Since their discovery just over 25 years ago, the single variable domain from heavy-chain-only antibodies plays a role in an increasing number of antibody-based applications. Structural and biophysical studies have revealed that the small, ∼15 kDa, single variable domain found in camelids displays versatility in target recognition. Such insight has served as the foundation to develop and engineer VHH domains with enhanced properties capable of targeting a range of therapeutically relevant protein antigens or low-molecular weight haptens. Furthermore, the modular nature of VHH domains allows them to be introduced into constructs that are simply not possible with conventional antibodies. Here, we review the structural and biophysical properties of VHH domains, highlight recent VHH-based therapeutics and diagnostics, and provide insight into VHH engineering that may pave the way to next-generation single domain antibody applications.
Impact statement
The development of novel antibody formats, beyond conventional antibodies, opens new possibilities in medical therapies, diagnostics, and general life science applications requiring affinity reagents. The camelid VHH domain, from heavy-chain-only antibodies, has emerged as a jack-of-all-trades module for novel affinity reagents. Applications include targeted cancer therapies, novel antimicrobial agents, conformation specific reagents, and tailor-made molecular switch entities. The breadth of unique uses for the VHH will continue to grow, opening new opportunities to treat and understand disease.
GriloALMantalarisA.The increasingly human and profitable monoclonal antibody market. Trends Biotechnol2019;
37:9–16
2.
Hamers-CastermanCAtarhouchTMuyldermansSRobinsonGHamersCSongaEBBendahmanNHamersR.Naturally occurring antibodies devoid of light chains. Nature1993;
363:446–8
3.
MuyldermansSCambillauCWynsL.Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci2001;
26:230–5
4.
MuyldermansS.Single domain camel antibodies: current status. J Biotechnol2001;
74:277–302
5.
GreenbergASAvilaDHughesMHughesAMcKinneyECFlajnikMF.A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature1995;
374:168–73
6.
FlajnikMFDeschachtNMuyldermansS.A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels?PLoS Biol2011;
9:e1001120
7.
ULC CCG.
Molecular Operating Environment (MOE). 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A2R7: Chemical Computing Group ULC, 2019
8.
NguyenVKMuyldermansSHamersR.The specific variable domain of camel heavy-chain antibodies is encoded in the germline. J Mol Biol1998;
275:413–8
9.
SpinelliSTegoniMFrenkenLvan VlietCCambillauC.Lateral recognition of a dye hapten by a llama VHH domain. J Mol Biol2001;
311:123–9
10.
MuyldermansSAtarhouchTSaldanhaJBarbosaJAHamersR.Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng Des Sel1994;
7:1129–35
11.
WuTTJohnsonGKabatEA.Length distribution of CDRH3 in antibodies. Proteins1993;
16:1–7
12.
LiangZWangTSunYYangWLiuZFeiJGuoYMaQPanQRenL.A comprehensive analysis of immunoglobulin heavy chain genes in the Bactrian camel (Camelus bactrianus). Front Agr Sci Eng2015;
2:249–59
13.
DecanniereKDesmyterALauwereysMGhahroudiMAMuyldermansSWynsL.A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops. Structure1999;
7:361–70
14.
DesmyterASpinelliSPayanFLauwereysMWynsLMuyldermansSCambillauC.Three camelid VHH domains in complex with porcine pancreatic alpha-amylase – inhibition and versatility of binding topology. J Biol Chem2002;
277:23645–50
15.
DesmyterATransueTRGhahroudiMAThiMHPoortmansFHamersRMuyldermansSWynsL.Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat Struct Biol1996;
3:803–11
16.
DeLanoWL.The PyMOL molecular graphics system.
San Carlos, CA, USA:
DeLano Scientific, 2002
17.
ConrathKELauwereysMGalleniMMatagneAFrereJMKinneJWynsLMuyldermansS.Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob Agents Chemother2001;
45:2807–12
18.
RasmussenSGFChoiH-JFungJJPardonECasarosaPChaePSDeVreeBTRosenbaumDMThianFSKobilkaTSSchnappAKonetzkiISunaharaRKGellmanSHPautschASteyaertJWeisWIKobilkaBK.Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature2011;
469:175
19.
KoideATereshkoVUysalSMargalefKKossiakoffAAKoideS.Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar K(D) of a single-domain antibody with a flat paratope. J Mol Biol2007;
373:941–53
20.
