gpcr-targeting nanobodies attractive research tools, diagnostics, and therapeutics

gpcr-targeting nanobodies attractive research tools, diagnostics, and therapeutics 文库 GPCR-targeting nanobodies attractive research tools, diagnostics, and therapeutics GPCR-targeting nanobodies: attractiveresearch tools, diagnostics, andtherapeuticsAzra Mujic ′-Delic ′*, Raymond H. de Wit*, Folkert Verkaar, and Martine J. SmitAmsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences,VU University Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The NetherlandsG-protein-coupled receptors (GPCRs) represent a majortherapeutic target class. A large proportion of marketeddrugs exert their effect through modulation of GPCRfunction, and GPCRs have been successfully targetedwith small molecules. Yet, the number of small newmolecular entities targeting GPCRs that has been ap-proved as therapeutics in the past decade has beenlimited. With new and improved immunization-relatedtechnologies and advances in GPCR purification andexpression techniques, antibody-based targeting ofGPCRs has gained attention. The serendipitous discov-ery of a unique class of heavy chain antibodies (hcAbs) inthe sera of camelids may provide novel GPCR-directedtherapies. Antigen-binding fragments of hcAbs, alsoreferred to as nanobodies, combine the advantages ofboth small molecules (e.g., molecular cavity binding, lowproduction costs) and monoclonal antibodies (e.g., highaffinity and specificity). Nanobodies are gaining groundas therapeutics and are also starting to find applicationas diagnostics and as high-quality tools in GPCR re-search. Herein, we review recent advances in the useof nanobodies in GPCR research.Towards nanobody-based targeting of GPCRsGPCRs (see Glossary) are seven transmembrane-spanningproteins that detect a vast repertoire of stimuli (rangingfrom light to large glycoproteins) and activate variousintracellular signaling pathways. Upon activation, GPCRsundergo a conformational change that enables the recruit-ment and activation of proteins that relay signals from theplasma membrane to the cell interior. These include sev-eral classes of G proteins that modulate effector proteinsand the scaffolding protein b-arrestin, which has beenimplicated in both termination of G protein-mediatedGPCR signaling as well as initiation of distinct GPCR-dependent signaling cascades [1].GPCR-induced signaling is a virtually universal meansof intercellular communication, and deregulated GPCRsignaling has been implicated in a multitude of diseases.An estimated 30% of marketed drugs exert their effectthrough modulation of GPCR function [2]. Consideringthat only a small proportion of these receptors are targetedby current therapies [3], the GPCR family provides a largeuntapped source of potential therapeutic interventions.Not surprisingly, many pharmaceutical companies haveactive programs aimed at identifying novel GPCR-inter-acting molecules. Low molecular weight molecules haveproven to be very successful as therapeutics. Yet, in thepast decade the number of small new molecular entitiestargeting GPCRs that were approved as therapeutics hasReviewGlossaryAffinity: a biochemical parameter representing the intrinsic ligand–targetbinding strength, generally described by the equilibrium constant (Kd) ofassociation (Kon) and dissociation rates (Koff).Avidity: functional affinity, representing the combined binding strength ofmultiple ligand–target interactions.Complementarity-determining region (CDR): the variable domains of anti-bodies 文库 文库 contain three hypervariable CDR loop regions, which primarily form theantigen-binding paratope.Chemokine receptors: a family of GPCRs that upon activation by chemokinesorchestrate directional migration of immune cells in homeostatic andinflammatory conditions.Clearance: a pharmacokinetic parameter depicting the rate at which asubstance is excreted from the body.Crystallization chaperone: a molecule that binds the crystallization target andincreases the probability of high-quality crystallization throughrestriction of conformation diversity.Epitope: the