot becomes visible within the nucleus due to the

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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binding of the lac repressor to the lac operator array. Coexpression of a second binding partner (antigen/antibody) comprising a distinguishable fluorescent marker results either in a diffuse distribution (no interaction) or both fluorescent markers are detectable at the nuclear spot (interaction) (Fig. 2). The interaction can be easily monitored using standard fluorescent microscopy without any further technical demands. One drawback of this approach is the redirection of the binding partners to the nucleus. However, most intracellular binding molecules are characterized by a low molecular size (50–60 kDa) and have access to the nuclear compartment by free diffusion throughout the nuclear pores. 4. Delivery systems One of the central issues in the application of intrabodies is the delivery of such molecules into living cells. Basically, there are two ways: (i) introduction of the genomic information encoded on expression vectors, or (ii) delivery of a binding molecule on the protein level. Currently gene-based expression constructs are preferred, however, increasing knowledge of protein transduction might provide a versatile and more flexible alternative for the delivery of intrabodies in the future. 4.1. Transfection/transduction of DNA Considering gene-based systems for intrabody expression conventional DNA transfection methodologies (including lipofection, electroporation or nanoparticle mediated transfection) are mainly used [57, 58]. Those methods are easy to adapt and well suited for a rapid analysis of large numbers of intrabodies. However, non-correlation between the amount of transfected DNA and the level of protein expression, high rates of toxicity, as well as the limitation to transfectable cell types makes it necessary to assess other technologies for the delivery of intrabody-coding expression vectors. A more differentiated approach

is the usage of viral delivery systems. Most viral vectors allow an efficient integration of a gene of interest in the genome of dividing and non-dividing cells, which facilitates the generation of stable cell lines expressing intrabodies in suitable amounts [59–61]. Viral vectors were successfully applied to transduce neurons [62], model cell lines or xenograft models [63] with target specific intrabodies for different functional studies [62–66]. 4.2. Cell penetrating peptides (CPPs) There is an ongoing debate whether it is possible to efficiently deliver intrabodies on the protein level into living cells. Obviously this would be the most flexible way to introduce intrabodies for numerous applications. However there are still significant technical hurdles, and ongoing efforts in the field lead to successful demonstrations of the first cell-penetrating intrabodies. The most common approach of protein transduction into cells is to use cell-penetrating peptides (CPPs). Since the first discovery of CPPs in 1988 with HIV-1 TAT protein [67,68] numerous natural and synthetic CPPs including derivates like transportan, polyarginines and polylysines [69–72] have been described (reviewed in Heitz et al. [73]). Although there is extensive research on protein transduction for more than twenty years, the precise mechanism of how CPPs enter the cell and facilitate the transport of a cargo across the plasma membrane is still under debate. Basically, the process can be divided into three steps: (i) interaction of CPPs with the cell membrane, (ii) stimulation of cellular uptake by endocytosis, and (iii) escape from endosomes into the cytoplasm (reviewed in van den Berg and Dowdy 2011) [74]. Over the last decade, CPP-based delivery of intrabodies has successfully been used in functional studies of cellular targets involved in cell proliferation, cancer and apoptosis. A purified scFv fragment directed against Bcl-2 and comprising a C-terminal TAT-peptide was successfully transduced into different cell lines including primary basophilic mast

Fig. 2. Schematic outline of the fluorescent 2-hybrid (F2H) assay. (A) The LacI domain of the antigen protein mediates binding to the chromosomally integrated lac operator array, which becomes visible as a fluorescent spot in nuclei of transfected cells. (B) If the differentially labeled VHH (nanobody) interacts with the antigen it becomes enriched at the same spot resulting in co-localization of fluorescent signals at the lac operator (visible as a yellow spot in the overlay image, top). If the VHH does not bind the antigen it remains dispersed in the nucleus, and the lac operator array is only visualized by the antigen (red spot, bottom). Scale bar: 10 μM.