KingMTHuhIShenaiABrooksTMBrooksCL.Structural basis of VHH-mediated neutralization of the food-borne pathogen Listeria monocytogenes. J Biol Chem2018;
293:13626–35
21.
KubalaMHKovtunOAlexandrovKCollinsBM.Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci2010;
19:2389–401
22.
De GenstESilenceKDecanniereKConrathKLorisRKinneJMuyldermansSWynsL.Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci USA2006;
103:4586–91
23.
KirchhoferAHelmaJSchmidthalsKFrauerCCuiSKarcherAPellisMMuyldermansSCasas-DelucchiCSCardosoMCLeonhardtHHopfnerKPRothbauerU.Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol2010;
17:133–8
24.
MitchellLSColwellLJ.Comparative analysis of nanobody sequence and structure data. Proteins2018;
86:697–706
25.
FrancoEJSonnesonGJDeLeggeTJHofstetterHHornJRHofstetterO.Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography. J Chromatograph B2010;
878:177–86
26.
GoldmanERLiuJLZabetakisDAndersonGP.Enhancing stability of camelid and shark single domain antibodies: an overview. Front Immunol2017;
8:865
27.
van der LindenRHFrenkenLGde GeusBHarmsenMMRuulsRCStokWde RonLWilsonSDavisPVerripsCT.Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta1999;
1431:37–46
28.
LadensonRCCrimminsDLLandtYLadensonJH.Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem2006;
78:4501–8
29.
KunzPZinnerKMückeNBartoschikTMuyldermansSHoheiselJD.The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep2018;
8:7934
30.
SteelandSVandenbrouckeRELibertC.Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today2016;
21:1076–113
31.
De VliegerDBallegeerMRosseyISchepensBSaelensX.Single-domain antibodies and their formatting to combat viral infections. Antibodies2018;
8:1
32.
KlarenbeekAEl MazouariKDesmyterABlanchetotCHultbergAde JongeNRooversRCCambillauCSpinelliSDel-FaveroJVerripsTde HaardHJAchourI.Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform. MAbs2015;
7:693–706
33.
SnoeckV.Current experience in immunogenicity assessment of next generation biologics nanobodies. In: European immunogenicity symposium, München, Germany, February 2013
34.
VinckeCLorisRSaerensDMartinez-RodriguezSMuyldermansSConrathK.General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem2009;
284:3273–84
35.
HarmsenMMVan SoltCBFijtenHPVan SettenMC.Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins. Vaccine2005;
23:4926–34
36.
HoefmanSOttevaereIBaumeisterJSargentini-MaierML.Pre-clinical intravenous serum pharmacokinetics of albumin binding and non-half-life extended nanobodies®. Antibodies2015;
4:141
37.
BeirnaertEDesmyterASpinelliSLauwereysMAardenLDreierTLorisRSilenceKPolletCCambillauCde HaardH.Bivalent llama single-domain antibody fragments against tumor necrosis factor have picomolar potencies due to intramolecular interactions. Front Immunol2017;
8:867
38.
DingLTianCFengSFidaGZhangCMaYAiGAchilefuSGuY.Small sized EGFR1 and HER2 specific bifunctional antibody for targeted cancer therapy. Theranostics2015;
5:378–98
39.
van WitteloostuijnSBPedersenSLJensenKJ.Half-Life extension of biopharmaceuticals using chemical methods: alternatives to PEGylation. Chem Med Chem2016;
11:2474–95
40.
WuYJiangSYingT.Single-Domain antibodies as therapeutics against human viral diseases. Front Immunol2017;
8:1802
41.
JamnaniFRRahbarizadehFShokrgozarMAMahboudiFAhmadvandDSharifzadehZParhamifarLMoghimiSM.T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: towards tumor-directed oligoclonal T cell therapy. Biochim Biophys Acta2014;
1840:378–86
42.
De MunterSIngelsJGoetgelukGBonteSPilleMWeeningKKerreTAbkenHVandekerckhoveB.Nanobody based dual specific CARs. Int J Mol Sci2018;
19:pii:E403
43.
CardosoFMIbanezLIVan den HoeckeSDe BaetsSSmetARooseKSchepensBDescampsFJFiersWMuyldermansSDepickerASaelensX.Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge. J Virol2014;
88:8278–96
44.