antigen surface that potentially is recognized by an antibody (fragment).Formatting: the procedure of multimerization to improve the pharmacokineticand/or pharmacodynamic properties of an antibody fragment.G protein-coupled receptors (GPCRs): a superfamily of seven transmembranedomain cell surface which are key modulators of signal transductionand well-established drug receptors, targets.Heavy chain antibody (hcAb): a fully functional antibody devoid of light chainsnaturally expressed in camelids.Nanobody (Nb): an antibody fragment consisting of the recombinant 12–15 kDa VHH domain. Nbs are the smallest functional antibody-based biologicsknown to date.Paratope: the interacting surface of the antibody (fragment) that binds theepitope.Positron emission tomography (PET): an indirect high resolution molecularimaging technique using a radiolabeled tracer.Single photon emission computed tomography (SPECT): a direct molecularimaging technique using a radiolabeled tracer.Tissue penetration: a pharmacokinetic parameter describing the propensity ofa molecule to penetrate deep into the tissue.Valency: the number of paratopes of an antibody (fragment).VHH: the variable domain of a heavy chain antibody, which is fully capable ofbinding an antigen.0165-6147/$ – see front matter? 2014 Published by Elsevier Ltd. : Smit, M.J. (mj.smit@vu.nl).Keywords: GPCRs; nanobodies; VHH; single-domain antibody; signaling.*These authors contributed equally to this work.Trends in Pharmacological Sciences, May 2014, Vol. 35, No. 5 247 been limited. The high attrition rate in preclinical andclinical trials, ascribed to, for example, toxicity, insufficientefficacy, or inadequate selectivity, is leading to an enor-mous increase in drug discovery costs [4]. In the meantime,the implementation of biologics as therapeutics has gainedconsiderable attention (Box 1). Monoclonal antibodies(mAbs) are attractive therapeutics as they are highlyselective and display extended half-lives compared withsmall molecules. With new and improved immunization-related technologies and advances in expression and/orpurification methods of GPCRs, there are ongoing effortsdirected at antibody-based targeting of GPCRs. Recently,mogamulizumab, a mAb targeting the chemokine receptorCCR4, was approved for treatment of adult T cell leukemiain Japan [5]. Currently, several therapeutic mAbs target-ing GPCRs for the treatment of various indications (e.g.,inflammation, oncology) are under investigation in clinicaltrials [3,6,7].Although proven effective for various indications, mAbsare large (150 kDa) heteromultimeric glycoproteins, whichare associated with the inability to target intracellularcomponents and molecular cavities, limited drug adminis-tration routes, and high production costs. Nanobodies(Nbs) represent a potential novel class of antibody-basedtherapeutics with some favorable characteristics. Theyhave already been successfully developed against severaldrug targets. Recently, we identified the first Nbs targetingGPCRs in vivo. Furthermore, the Nb platform has led tosignificant breakthroughs in basic research on GPCRstructure and signaling. In this review, we will focus 文库 文库 onthe exciting progress in the field of GPCR-targeting Nbs.Unique characteristics and therapeutic potential of NbsDiscovery and advantages of NbsIn the early 1990s, Hamers-Casterman et al. discoveredthat camel sera contain a unique class of antibodies devoidof light chains and composed only of heavy chains [8].Besides camels, other closely related members from theBox 1. Conventional mAbs and fragmentsThe immune system contains a diverse repertoire of tools to protectthe host organism against harmful pathogens and xenobiotics.Antibodies, which are glycoprotein complexes secreted by B cells,are one of these tools that provide humoral (i.e., antibody-mediated)immunity by binding and subsequent neutralization of targetmolecules. The most abundantly expressed antibodies are theimmunoglobulin-g (IgG) proteins, which are the origin of conven-tional mAbs. The overall kDa molecules isvery complex, consisting of four peptides made up