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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cells. It was shown that this cell penetrating intrabody neutralizes Bcl-2 and therefore induces apoptosis [75]. Another cell penetrating scFv was directed against c-Myc and fused to the internalization domain of Antennapedia (penetratin). This construct was shown to interfere with cell proliferation by blocking endogenous c-Myc after transduction in living colon cancer cells (HCT116) [76]. Although cell-penetrating intrabodies have been used in functional assays, most microscopic analyses reveal a characteristic vesicular distribution of the transduced intrabodies within the cytoplasm indicating a limited endosomal escape of transduced intrabodies [77]. Another approach to transduce antibodies is adsorptive-mediated endocytosis. A conversion of surface exposed carboxyl groups into extended primary amino groups, increases the isoelectric point (pI) of an antibody. The “cationized” antibody adsorbs to anionic charges at the cell surface and triggers its cellular uptake [78–81]. Recently, this method has been used to generate cell penetrating sdAbs directed against glial fibrillary acidic protein (GFAP). For microscopic analysis the sdAb was genetically coupled to GFP connected by a linker comprising a set of positively charged amino acids, which resulted in a pI of 9.3. It was shown that only those constructs with a pI of 9.3 and higher were effectively transduced in cultured astrocytoma U373 cells. Moreover, the authors demonstrated that such a fluorescently labeled construct is able to cross the blood brain barrier (BBB) in vivo after injection in mice where it recognizes and binds GFAP within astrocytes [82]. However, the efficiency of the cellular passage of the recombinant binder (7.8% after 60 min) is still limited and further studies are needed to improve the transduction efficiency. Nevertheless, the possibility to generate cell penetrating and BBB-crossing intrabodies is a promising approach to develop new strategies for imaging-based assays and drug development. 5. Functionality of intracellular antibodies Currently most of the target discovery and validation arena is still covered by RNAi and gene editing technologies [83,84]. However, target related factors, such as fast mRNA degradation and slow protein turnover can interfere with the effect of RNAi treatment [85]. Gene-based knockdowns mostly provide a comparison of gene-high versus genelow status, whereas intracellular expressed antibody fragments functionally address their targets on the protein level. Specific binding to relevant epitopes of proteinaceous targets enables the selective influence of discrete protein functions. This mode of action has been successfully shown for intrabodies in different applications. 5.1. Enzyme modulation sdAbs derived from heavy chain antibodies of camelids preferentially bind concave surface structures. Due to their convex-shaped structure, such paratopes are able to insert into surface grooves of target proteins [47]. This structural advantage makes such sdAbs usable to act as enzyme modulators [86]. A first report on immunomodulation of enzymatic activity in living cells using sdAbs was published ten years ago. sdAbs specific for potato starch branching enzyme A (SBE-A) were selected by phage display from a randomized library and expressed in the plastids of potato plants. The enzymatic activity of SBE-A in transgenic potato lines was analyzed and a significant inhibitory effect was detected (b 50%). Moreover, the induced phenotype was stronger compared to RNAi mediated knockdown. Albeit the binding epitopes of the selected sdAbs were not evaluated, interaction close to the active site of the enzyme was presumed [48]. Another example further demonstrated that sdAbs are potent and selective non-competitive enzymatic inhibitors in mammalian cells. sdAbs directed against the proprotein convertase furin expressed in HEK293T cells protect cells from diphtheria-toxin-induced cytotoxicity. The observed cell viability is caused by the inhibition of the furin mediated cleavage of the diphtheria toxin [87].

