Antibodies represent a well-established class of clinical diagnostics for medical applications as well as essential research and biotechnological tools. Although both polyclonal and monoclonal antibodies are indispensable reagents in basic research and diagnostics but both of them have their limitations. Hence, there is urgent need to develop strategies aimed at production of alternative scaffolds and recombinant antibodies of smaller dimensions that could be easily produced, selected and manipulated. Unlike conventional antibodies, members of Camelidae and sharks produce antibodies composed only of heavy chains with small size, high solubility, thermal stability, refolding capacity and good tissue penetration in vivo. The discovery of these naturally occurring antibodies having only heavy-chain in Camelidae family and their further development into small recombinant nanobodies represents an attractive alternative in drug delivery, diagnostics and imaging. Nanobody derivatives are soluble, stable, versatile, have unique refolding capacities, reduced aggregation tendencies and high-target binding capabilities. They can be genetically customized to target enzymes, transmembrane proteins or molecular interactions. Their ability to recognize recessed antigenic sites has been attributed to their smaller size and the ability of the extended CDR3 loop to quickly penetrate into such epitopes. With the advent of molecular engineering and phage display technology, they can be of potential use in molecular imaging, drug delivery and therapeutics for several major diseases. In this review we present the recent advances in nanobodies for modulating immune functions, for targeting cancers, viruses, toxins and microbes as well as their utility as diagnostic and biosensor tools.
MurailleE., Redefining the immune system as a social interface for cooperative processes, PLoS Pathogens9(3) (2013), e1003203.
2.
AbbasA.K.LichtmanA.H. et al., Basic immunology: functions and disorders of the immune system: Elsevier Health Sciences, 2014.
3.
Riera RomoM.Pérez-MartínezD. et al., Innate immunity in vertebrates: an overview, Immunology148(2) (2016), 125–139.
4.
KurosakiT.KometaniK. et al., Memory B cells, Nature Reviews Immunology15(3) (2015), 149.
5.
FlajnikM.F. and KasaharaM., Origin and evolution of the adaptive immune system: genetic events and selective pressures, Nature Reviews Genetics11(1) (2010), 47.
6.
SesterhennF.GallouxM. et al., Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen, PLoS Biology17(2) (2019), e3000164.
7.
ParolaC.NeumeierD. et al., Integrating high-throughput screening and sequencing for monoclonal antibody discovery and engineering, Immunology153(1) (2018), 31–41.
8.
BannasP.HambachJ. et al., Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics, Frontiers in Immunology8 (2017), 1603.
9.
WesolowskiJ.AlzogarayV. et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity, Medical Microbiology Immunology198(3) (2009), 157–174.
10.
KönningD.ZielonkaS. et al., Camelid and shark single domain antibodies: structural features and therapeutic potential, Current Opinion in Structural Biology45 (2017), 10–16.
11.
EyerL. and HruskaK., Single-domain antibody fragments derived from heavy-chain antibodies: a review, Vet Med57(9) (2012), 439–513.
12.
RathoreA.S.SarkerA. et al., Recent developments toward antibody engineering and affinity maturation, Protein and Peptide Letters25(10) (2018), 886–896.
13.
KrahS.SchröterC. et al., Single-domain antibodies for biomedical applications, Immunopharmacology Immunotoxicology38(1) (2016), 21–28.
14.
IezziM.E.PolicastroL. et al., Single-domain antibodies and the promise of modular targeting in cancer imaging and treatment, Frontiers in Immunology9 (2018), 273.
15.
SpurrellE.L. and LockleyM., Adaptive immunity in cancer immunology and therapeutics, Ecancermedicalscience (2014), 8.
NezlinR., Dynamic aspects of the immunoglobulin structure, Immunological Investigations (2019), 1–10.
18.
WangX.-z.ColjeeV.W. et al., Back to the future: recombinant polyclonal antibody therapeutics, Current Opinion in Chemical Engineering2(4) (2013), 405–415.
19.
WilsonI.A. and StanfieldR.L., Antibody-antigen interactions: new structures and new conformational changes, Current Opinion in Structural Biology4(6) (1994), 857–867.
20.
ArtisD. and SpitsH., The biology of innate lymphoid cells, Nature517(7534) (2015), 293.
21.
CooperM.D., The early history of B cells, Nature Reviews Immunology15(3) (2015), 191–197.
22.
GavilondoJ. and LarrickJ., Antibody production technology in the millennium, Biotechniques29(1) (2000), 128–145.
23.
DeyevS.M.LebedenkoE.N. et al., Man-made antibodies and immunoconjugates with desired properties: function optimization using structural engineering, Russian Chemical Reviews84(1) (2015), 1.