structure of these 150 from twoidentical heavy (H) and two identical light (L) chains togetherassembling in a tetrameric Y-shaped structure (see Figure 1 in maintext). Both chains of conventional mAbs consist of several differentfunctional domains. Whereas the heavy chains consist of onevariable domain (VH) and three constant domains (CH1–3), the lightchains consist of only two domains, one variable (VL) and oneconstant domain (CL). The constant domains CH1 and CL togetherwith the variable domains comprise the two Fab regions, both ableto bind one antigen each. The bivalent structure of an antibody isbelieved to contribute to increased avidity, thereby conferring highretention times. The remaining part of the antibody complexconsisting of the CH2–CH3 homodimer is termed the Fc region[86]. The specific antigen-binding characteristics of a mAb aredependent on the complementarity-determining (CDRs)encoded by both variable domains (VH and VL), which togetherconstitute the regions variable fragment (Fv). The constant domains in theFc region are not directly involved in binding but are involved inmediating additional immune responses upon target binding.To improve the pharmacokinetics of mAbs, new formats ofconventional antibody-based therapeutics were developed.Through removal of the Fc region and multimerization of theantigen-specific (VH and VL) domains, several types of antibody-based fragments of relatively small size were produced. The 55 kDamonovalent Fab fragments consist, as the name suggests, solely ofone functional Fab region and thus also contain, besides thevariable domains, the CH1 and CL constant domains. This is incontrast to the single chain Fv (scFv) fragments, which are the twovariable domains directly connected by a peptide linker resulting ina 30 kDa size. Furthermore, several modulatory approaches areapplied to these two functional antibody-based building blocks togenerate multivalent constructs [34,87]. With the design of conven-tional IgG-based antibody fragments, tissue penetration is in-creased, whereas immunogenicity is decreased; however, thiscomes with the cost of increased blood clearance [88].Conven?onal an?body150 kDaHeavy chain an?body90 kDaFab fragment50 kDaVHH/Nanobody15 kDaCH2CH3CH2VHHVHCH1CLVL,15 nm,2.5 nmCH3TRENDS in Pharmacological Sciences Figure 1. Structural features of different antibodies. Conventional g-immunoglobulins consist of two heavy chains, each containing three invariant (constant) domains(CH1–CH3) and a single variable region (VH), and two light chains composed of a constant region (CL) and a variable region (VL). The VH and VL regions together form theparatope, and it is this antigen-recognizing portion of the antibody that is retained in Fab fragments. By contrast, camelid heavy chain antibodies are composed of ahomodimer composed of two heavy chains containing two constant regions (i.e., they are lacking the CH1 domain) and a single variable (VHH) domain harboring threecomplementarity determining regions (rectangles). Nanobodies are the smallest functional single chain 文库 文库 antigen-recognizing polypeptides known to date, with a molecularweight of 12–15 kDa and 1.5–2.5 nm dimensions.ReviewTrends in Pharmacological Sciences May 2014, Vol. 35, No. 5248 Camelidae family (e.g., llamas and alpacas) naturally ex-press the same type of 90 kDa heavy chain antibodies(hcAbs) (Figure 1). Interestingly, another class of evolu-tionary distinct hcAbs (immunoglobulin new antigen re-ceptor, Ig-NAR) was discovered in cartilaginous fish [9–11].The antigen-binding properties of hcAbs are solelydependent on a single 12–15 kDa variable domain of theheavy chain antibody (VHH) (Figure 1 and Box 2). Therecombinant equivalents of these VHH domains, termedNbs, retain full antigen-binding characteristics. The bio-chemical properties of Nbs provide advantages over conven-tional antibody-based molecules, thus facilitating manybiotechnological and therapeutic applications. In particular,their small size, single domain nature, and hydrophiliccharacteristics contribute greatly to the excellent biochemi-cal and pharmaceutical