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5.2. Influencing protein properties and functional knockdowns caused by intrabodies Potential target structures known to influence cellular processes also include domains without intrinsic enzymatic activity. Masking crucial interaction or essential posttranslational modification sites can lead to subsequent modulation of protein properties, functionality or even loss of function. Binding often triggers conformational changes within the involved protein structures. Concerning the latter, sdAbs specific for GFP were selected by phage display and screened for those that can modulate GFP activity in vitro and in living cells. Two sdAbs with slightly overlapping epitopes have been identified, that elicit either a fourfold increase or a fivefold reduction of GFP fluorescence upon binding. Structural analyses revealed that binding of the sdAbs induces subtle opposing changes in the chromophore. Whereas one sdAb stabilizes the negatively charged phenolate state of the chromophore increasing the absorption maxima at 488 nm, binding of the other sdAb minimizes the absorption spectra accordingly. This mechanism was recapitulated in living cells and used for a number of in vivo applications including monitoring the trafficking of GFP-tagged estrogen receptors in HeLa cells [88]. Besides protein modulation, recently, sdAbs have been shown to inhibit protein interaction in vivo. Intracellular sdAbs derived from conventional VHs and VLs, respectively, mask the interaction sites of the tyrosin protein kinase Fyn in Wiskott–Aldrich syndrome protein (WASP) upon transgenic expression in mice resulting in a functional knockdown [34]. Another example shows the targeted inhibition of protein function using sdAbs. Intracellular expressed sdAbs directed against Bax – a proapoptotic protein implicated in cell death – have been observed to prevent the collapse of the mitochondrial membrane after induction of apoptosis in the human neuroblastoma cell line SH-SY5Y. Consequently, cells stably expressing the Bax specific sdAbs were highly resistant to oxidative stress-induced apoptosis [89]. Neutralization of protein function at the post-translational level could be furthermore realized by the retention of proteins in subcellular compartments that are spatially separated from their final site of action. As an example antibody fragments, extended by the ER retention peptide KDEL have been shown to reside inside the ER. Such binding molecules were successfully used to trap target proteins within this compartment and, in consequence, efficiently block the translocation of secreted and cell surface molecules [90,91]. Recently it has been demonstrated that expression of such intrabodies mediates the downregulation of target proteins in living cells. Successfully shown by flow cytometry, ER retained scFvs directed against the p75 neurotrophin receptor (p75NTR) suppress its surface expression in neuron-like rat pheochromocytoma cell line PC12 cells and mouse neuroblastoma × mouse spinal cord hybrid cell line NSC19. As a functional consequence the anti p75NTR ER scFv inhibited the neurite outgrowth after treatment with nerve growth factor (NDF) in vitro [92]. Intrabodies recognizing conformational isoforms of distinct proteins are valuable tools to study protein aggregation in living cells. In this context a scFv-based intrabody slowing protein aggregation in Huntington disease (HD) was initially described by Lecerf et al. in 2001 [93]. HD pathogenesis is phenotypically characterized by intracellular aggregation of mutant Huntingtin (htt) protein. The established scFv has a preference to selectively bind N-terminal mutated forms of htt. Therefore, the scFv enhances the solubility of targeted htt protein and circumvents undesired protein aggregation. A reduction in mutant htt aggregation could be shown in both cell culture and in Drosophila as well as mouse models [94–96]. Recently, bifunctional intrabodies were developed mediating targeted degradation of misfolded htt. Coupling the anti-mutant htt scFv to a proteolytic PEST signal sequence redirects the antigen–antibody complex to the proteasome and enhances degradation of the mutant htt protein [97]. A similar approach was used to deplete GFP-fusion proteins from living cells and organisms. Here, an anti-GFP sdAb was fused to the F-box domain of the Drosophila melanogaster protein Slmb. The genetically