24.
LoosA. and SteinkellnerH., IgG-Fc glycoengineering in non-mammalian expression hosts, Archives of Biochemistry Biophysics526(2) (2012), 167–173.
25.
HoogenboomH.R. and ChamesP., Natural and designer binding sites made by phage display technology, Immunology Today21(8) (2000), 371–378.
26.
ScottA.M.WolchokJ.D. et al., Antibody therapy of cancer, Nature Reviews Cancer12(4) (2012), 278.
27.
SafdariY.FarajniaS. et al., Antibody humanization methods – a review and update, Biotechnology Genetic Engineering Reviews29(2) (2013), 175–186.
28.
HardingF.A.SticklerM.M. et al., eds, The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions, MAbs; 2010: Taylor & Francis.
29.
TsumotoK.IsozakiY. et al., Future perspectives of therapeutic monoclonal antibodies, Immunotherapy11(2) (2019), 119–127.
30.
CunninghamD.HumbletY. et al., Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer, New England Journal of Medicine351(4) (2004), 337–345.
31.
HurwitzH.FehrenbacherL. et al., Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer, New England Journal of Medicine350(23) (2004), 2335–2342.
32.
SlamonD.J.Leyland-JonesB. et al., Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2, New England Journal of Medicine344(11) (2001), 783–792.
33.
TappendenP.JonesR. et al., Systematic review and economic evaluation of bevacizumab and cetuximab for the treatment of metastatic colorectal cancer, NIHR Health Technology Assessment programme: Executive Summaries: NIHR Journals Library, 2007.
34.
CiardielloF.TroianiT. et al., Interaction between the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) pathways: a rational approach for multi-target anticancer therapy, Annals of Oncology17(suppl_7) (2006), vii109–vii14.
35.
TijinkB.M.NeriD. et al., Radioimmunotherapy of head and neck cancer xenografts using 131I-labeled antibody L19-SIP for selective targeting of tumor vasculature, Journal of Nuclear Medicine47(7) (2006), 1127–1135.
36.
EngelmanJ.A.ZejnullahuK. et al., MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling, Science316(5827) (2007), 1039–1043.
37.
YokotaT.MilenicD.E. et al., Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms, Cancer Research52(12) (1992), 3402–3408.
38.
HudsonP.J. and SouriauC., Engineered antibodies, Nature Medicine9(1) (2003), 129.
39.
KhodabakhshF.BehdaniM. et al., Single-domain antibodies or nanobodies: a class of next-generation antibodies, International Reviews of Immunology (2019), 1–7.
40.
Hamers-CastermanC.AtarhouchT. et al., Naturally occurring antibodies devoid of light chains, Nature363(6428) (1993), 446.
41.
SockoloskyJ.T.DouganM. et al., Durable antitumor responses to CD47 blockade require adaptive immune stimulation, Proceedings of the National Academy of Sciences113(19) (2016), E2646–E54.
42.
VuchelenA.O’DayE. et al., 1 H, 13 C and 15 N assignments of a camelid nanobody directed against human α-synuclein, Biomolecular NMR Assignments3(2) (2009), 231.
43.
KumarH.Finer-MooreJ.S. et al., Crystal structure of a ligand-bound LacY-nanobody complex, Proceedings of the National Academy of Sciences115(35) (2018), 8769–8774.
44.
RobertB.DorvilliusM. et al., Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA), International Journal of Cancer81(2) (1999), 285–291.
45.
ConrathK.E.LauwereysM. et al., Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs, Journal of Biological Chemistry276(10) (2001), 7346–7350.
46.
SedykhS.E.PrinzV.V. et al., Bispecific antibodies: design, therapy, perspectives, Drug Design, Development Therapy12 (2018), 195.
47.
DaviesJ. and RiechmannL., Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability, Protein Engineering, Design Selection9(6) (1996), 531–537.
48.
GhahroudiM.A.DesmyterA. et al., Selection and identification of single domain antibody fragments from camel heavy-chain antibodies, FEBS Letters414(3) (1997), 521–526.
49.
PérezJ.M.RenisioJ.G. et al., Thermal unfolding of a llama antibody fragment: a two-state reversible process, Biochemistry40(1) (2001), 74–83.
50.
DumoulinM.ConrathK. et al., Single-domain antibody fragments with high conformational stability, Protein Science11(3) (2002), 500–515.
51.
WolfgangW.J.MillerT.W. et al., Suppression of huntington’s disease pathology in drosophila by human single-chain Fv antibodies, Proceedings of the National Academy of Sciences102(32) (2005), 11563–11568.