RosseyIGilmanMSAKabecheSCSedeynKWrappDKanekiyoMChenMMasVSpitaelsJMeleroJAGrahamBSSchepensBMcLellanJSSaelensX.Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state. Nat Commun2017;
8:14158
45.
LaursenNSFriesenRHEZhuXJongeneelenMBloklandSVermondJvan EijgenATangCvan DiepenHObmolovaGvan der Neut KolfschotenMZuijdgeestDStraetemansRHoffmanRMBNieusmaTPallesenJTurnerHLBernardSMWardABLuoJPoonLLMTretiakovaAPWilsonJMLimberisMPVogelsRBrandenburgBKolkmanJAWilsonIA.Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin. Science2018;
362:598–602
46.
HarmsenMMvan SoltCBvan Zijderveld-van BemmelAMNiewoldTAvan ZijderveldFG.Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol2006;
72:544–51
47.
HussackGRiaziARyanSvan FaassenHMacKenzieRTanhaJArbabi-GhahroudiM.Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility. Protein Eng Des Sel2014;
27:191–8
48.
CroweJSRobertsKJCarltonTMMaggioreLCubittMFClareSHarcourtKRecklessJMacDonaldTTRayKPVossenkämperAWestMR.Preclinical development of a novel, orally-administered anti-tumour necrosis factor domain antibody for the treatment of inflammatory bowel disease. Sci Rep2018;
8:4941
49.
FanningSWHornJR.An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop. Protein Sci2011;
20:1196–207
50.
SpinelliSFrenkenLGJHermansPVerripsTBrownKTegoniMCambillauC.Camelid heavy-chain variable domains provide efficient combining sites to haptens. Biochemistry2000;
39:1217–22
51.
SonnesonGJHornJR.Hapten-Induced dimerization of a Single-Domain VHH camelid antibody. Biochemistry2009;
48:6693–5
52.
LesneJChangH-JDe VischAPaloniMBarthePGuichouJ-FMayonovePBarducciALabesseGBonnetJCohen-GonsaudM.Structural basis for chemically-induced homodimerization of a single domain antibody. Sci Rep2019;
9:1840
53.
Alvarez-RuedaNBeharGFerreVPugniereMRoquetFGastinelLJacquotCAubryJBatyDBarbetJBirkleS.Generation of llama single-domain antibodies against methotrexate, a prototypical hapten. Mol Immunol2007;
44:1680–90
54.
Tabares-da RosaSWogulisLAWogulisMDGonzalez-SapienzaGWilsonDK.Structure and specificity of several triclocarban-binding single domain camelid antibody fragments. J Mol Recognit2019;
32:e2755
55.
DingLWangZZhongPJiangHZhaoZZhangYRenZDingY.Structural insights into the mechanism of single domain VHH antibody binding to cortisol. FEBS Lett2019;
593:1248–56
56.
SaerensDPellisMLorisRPardonEDumoulinMMatagneAWynsLMuyldermansSConrathK.Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol2005;
352:597–607
57.
WagnerHJWehrleSWeissECavallariMWeberW.A Two-Step approach for the design and generation of nanobodies. Int J Mol Sci2018;
19:pii:E3444
58.
HagiharaYMineSUegakiK.Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region. J Biol Chem2007;
282:36489–95
59.
AndersonGPLiuJHZabetakisDLiuJLGoldmanER.Thermal stabilization of anti-alpha-cobratoxin single domain antibodies. Toxicon2017;
129:68–73
60.
ShahiBMousavi GargariSLRasooliIRajabi BazlMHoseinpoorR.Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library. J Appl Microbiol2014;
117:528–36
61.
FreiJCLaiJR.Protein and antibody engineering by phage display. Meth Enzymol2016;
580:45–87
62.
TabasinezhadMTalebkhanYWenzelWRahimiHOmidiniaEMahboudiF.Trends in therapeutic antibody affinity maturation: from in-vitro towards next-generation sequencing approaches. Immunol Lett2019;
212:106–13
63.
LimCCChoongYSLimTS.Cognizance of molecular methods for the generation of mutagenic phage display antibody libraries for affinity maturation. Int J Mol Sci2019;
20:1861
64.
TillerKEChowdhuryRLiTLudwigSDSenSMaranasCDTessierPM.Facile affinity maturation of antibody variable domains using natural diversity mutagenesis. Front Immunol2017;
8:986
65.