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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engineered chimeric F-box binding protein (Slmb/anti-GFP) completes E3 ubiquitin ligase complexes. Consequently, endogenously expressed GFP proteins are marked for degradation. Regulating the intracellular expression of Slmb/anti-GFP by appropriate promotors, spatiotemporal protein knockouts in Drosophila embryos carrying fluorescently labeled proteins can be induced [98]. 6. Intrabodies for live cell imaging There are multitudes of biochemical and cell-based methods available to study proteins and their impact on cellular processes. For imaging-based analysis fluorescent fusion proteins are the most widespread and popular approach to study localization and dynamic changes of proteins in living cells. These fusion proteins consist of the protein of interest fused to a fluorescent protein, like the green fluorescent protein (GFP) [99,100]. Commercially available GFP-based live cell assays enable the investigation of relevant cellular processes like cell proliferation, apoptosis or DNA-damage. However, this approach is limited to the visualization of artificially introduced fluorescent fusion proteins, whereas the endogenous proteins remain invisible. Importantly, artificial fusion proteins may considerably differ from their endogenous counterparts in terms of expression level, activity, and localization [101,102]. Finally, fluorescent fusions are limited to proteins per se and do not provide information about conformational changes or posttranslational modifications. Intrabodies provide a versatile alternative to fluorescent fusion proteins especially as they can visualize endogenous cellular components. During the last decade an increasing number of intracellular functional binding molecules have been developed and successfully tested for live cell imaging in real time. One of the smallest structures currently used for live cell visualization is a peptide comprising 17 aa derived from the actin-binding protein Abp140, of Saccharomyces cerevisiae. This peptide – called lifeact – fused to the N-terminus of fluorescent proteins stains filamentous actin in fixed cells and tissues but also enables the visualization of actin dynamics without functional interference [103]. Besides tracing dynamic changes of the cytoskeleton on the cellular level, lifeact has been demonstrated to visualize actin filaments in whole organisms including mice [104]. Another non-antibody scaffold for cellular imaging was recently demonstrated by using a fibronectin derived fluorescent intrabody to visualize synaptic proteins. The so called FingRs (fibronectin intrabodies generated with mRNA display) specific for the synaptic structural proteins Gephyrin and PSD95 were selected from a library based on the scaffold of the 10th domain of human fibronectin type III. For visualization FingRs were genetically fused to GFP and transfected into neurons. A remarkable feedback system was implemented to minimize the amount of unbound intrabody. By introducing the transcription repressor KRAB(A) fused to a Zinc-finger domain in the respective expression constructs the transcription of FingRs is negatively controlled by the unbound and freely diffusible FingRs. This significantly reduces excessive background fluorescence of unbound intrabody and enables the visualization of Gephyrin and PSD95 in excitatory and inhibitory synapses in living neurons [55]. Besides alternative binding molecules, antibody fragments have also been used for live cell imaging. In an early study a fluorescently labeled anti-p21Ras scFv was expressed in COS7 cells to study the fate of the intracellular antigen–antibody complex. Microscopic analysis revealed a high tendency for unspecific aggregation of the detectable scFv indicating that the main fraction does not fold properly in the reducing environment of the cytoplasm [105]. One of the first soluble, intracellularly functional and fluorescently labeled scFvs was directed against Giantin (TA10-YFP). After expression in HeLa cells this scFv outlines the characteristic Golgi pattern as observed by classical immunodetection and traces dynamic changes of Giantin after treatment with Brefeldin A (BFA) [106]. Similarly, a scFv (AA2) was selected against the Golgilocalized small GTPase Rab6A. The YFP-tagged AA2 was shown to specifically bind the GTP-bound state of Rab6 and can be used to trace tubulovesicular transport processes of endogenous Rab6 in HeLa cells