52.
WangJ.MukhtarH. et al., VHH antibodies: reagents for mycotoxin detection in food products, Sensors18(2) (2018), 485.
53.
McMurphyT.XiaoR. et al., The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma, PLoS One9(2) (2014), e89895.
54.
BlickS.K. and CurranM.P., Certolizumab pegol: in Crohn’s disease BioDrugs: clinical immunotherapeutics, Biopharmaceuticals and Gene Therapy21(3) (2007), 195–201; discussion 2–3.
55.
PadlanE.A., X-ray crystallography of antibodies, Advances in Protein Chemistry49 (1996), 57–134.
56.
ConrathK.E.LauwereysM. et al., β-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae, Antimicrobial Agents Chemotherapy45(10) (2001), 2807–2812.
57.
LauwereysM.GhahroudiM.A. et al., Potent enzyme inhibitors derived from dromedary heavy-chain antibodies, The EMBO Journal17(13) (1998), 3512–3520.
58.
VinckeC.LorisR. et al., General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold, Journal of Biological Chemistry284(5) (2009), 3273–3284.
59.
LiuW.SongH. et al., Recent advances in the selection and identification of antigen-specific nanobodies, Molecular Immunology96 (2018), 37–47.
60.
SalvadorJ.-P.VilaplanaL. et al., Nanobody: outstanding features for diagnostic and therapeutic applications, Analytical Bioanalytical Chemistry (2019), 1–11.
61.
UtsumiS. and KarushF., The subunits of purified rabbit antibody, Biochemistry3(9) (1964), 1329–1338.
62.
YooT.RoholtO. et al., Specific binding activity of isolated light chains of antibodies, Science157(3789) (1967), 707–709.
63.
WardE.S.GüssowD. et al., Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli, Nature341(6242) (1989), 544.
64.
SundbergE.J. and MariuzzaR.A., Molecular recognition in antibody-antigen complexes, Advances in Protein Chemistry61 (2002), 119–160.
65.
SmolarekD.BertrandO. et al., Variable fragments of heavy chain antibodies (VHHs): a new magic bullet molecule of medicine? Advances in Hygiene & Experimental Medicine/ Postepy Higieny i Medycyny Doswiadczalnej, 2012, 66.
66.
RevetsH.De BaetselierP. et al., Nanobodies as novel agents for cancer therapy, Expert Opinion on Biological Therapy5(1) (2005), 111–124.
67.
Hassanzadeh-GhassabehG.DevoogdtN. et al., Nanobodies and their potential applications, Nanomedicine (Lond)8 (2013), 1013–1026.
68.
OliveiraS.HeukersR. et al., Targeting tumors with nanobodies for cancer imaging and therapy, Journal of Controlled Release172(3) (2013), 607–617.
69.
VaneyckenI.DevoogdtN. et al., Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer, The FASEB Journal25(7) (2011), 2433–2446.
70.
HarmsenM. and De HaardH., Properties, production, and applications of camelid single-domain antibody fragments, Applied Microbiology Biotechnology77(1) (2007), 13–22.
71.
SiontorouC.G., Nanobodies as novel agents for disease diagnosis and therapy, International Journal of Nanomedicine8 (2013), 4215.
72.
GreenbergA.S.AvilaD. et al., A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks, Nature374(6518) (1995), 168.
73.
van der LindenR.de GeusB. et al., Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama, Journal of Immunological Methods240(1–2) (2000), 185–195.
74.
TanhaJ.DubucG. et al., Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH properties, Journal of Immunological Methods263(1–2) (2002), 97–109.
75.
YauK.Y.GrovesM.A. et al., Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library, Journal of Immunological Methods281(1–2) (2003), 161–175.
76.
VerheesenP.RoussisA. et al., Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation, Biochimica et Biophysica Acta-Proteins Proteomics1764(8) (2006), 1307–1319.
77.
GoldmanE.R.AndersonG.P. et al., Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library, Analytical Chemistry78(24) (2006), 8245–8255.
78.
MuyldermansS., Single domain camel antibodies: current status, Reviews in Molecular Biotechnology74(4) (2001), 277–302.
79.
HolligerP. and HudsonP.J.J.N.b., Engineered antibody fragments and the rise of single domains, Nature Biotechnology23(9) (2005), 1126.
80.
DesmyterA.TransueT.R. et al., Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme, Nature Structural Biology3(9) (1996), 803.
81.
De GenstE.SilenceK. et al., Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies, Proceedings of the National Academy of Sciences of the United States of America103(12) (2006), 4586–4591.