ChengXWangJKangGHuMYuanBZhangYHuangH.Homology Modeling-Based in silico affinity maturation improves the affinity of a nanobody. Int J Mol Sci2019;
20:4187
66.
RizkSSPaduchMHeithausJHDuguidEMSandstromAKossiakoffAA.Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins. Nat Struct Mol Biol2011;
18:437–42
67.
MukherjeeSGriffinDHHornJRRizkSSNocula-LugowskaMMalmqvistMKimSSKossiakoffAA.Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins. J Biol Chem2018;293:2815–28
68.
YanJLiGHuYOuWWanY.Construction of a synthetic phage-displayed nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J Transl Med2014;
12:343
69.
ZimmermannIEgloffPHutterCAJArnoldFMStohlerPBocquetNHugMNHuberSSiegristMHetemannLGeraJGmürSSpiesPGygaxDGeertsmaERDawsonRJPSeegerMA.Synthetic single domain antibodies for the conformational trapping of membrane proteins. eLife2018;
7:e34317
70.
MoutelSBeryNBernardVKellerLLemesreEde MarcoALigatLRainJCFavreGOlichonAPerezF.NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife2016;
5:pii:e16228
71.
McMahonCBaierASPascoluttiRWegreckiMZhengSOngJXErlandsonSCHilgerDRasmussenSGFRingAMManglikAKruseAC.Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol2018;
25:289–96
72.
ManglikAKobilkaBKSteyaertJ.Nanobodies to study G protein–coupled receptor structure and function. Annu Rev Pharmacol Toxicol2017;
57:19–37
73.
CheTMajumdarSZaidiSAOndachiPMcCorvyJDWangSMosierPDUpretyRVardyEKrummBEHanGWLeeM-YPardonESteyaertJHuangX-PStrachanRTTriboARPasternakGWCarrollFIStevensRCCherezovVKatritchVWackerDRothBL.Structure of the Nanobody-Stabilized active state of the kappa opioid receptor. Cell2018;
172:55–67.e15
74.
WinglerLMMcMahonCStausDPLefkowitzRJKruseAC.Distinctive activation mechanism for angiotensin receptor revealed by a synthetic nanobody. Cell2019;
176:479–90.e12
75.
Kromann-HansenTLouise LangeEPeter SørensenHHassanzadeh-GhassabehGHuangMJensenJKMuyldermansSDeclerckPJKomivesEAAndreasenPA.Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator. Sci Rep2017;
7:3385
76.
De GroofTWMBobkovVHeukersRSmitMJ.Nanobodies: new avenues for imaging, stabilizing and modulating GPCRs. Mol Cell Endocrinol2019;
484:15–24
77.
SchonichenAWebbBAJacobsonMPBarberDL.Considering protonation as a posttranslational modification regulating protein structure and function. Annu Rev Biophys2013;
42:289–314
78.
ItoWSakatoNFujioHYutaniKArataYKurosawaY.The his-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values. FEBS Lett1992;
309:85–8
79.
Chaparro-RiggersJLiangHDeVayRMBaiLSuttonJEChenWGengTLindquistKCasasMGBoustanyLMBrownCLChabotJGomesBGarzonePRossiAStropPSheltonDPonsJRajpalA.Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9. J Biol Chem2012;
287:11090–7
80.
IgawaTIshiiSTachibanaTMaedaAHiguchiYShimaokaSMoriyamaCWatanabeTTakuboRDoiYWakabayashiTHayasakaAKadonoSMiyazakiTHarayaKSekimoriYKojimaTNabuchiYAsoYKawabeYHattoriK.Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol2010;
28:1203–7
81.
MurtaughMLFanningSWSharmaTMTerryAMHornJR.A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches. Protein Sci2011;
20:1619–31
82.
DavenportKRSmithCAHofstetterHHornJRHofstetterO.Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns. J Chromatogr B Analyt Technol Biomed Life Sci2016;
1021:114–21
83.
FanningSWMurtaughMLHornJR.A combinatorial approach to engineering a dual-specific metal switch antibody. Biochemistry2011;
50:5093–5
84.
FanningSWWalterRHornJR.Structural basis of an engineered dual-specific antibody: conformational diversity leads to a hypervariable loop metal-binding site. Protein Eng Des Sel2014;
27:391–7