properties of Nbs, which production, affinity, and formatting.The straightforward and includestability, cost-effective production of Nbsis an important advantage compared with the high produc-tion costs of mAb-based biologics. Although microbial pro-duction of conventional antibody-based fragments can beachieved, their multidomain structure, glycosylation, andhydrophobic characteristics result in low yields and forma-tion of aggregates. Fortunately, the production of Nbs isstraightforward. The monomeric nature, absence of post-translational modifications and high aqueous solubilitycontribute to the cost-effective production of Nbs in a varietyof cell expression systems of bacterial, yeast, plant, ormammalian origin [12]. Nbs have low propensity for aggre-gation and are highly stable [13–17]. They retain full anti-gen-binding capacity at harsh thermal (60–808C) andchemical (2–3 M guanidinium chloride) denaturing condi-tions and furthermore display high refolding capability[16,17]. Moreover, the small proteins can be resistant toproteolytic degradation in the gastrointestinal tract, whichpotentially permits effective oral administration of Nbs [18].Major strengths of antibody-based biologics, includingNbs, are their high affinity and specificity for their target.The affinity of Nbs is in the nanomolar–picomolar range,and as such they are highly suited for research, diagnostic,and therapeutic purposes [14,19–21]. The antigen-inter-acting surface of a conventional antibody has a flat orconcave (i.e., hollow) shape, whereas Nbs can adopt a moreflexible and often convex (i.e., protruding) conformation.Overall, the unique flexible paratope structure and smallsize enables Nbs to bind molecular clefts and cavities, forinstance, receptor-binding pockets or enzyme active sites.This is supported by X-ray crystallography studies of Nb-bound lysozyme or epidermal growth factor receptor(EGFR), revealing buried epitopes that are solely targetedby Nbs and not by other antibody-based molecules [21,22].The small size also has a major impact on clearance.Because the Nb size is below the renal cut-off size of 50–60 kDa, the proteins are rapidly cleared from the body. Onaverage, the half-life of a Nb does not exceed 2 h, which iscomparable to other antibody fragments, although a lotshorter than mAbs. A short half-life is ideal for a number ofpurposes, including stem cell mobilization, toxin delivery,and noninvasive in vivo imaging [23–25]. However, forchronic treatment, prolonged presence of Nbs is desired.This can be achieved by formatting the monomeric Nbs,generating multimeric constructs. Half-life extensionmethods used for conventional antibody fragments, suchas PEGylation or fusion to Nbs targeting serum albumincan be used to tailor the half-life of Nbs from <2 h toseveral weeks [26–29]. This broadens their applicabilityas therapeutics for both acute and chronic treatments.The Nb platform allows a plug-and-play 文库 文库 approach withmultiple possibilities not only to tailor half-life but also toincrease avidity and to create multispecific Nbs. Throughcoupling of two identical (bivalent) or distinct Nbs thatrecognize different epitopes on the same antigen (bipar-atopic) spectacular, up to 4000-fold, increases in avidityand potency compared with their monovalent counterpartsare observed [19,20,27,30].Diagnostics and therapeutic potential of NbsNbs are highly suitable as diagnostic tools and have beenevaluated as biosensors and in vivo imaging tracers. Immu-noimaging is a valuable tool for noninvasive visualization ofbiomarkers in vivo. Currently, radiolabeled biomarker-spe-cific mAbs are used in the clinic to image tumors and forpatient stratification. With their high affinity and specifici-ty, proper tissue penetration and rapid clearance [26,31],Nbs provide advantages over mAbs and meet most char-acteristics of an ideal noninvasive molecular imaging probe[32–34]. An array of Nbs targeting the human epidermalBox 2. Structural properties of hcAbs and NbsIn addition to conventional antibodies, camelids naturally expresshcAbs. The camelid hcAb is a