82.
Te WuT.JohnsonG. et al., Length distribution of CDRH3 in antibodies, Proteins: Structure, Function, Bioinformatics16(1) (1993), 1–7.
83.
PiniA. and BracciL., Phage display of antibody fragments, Current Protein Peptide Science1(2) (2000), 155–169.
Cortez-RetamozoV.LauwereysM. et al., Efficient tumor targeting by single-domain antibody fragments of camels, International Journal of Cancer98(3) (2002), 456–462.
86.
DumoulinM.LastA.M. et al., A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme, Nature424(6950) (2003), 783.
87.
RothbauerU.ZolghadrK. et al., Targeting and tracing antigens in live cells with fluorescent nanobodies, Nature Methods3(11) (2006), 887.
88.
OlichonA. and SurreyT., Selection of genetically encoded fluorescent single domain antibodies engineered for efficient expression in Escherichia coli, Journal of Biological Chemistry282(50) (2007), 36314–36320.
89.
CoppietersK.DreierT. et al., Formatted anti-tumor necrosis factor α VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis, Arthritis Rheumatism54(6) (2006), 1856–1866.
90.
PantN.HultbergA. et al., Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea, The Journal of Infectious Diseases194(11) (2006), 1580–1588.
91.
GueorguievaD.LiS. et al., Identification of single-domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis, The FASEB Journal20(14) (2006), 2636–2638.
92.
JoblingS.A.JarmanC. et al., Immunomodulation of enzyme function in plants by single-domain antibody fragments, Nature Biotechnology21(1) (2003), 77.
93.
SerruysB.Van HoutteF. et al., Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice, Hepatology49(1) (2009), 39–49.
94.
LonbergN., Fully human antibodies from transgenic mouse and phage display platforms, Current Opinion in Immunology20(4) (2008), 450–459.
95.
PaalanenM.M.EkokoskiE. et al., The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon, European Journal of Pharmaceutical Sciences42(4) (2011), 332–339.
96.
VercruysseT.PardonE. et al., An intrabody based on a llama single-domain antibody targeting the N-terminal α-helical multimerization domain of HIV-1 Rev prevents viral production, Journal of Biological Chemistry285(28) (2010), 21768–21780.
97.
MarschallA.L. and DübelS., Antibodies inside of a cell can change its outside: can intrabodies provide a new therapeutic paradigm? Computational Structural Biotechnology Journal14 (2016), 304–308.
98.
VandenbrouckeK.De HaardH. et al., Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis, Mucosal Immunology3(1) (2010), 49.
99.
StoneE.HiramaT. et al., The assembly of single domain antibodies into bispecific decavalent molecules, Journal of Immunological Methods318(1–2) (2007), 88–94.
100.
RutgersK.NabuursR. et al., Transmigration of beta amyloid specific heavy chain antibody fragments across the in vitro blood-brain barrier, Neuroscience190 (2011), 37–42.
101.
Kazemi-LomedashtF.BehdaniM. et al., Selection and characterization of specific Nanobody against human immunoglobulin G, Monoclonal Antibodies in Immunodiagnosis Immunotherapy34(3) (2015), 201–205.
102.
Kazemi-LomedashtF.Pooshang-BagheriK. et al., In vivo immunotherapy of lung cancer using cross-species reactive vascular endothelial growth factor nanobodies, Iranian Journal of Basic Medical Sciences20(5) (2017), 489.
103.
HomayouniV.Ganjalikhani-HakemiM. et al., Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3), Iranian Journal of Basic Medical Sciences19(11) (2016), 1201.
104.
BagheriM.BabaeiE. et al., Development of a recombinant camelid specific diabody against the heminecrolysin fraction of Hemiscorpius lepturus scorpion, Toxin Reviews36(1) (2017), 7–11.
105.
Kazemi-LomedashtF.BehdaniM. et al., Production and characterization of novel camel single domain antibody targeting mouse vascular endothelial growth factor, Monoclonal Antibodies in Immunodiagnosis Immunotherapy35(3) (2016), 167–171.
106.
Kazemi-LomedashtF.BehdaniM. et al., Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody, Molecular Immunology65(1) (2015), 58–67.
107.
AlirahimiE.AshkiyanA. et al., Intrabody targeting vascular endothelial growth factor receptor-2 mediates downregulation of surface localization, Cancer Gene Therapy24(1) (2017), 33.
108.
AbskharonR.N.SororS.H. et al., Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes, Protein Engineering, Design Selection24(9) (2011), 737–741.
109.