in real time [107]. For live cell imaging of components of the microtubule network an α-tubulin specific scFv 2G4-GFP has been shown to track the intracellular dynamics of microtubule formation in fixed and living cells. Derived from a hyperstable human scFv library this intrabody labels its target uniformly along microtubules, differentiates between posttranslational modifications and preferentially binds to tyrosinated α-tubulin [108]. Furthermore a genetically encoded system to track dynamic posttranslational histone modifications in living cells was reported recently. In this approach scFvs were cloned from mouse monoclonal antibodies which are specific for the acetylated histone 3 at lysine 9. The scFvs were fused to the N-terminus of EGFP generating so-called modification-specific intracellular antibodies (mintbodies). It has been shown that mintbodies recognize H3K9 acetylation in hTERT-RPE1 and stable U2OS cells by co-staining with Hoechst. Fluorescent recovery after photobleaching (FRAP) analysis revealed an accelerated recovery of the fluorescent mintbody signal upon inhibition of histone deacetylases. In addition, a transgenic mintbody Drosophila line showed euchromatic banding patterns on polytene chromosomes. Normal development and unaffected fertility of transgenic zebrafish further indicate that mintbodies can be applied for in vivo imaging of H3K9ac in vertebrates [109]. Another conformation-specific intrabody has been developed by Fukata et al. to study palmitoylation-regulated membrane targeting of PSD-95 in neural postsynaptic densities (PSDs). The scFv PF11 was selected by phage display against purified PSD-95-GFP palmitoylated by the palmityl transferase DHHC15. In intracellular studies using HEK293T cells and primary neurons they showed that fluorescently labeled PF11 recognizes PSD-95 exclusively after palmitoylation at the plasma membrane. After treatment with the palmitoylation inhibitor BP-2 they could follow the dynamic depalmitoylation in time lapse analysis. Finally, they performed dualcolor stimulated emission depletion (2C-STED) high resolution microscopy using the PF11-GFP intrabody as well as PSD-95-GFP and proposed a subdomain model for PSD organization, based on an ordered assembly of PSD-95 nanodomains, initiated by local DHHC2 [110]. Recently scFvs have been developed acting as fluorogen activating proteins (FAPs) which are functional for high resolution microscopy in living cells. Szent-Gyorgyi et al. selected scFvs specific for the fluorogens thiazole orange (TO) and malachite green (MG). They showed that upon binding the fluorescence signals of TO and MG are dramatically increased up to 2600 fold and 18,000 fold respectively [111]. Initially used to visualize yeast surface-displayed fluorogen activating scFvs, up to now advanced disulfide free scFvs that are functional within the reducing environment of living cells become available [112]. Such FAPs have been successfully used as genetically targetable far-red fluorescent probes for STED microscopy based on their quantum yields and spectral characteristics which are comparable to ATTO dyes [112]. Modified MG specific FAPs have been genetically fused to actin and transiently expressed in living HeLa cells. Using STED microscopy of living cells it has been demonstrated that those constructs can visualize the cytoskeletal morphology in the nanometer range [113]. These kinds of targetable probes with reduced phototoxicity in the far red combined with STED high resolution microscopy will expand live cell microscopy. Although scFvs represent the most prominent recombinant antibody format and a multitude of scFvs directed against intracellular components are available only a minor proportion of those molecules are currently used for intracellular imaging applications. This might be due to the fact that most of the scFvs have the tendency to aggregate as a result of inefficient folding and disulfide bond formation in the reducing environment of the cytoplasm. Using fluorescently labeled scFvs such aggregates become visible and interfere with downstream microscopic analysis. 7. Chromobody technology As described earlier in this review single domain antibody fragments from camelids provide a versatile and reliable alternative to conventional

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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antibodies. According to their robustness and structural simplicity VHHs can be efficiently selected for functional analysis in living cells. Based on these unique characteristics, a new class of cellular biomarkers has been established by genetically fusing VHHs with fluorescent proteins for visualization of antigens in living mammalian cells. Due to their chimeric structure these recombinant derivatives were termed “chromobodies”. In a first proof-of-concept study a chromobody directed against GFP has been generated, which is diffusely distributed in the absence of the antigen. In a cell-based fluorescence co-localization assay expressing the red fluorescent GFP-chromobody in combination with different GFP-labeled marker proteins (components of the cytoskeleton, the nuclear lamina or the chromatin) a significant overlap of the fluorescence intensities of antigen and chromobody (90%–97%) was observed. Moreover, it has been shown that the GFP-chromobody can trace dynamic changes of chromatin associated proteins (e.g. H2B-GFP) throughout mitosis [42]. Additionally, the GFP-chromobody has been used for functional analysis in plants. Expression of the GFP-chromobody in Nicotiana benthamiana traps GFP-fusion constructs, e.g. the v-SNARE vesicle (GFP-VAMP722) and interferes with the correct localization at the endomembrane compartments. This was shown to alter the phenotype of the plants mediated by trapping the targeted proteins to the cytoplasm [114]. In the following chromobodies against the endogenous proteins cytokeratin-8 and the nuclear lamina have been generated. It was demonstrated, that the green fluorescent lamin-chromobody detects and visualizes the nuclear envelope in living cells. In a more detailed analysis it was shown by FRAP analyses that the lamin-chromobody binds to the nuclear lamina in a very transient manner [42]. However, this binding is sufficient to visualize the nuclear envelope and – in contrast to GFP-laminB1 fusion proteins – neither influences the nuclear morphology nor hampers the progression of the cell cycle [115]. Since the nuclear morphology is a relevant biomarker to trace biological processes like apoptosis, a HeLa cell line stably expressing the lamin-chromobody was established to monitor apoptosis in real time. Using automated image acquisition and computational pattern recognition of the chromobody signal, a detailed temporal analysis on the onset of apoptosis and ratio of apoptotic cells in relation to treatment dosages was performed [116]. With the fluorescent-2 hybrid assay functional chromobodies against components of the replication machinery have been selected and a chromobody specific for the endogenous proliferating cell nuclear antigen (PCNA) was identified. Similar to the lamin-chromobody, the PCNA-chromobody shows only a very low binding affinity (unpublished observations). Transient binding of the PCNA-chromobody seems to minimize functional interference and allows tracing of PCNA throughout different cycle stages (Fig. 3). Following the dynamic formation of