homodimer composed of H chainsthat individually fold in three domains, an N terminal variabledomain (VHH) and two constant domains (CH2 and CH3) (seeFigure 1 in main text). Thus, these distinctive antibodies lack the CH1domain, which is present in conventional antibodies. Because theCH1 domain is involved with L chain interactions, it is believed thatloss of this domain impairs L chain coupling. Owing to the lack oflight chains, the antigen-binding properties of these distinctiveimmunoglobulins are dependent on a single 12–15 kDa variabledomain of the heavy chain antibody (VHH) (see Figure 1 in maintext). As a result of the simple structural nature of these VHHs, therecombinant equivalents of these natural domains, termed Nbs,retain full antigen-binding characteristics. Similar to a human VHdomain, the camelid VHH domain is organized in three hypervari-able complementarity-determining regions (CDR1–3) separated byfour more conserved framework regions (FR1–4), folding in four-and five-stranded antiparallel b-sheets. The hypervariable CDRs arelocated within the loops of this structure and cluster at one side ofthe domain, hence forming a paratope [15,89]. Although the fold ofa VHH domain largely overlaps with VH, there are some distinctivedifferences in CDRs and FRs, both contributing to the uniqueproperties of the VHH domain. To support dimerization with VL, theFR2 of VH contains a number of conserved hydrophobic residues(Val37, Gly44, Leu45, and Trp47 according to Kabat numbering). Ahallmark of VHHs is that these residues are often substituted tomore hydrophilic or smaller residues, thus abrogating VL interactionand resulting in a strict monomeric antigen-binding state andincreased solubility of the domain [14,15,90]. Other critical differ-ences between VH and VHH lie within the CDRs. For example, the Nterminal region of CDR1 tends to be more variable in VHH resultingin more conformational diversity of this loop [10]. Moreover,because CDRs of the VHH are not restricted to pair with anotherdomain form a paratope, this allows the variable regions of VHHs toadopt a more flexible and often convex (i.e., protruding) conforma-tion. Overall, the unique flexible paratope structure and small sizeenables VHHs (and Nbs as their recombinant equivalents) to bindmolecular clefts and cavities [21,22].ReviewTrends in Pharmacological Sciences May 2014, Vol. 35, No. 5249 growth factor receptors 1 and 2 (HER1/HER2) [31,35,36],the macrophage mannose receptor [37], and the vascular celladhesion molecule 1 (VCAM1) [38] have been successfullylabeled with radionuclides for positron emission tomogra-phy (PET) and/or single photon emission computed tomog-raphy (SPECT) imaging purposes, to image tumors,inflammation, and atherosclerotic lesions, respectively.The preclinical results indicate that high signal-to-noiseratio 文库 文库 images are obtained within a few hours after adminis-tration [31,38]. These results demonstrate the potential ofNb-linked tracers as a new class of diagnostic imaging tools.In addition, Nbs have been described that are able totraverse the blood–brain barrier (BBB) [39–41]. This is avery promising characteristic, because targeted transport ofmolecules across the BBB remains a major challenge inmodern drug and diagnostics development. By coupling ofBBB crossing Nbs to therapeutics or imaging probes, awindow of opportunity is opened for brain tissue-targeteddrug delivery and neuroimaging.Although no Nbs have found their way to the marketyet, various humanized and sequence optimized Nbs haveentered the clinical phases of drug research. Most ad-vanced are Nbs targeting tumor necrosis factor (TNF)a[27] and interleukin-6 receptor (IL-6R) [42] for the treat-ment of rheumatoid -6R display excellent efficacy in in vitro models arthritis (RA). Nbs against TNFa andIL andmouse models of RA and have successfully progressed intoPhase IIb clinical testing. Nbs generated and characterizedpreclinically targeting receptor tyrosine kinases such asEGFR [43], hepatocyte growth factor receptor (HGFR/c-Met) [28], and vascular endothelial growth factor receptor2 (VEGFR2) [44] represent promising therapeutic candi-dates for the treatment of cancer.Owing to the high sequence similarity of the camelidVHH and the human VH framework regions, it is unlikelythat Nbs will be highly immunogenic [14,15], which issupported by recent findings showing no immunogenicityin murine and human studies [15,27,45]. Additionally, tofurther reduce risk of immunogenicity, Vincke et al. for-mulated a general strategy for Nb humanization [46].Hence, it seems unlikely that immunogenicity will posea major concern in the development of Nbs for diagnostic ortherapeutic applications.GPCR-targeting nanobodiesModulation of GPCR function by NbsThe first Nbs targeting GPCRs, namely chemokine recep-tors and their ligands, modulating GPCR function wererecently described [19,20]. Chemokine receptors areexpressed on immune cells and their main function is toguide leukocytes towards chemokine gradients upon in-flammation [47]. This receptor family is implicated in(A)(B)(C)NbsSignaling-deficientchemokinemutantsGAG-binding-deficientchemokine mutantsMAbsSmall moleculesChemokine receptorPlasma membraneGαiChemokineGlycosaminoglycan (GAG)HeparinMAbsSmall moleculesNbsHNHNNNNHNHNHHNTRENDS in Pharmacological Sciences Figure 2. Potential therapeutic strategies for targeting of the chemokine system. (A) Much effort from both industrial and academic laboratories has centered around thedevelopment of antibody- and small molecule-based inhibition of chemokine receptors [6,47]. Additionally, several nanobodies have been described that inhibit chemokinebinding to the CXCR4 and CXCR7 chemokine receptors [19,20]. Lastly, chemokine binding can be competitively inhibited by N terminal truncation mutants of chemokinesthat have retained chemokine receptor binding but are deficient in receptor activation [91]. (B) Although chemokine-binding antibodies and nanobodies form the mainstayof potential therapeutic options targeting chemokines [6,59], there are also examples of small molecules that bind CXCL12 and prevent binding to its cognate receptorCXCR4 [92,93]. (C) Finally, chemokine function can be modulated through disruption of chemokine gradient formation. Such gradients are pivotal for the directed migrationof leukocytes and require the interaction of chemokines to cell-associated glycosaminoglycans (GAGs) [94]. Chemokine gradient formation can be altered by heparin (ahighly sulfated soluble GAG subtype) [95] and by chemokine mutants that are defective in GAG binding, but can still bind and activate chemokine receptors [96–98].ReviewTrends in Pharmacological Sciences May 2014, Vol. 35, No. 5250 文库 文库 several pathologies, such as inflammatory diseases, can-cer, and HIV [48–50]. Targeting of these receptors andtheir ligands has been intensively investigated in the pastyears (Figure 2), leading to the FDA approval of two smallmolecule drugs specifically targeting this receptor family.Maraviroc, a CCR5 antagonist, is successfully used forseveral years now in the treatment of CCR5 tropic HIV-1 infection, and Plerixafor (AMD3100), a CXCR4 antago-nist, has recently been approved for hematopoietic stemcell mobilization [51,52].One of the most-investigated chemokine–chemokinereceptor pairs is the CXCL12/CXCR4 axis, because of itsrole in cancer and HIV entry [53,54]. Using a time-efficientwhole cell immunization of llamas with intact CXCR4-expressing HEK293T cells, followed by phage displayand counterselection, Nbs against CXCR4 were identified[19]. The reported CXCR4–Nbs bind with high affinity andspecificity to the extracellular loop 2 (ECL2) and potentlyinhibit CXCL12-induced signaling (Figure 3A). Biparatopic CXCR4–Nbs, which have distinctand partially overlapping epitopes, show enhanced affini-ties and potencies (Figure 3A). Interestingly, these bipar-atopic Nbs act as inverse agonists on CXCR4, whereasAMD3100 acts a neutral antagonist [19]. Because CXCR4is overexpressed in a large number of tumors [55], which isand chemotaxisoften associated with increased levels of basal activity, theuse of inverse agonistic CXCR4 biparatopic Nbs could bebeneficial. The two identified Nbs targeting CXCR4 arehighly selective for human but not murine CXCR4, indi-cating that minor changes (five additional amino acids) inECL2 are sufficient to affect Nb affinity for CXCR4. Thisunderlines the exquisite selectivity that can be achieved byNbs. Of therapeutic interest, CXCR4–Nbs were demon-strated to inhibit HIV-1 virus replication in vitro as well asto dose-dependently induce stem cell mobilization in cyno-molgus monkeys, which underscores their therapeutic po-tential (Figure 3B) [19].Similar to CXCR4, CXCR7 binds CXCL12 and plays animportant role in proliferation, angiogenesis, and metas-tasis [56]. However, CXCR7 is an atypical chemokinereceptor as it does not signal through G proteins butspecifically activates signaling pathways through recruit-ment of b-arrestin [57,58]. Specific Nbs binding to thisreceptor were recently developed and characterized [20].CXCR7–Nbs display high affinity and antagonistic proper-ties, as they are able to inhibit CXCL12-induced recruit-ment of b-arrestin 2 to CXCR7 [20]. Moreover, CXCR7–Nbs were shown to inhibit head and neck cancer tumorgrowth in a mouse xenograft model (Figure 3C) throughinhibition of angiogenesis [20]. The use of CXCR7–Nbs(A)120250200150100500020406080100120–10–10–9–8–7–601020304050*100806040 200100Key:Key:Key:Key:8060402000 6AMD3100Biparatopic CXCR4 NbMonovalent CXCR4 Nb 1Monovalent CXCR4 Nb 2Biparatopic CXCR4 Nb (1+2)PBSBiparatopic CXCR7 NbMonovalent CXCL12 Nb121824–12 –11 –10–9log [Nb]log [Nb]Treatment dura?on (days)Tumor size (mm3)Migrated cells (%)Time post–injec?on (h)–8–7–6Specific 125I–CXCL12 binding(%)CD34+ cells/ μl blood(B)(C) (D)TRENDS in Pharmacological Sciences Figure 3. Nanobodies targeting the chemokine system effectively inhibit chemokine function in vitro and in vivo. (A) A biparatopic nanobody (Nb) targeting the CXCR4chemokine receptor displays increased binding affinity in a CXCL12 radioligand displacement assay compared with the individual monovalent Nbs from which it iscomposed. Adapted from [19]. (B) The biparatopic CXCR4-targeting Nb induces the mobilization of CD34-positive stem cells in the blood of macaque monkeys to a similarextent as the small molecule CXCR4 antagonist AMD3100. Adapted from [19]. (C) A biparatopic CXCR7-targeting Nb inhibits growth in mice xenografted with the head-and-neck cancer cell line UM-SCC-22A. Adapted from [20]. (D) A CXCL12-binding Nb 文库 文库 concentration-dependently inhibits CXCL12-induced chemotaxis of murine pre-B L1.2 cellsin a transwell migration assay. Adapted from [59].ReviewTrends in Pharmacological Sciences May 2014, Vol. 35, No. 5251 might thus be of therapeutic interest in this cancer type,but also other cancers showing high expression of CXCR7might be effectively targeted by CXCR7–Nbs.Finally, generation of Nbs targeting chemokines havealso been recently reported [59]. Inflammatory chemokinesCCL2 (binding to CCR2), CCL5 (binding to CCR1, CCR3,and CCR5), and CXCL11 (binding to CXCR3 and CXCR7),as well as the homeostatic chemokine CXCL12 (binding toCXCR4 and CXCR7), were successfully targeted by che-mokine-specific Nbs. All these Nbs inhibit chemokine bind-ing their cognate chemokine receptor (Figure 2).Furthermore, the CXCL11- and CXCL12-specific to Nbs in-hibit Gaisignaling and chemotaxis through CXCR3 andCXCR4, respectively (Figure 3D). All chemokine–chemo-kine receptor pairs mentioned above have been implicatedin cancer [49,60,61]. Additionally, inflammatory diseasessuch as RA [62,63] and multiple sclerosis (MS) [64,65] aredependent on CXCL11–CXCR3- and CCL2–CCR2-inducedcellular signaling. The chemokine-targeting Nbs mightprove therapeutically beneficial for these indications.Collectively, Nbs against CXCR4 and CXCR7, as well asagainst a panel of chemokines, show great promise astherapeutics and research tools to further characterizethe chemokine receptor system.GPCR-targeting Nbs as research toolsAlthough Nbs have received a great deal of attention for theirtherapeutic potential, their use as biomolecular tools in basicGPCR research is also gaining momentum. One applicationof Nbs in biomolecular