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distinct foci reflecting the sites of DNA replication during S phase, a detailed time lapse analysis of the S phase becomes feasible. In addition, mitosis can be detected by the diffuse, non-bound fraction of the chromobody outlining the cellular area. In contrast to fluorescent fusion proteins (e.g. GFP-PCNA), the expression of the chromobody does not influence the progression or duration of the cell cycle (Fig. 3). Recently, this was highlighted by quantitative live imaging of endogenous DNA replication in human cells. Using a HeLa cell line stably expressing the green fluorescent PCNA-chromobody active DNA replication sites can be observed based on the characteristic speckled chromobody signal. Moreover, the precise duration of early, mid and late S phase could be determined which was not possible with fluorescent fusion protein. Furthermore, it was shown that treatment with DNA damaging reagents results in a reduction in both number and volume of PCNA foci and an elongated S phase. This example highlights the unique advantages of chromobodies for imaging endogenous cellular components [117]. Chromobodies can also address non-endogenous components. To visualize the morphogenesis of HIV in living cells without genetic modification of the virus, a chromobody specifically recognizing the CA-harboring HIV-1 Gag precursor protein has been generated. In combination with live cell and super resolution microscopy the assembly of individual viral particles can be traced from their formation in the cytoplasm to their assembly into virions at the plasma membrane reflecting the different organizational states of HIV-1 Gag molecules during virion morphogenesis [118]. Virion assembly processes, visualized by a CA chromobody are suited for automated image analysis in HCA assay systems and thus provide a new read-out for e.g. targeted antiviral compound screens. 8. Outlook With chromobodies a novel type of fluorescent, antigen-binding proteins is available, which is suitable for targeting and tracing antigens in different subcellular compartments and structures in living cells. With its unique small size and compact structure, the antigen-binding domain of the chromobody supports intracellular stability and minimizes steric hindrances. Immunized, naïve or synthetic libraries provide versatile sources for chromobody generation. By combining conventional screening protocols (e.g. phage display) with new intracellular selection technologies (e.g. the fluorescent 2-hybrid screen) functional chromobodies can easily be identified. The unique ability of chromobodies to detect practically any antigen in living cells without affecting its function makes them ideally suited for high content analyses (HCA) of cellular processes and redistribution assays of endogenous components. Furthermore even specific chromobodies which interfere with distinct protein function or interactions can be selected and applied for functional studies within living cells. This versatile approach

Fig. 3. (A) PCNA-chromobody signal during the cell cycle. Confocal images of time lapse analyses are shown. The chromobody signal starts as a homogeneous distribution throughout the nucleus and cytoplasm. Over time the nucleus begins to appear granular and forms spots, until finally the granularity disappears and the cell divides (indicated by arrows). (B) Following a single cell through a full cell cycle. The texture parameters were used to measure the PCNA-chromobody signal in the nucleus in order to identify cells in S phase (square). Measurement of the cytoplasmic area was used to identify cells rounding up and entering mitosis (triangles).

Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019

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offers new perspectives for target identification, validation and visualization in living cells. We and others are currently developing chromobodies and cellular assays to target and trace morphological markers (including the cytoskeleton), components of signaling pathways, posttranslational modifications and non-protein components. In combination with new disease relevant cellular models, chromobodies will become reliable and important research tools with an impact in the fields of phenotypic screening, epigenetic regulation, DNA repair and signal transduction. Acknowledgement We thank Kourosh Zolghadr for helping us with the figures and Christian Schmees for helpful discussions and critical reading of the manuscript. This work was supported by the Ministry of Science, Research and the Arts of Baden-Würtemberg (V.1.4.-H3-1403-74). References [1] A. Koide, C.W. Bailey, X. Huang, S. Koide, The fibronectin type III domain as a scaffold for novel binding proteins, J. Mol. 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Please cite this article as: P.D. Kaiser, et al., Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.04.019