research is their use as crystallizationchaperones. A major hurdle for successful crystallization ofGPCRs has been their hydrophobic and thermolabile nature.Through in-frame insertion of hydrophilic crystallizationchaperones (such as T4 lysozyme), addition of high-affinityinverse agonists, or antagonists, with long residence times orthrough mutations that increase thermal stability, severalstructures of GPCRs in their inactive state have now beenpublished [66–69]. Crystallization of the agonist-bound ac-tive state of a GPCR has provided an even bigger challenge,because this receptor conformation appears to be intrinsi-cally less stable than the inactive state [70]. Crystallizationof such energetically unfavorable protein conformations hasbeen achieved through co-crystallization with antibodies,antibody fragments, or Nbs [71]. Indeed, co-crystallizationof the b2 adrenergic receptor (b2AR) and more recently theM2 muscarinic acetylcholine receptor (M2R) with Nbs thatspecifically recognize the active receptor conformation led tothe unveiling of the first active state GPCR structures[72,73]. These GPCR-targeting Nbs act as so-called ‘G pro-tein mimetics’, enhancing agonist affinities indicating thatthey stabilize active states of the receptor. In addition, a Nbdirected against the Gasheterotrimer was used to stabilize aco-crystal composed of the active b2AR in complex with its Gprotein [74].Another important asset of GPCR-targeting Nbs inGPCR research is their use as conformation-specific biosen-sors. Fluorescently tagged Nbs provide a means of trackingproteins in live cells with unprecedented resolution [75,76].Recent efforts [77] harnessed this knowledge to study thespatial localization of b2AR signaling using Nbs that recog-nized activated b2AR [72] and nucleotide-free (active) Gas[78], respectively. By genetically fusing these Nbs to GFPand expressing them in b2AR-expressing cells, followed bymicroscopic examination of GFP localization followingb2AR activation, direct evidence for G protein activationfrom internalized GPCRs was provided [77], a phenomenonthat has been suggested to occur for multiple GPCRs butwas never directly 文库 文库 ascertained [79–81].In addition, these intracellularly expressed Nbs, alsotermed intrabodies, can also be utilized to modulate recep-tor-dependent signaling. A recent study that con-formational-specific b2AR intrabodies inhibit downstreamsignal showed transduction, including G protein activation, Gprotein-coupled receptor kinase (GRK)-mediated receptorphosphorylation, b-arrestin recruitment, and receptor in-ternalization [82]. These findings illustrate the potential ofNbs as research tools to study receptor-specific GPCRsignaling.Future perspectivesConformation-selective NbsConformation-selective Nbs, that target the intracellulardomain of GPCRs, recognize and stabilize conformation-specific states, such as the active state of b2AR and M2R[72,73]. Nbs that target the extracellular domain of GPCRsand effectively inhibit GPCR function bind to the ECLepitopes, in particular ECL2 [19,20], indicating that theseNbs also recognize conformation-dependent structures.Immunization of camelids with intact cells or membranesoverexpressing GPCRs allow recognition of GPCR extra-cellular domains in their native conformation with appro-priate post-translational modifications. particular interest in view of the emerging concept thatone can selectively modulate GPCR-induced signalingpathways by means of biased ligands [1]. By binding todistinct GPCR conformations, biased ligands may selec-tively modulate downstream signaling pathways. Biasedligands may prove to be superior as therapeutics, minimiz-ing activation of pathways that cause adverse side effectsin some diseases [1]. To obtain biased conformation-specificNbs, one can use genetically engineered GPCRs favoring(in)activation of a given signal transduction pathway [e.g.,constitutively (in)active GPCRs, stabilized receptors inspecific state (StaRs)] overexpressed in cells or viral par-ticles, or purified GPCRs (e.g., in nanodiscs) in the presenceof high-affinity co... 文库