FrancoE.J.SonnesonG.J. et al., Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography, Journal of Chromatography B878(2) (2010), 177–186.
110.
Kazemi-LomedashtF.MuyldermansS. et al., Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function, Iranian Journal of Basic Medical Sciences21(3) (2018), 260.
111.
GuraT., Therapeutic antibodies: magic bullets hit the target, Nature417(6889) (2002), 584–587.
112.
StrebhardtK. and UllrichA., Paul ehrlich’s magic bullet concept: 100 years of progress, Nature Reviews Cancer8(6) (2008), 473.
113.
FangR.H.LukB.T. et al., Engineered nanoparticles mimicking cell membranes for toxin neutralization, Advanced Drug Delivery Reviews90 (2015), 69–80.
114.
PirazziniM. and RossettoO., Challenges in searching for therapeutics against botulinum neurotoxins, Expert Opinion on Drug Discovery12(5) (2017), 497–510.
115.
BuschC.SchömigK. et al., Characterization of the catalytic domain of clostridium novyi Alpha-Toxin, Infection Immunity68(11) (2000), 6378–6383.
116.
LiuJ.L.AndersonG.P. et al., Selection of cholera toxin specific IgNAR single-domain antibodies from a naive shark library, Molecular Immunology44(7) (2007), 1775–1783.
117.
GoldmanE.R.AndersonG.P. et al., Thermostable llama single domain antibodies for detection of botulinum a neurotoxin complex, Analytical Chemistry80(22) (2008), 8583–8591.
118.
El KhattabiM.AdamsH. et al., Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: prospects for therapeutic applications, Clinical Vaccine Immunology13(10) (2006), 1079–1086.
119.
HarrisonR. and WerneryU., The unique properties of camelid IgG have potential to improve the treatment of snake bite, Journal of Camel Practice Research14(1) (2007), 15–16.
120.
Meddeb-MouelhiF.Bouhaouala-ZaharB. et al., Immunized camel sera and derived immunoglobulin subclasses neutralizing androctonus australis hector scorpion toxins, Toxicon42(7) (2003), 785–791.
121.
HarrisonR.HassonS. et al., Neutralisation of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera, Toxicon47(3) (2006), 364–368.
122.
StewartC.S.MacKenzieC.R. et al., Isolation, characterization and pentamerization of α-cobrotoxin specific single-domain antibodies from a naïve phage display library: preliminary findings for antivenom development, Toxicon49(5) (2007), 699–709.
123.
HmilaI.SaerensD. et al., VHH, bivalent domains and chimeric heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI’, Molecular Immunology45(14) (2008), 3847–3856.
124.
ForsmanA.BeirnaertE. et al., Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120, Journal of Virology82(24) (2008), 12069–12081.
125.
GaraicoecheaL.OlichonA. et al., Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice, Journal of Virology82(19) (2008), 9753–9764.
126.
HarmsenM.Van SoltC. et al., Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease, Veterinary Microbiology120(3–4) (2007), 193–206.
127.
HarmsenM.FijtenH. et al., Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin, Veterinary Microbiology132(1–2) (2008), 56–64.
De HaardH.J.BezemerS. et al., Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection, Journal of Bacteriology187(13) (2005), 4531–4541.
130.
LedeboerA.BezemerS. et al., Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama, Journal of Dairy Science85(6) (2002), 1376–1382.
131.
DekkerS.ToussaintW. et al., Intracellularly expressed single-domain antibody against p15 matrix protein prevents the production of porcine retroviruses, Journal of Virology77(22) (2003), 12132–12139.
132.
SherwoodL.J.OsbornR.C., Jr, et al., Rapid assembly of sensitive antigen-capture assays for Marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level, The Journal of Infectious Diseases196(Supplement_2) (2007), S213–S9.
133.
HarmsenM.Van SoltC. et al., Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy, Applied Microbiology Biotechnology72(3) (2006), 544–551.
134.
SzynolA.De SoetJ. et al., Bactericidal effects of a fusion protein of llama heavy-chain antibodies coupled to glucose oxidase on oral bacteria, Antimicrobial Agents Chemotherapy48(9) (2004), 3390–3395.
135.
KrügerC.HultbergA. et al., Therapeutic effect of llama derived VHH fragments against streptococcus mutans on the development of dental caries, Applied Microbiology Biotechnology72(4) (2006), 732–737.
136.
KorotkovK.V.PardonE. et al., Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody, Structure17(2) (2009), 255–265.
137.
LamA.Y.PardonE. et al., Nanobody-aided structure determination of the EpsI: EpsJ pseudopilin heterodimer from Vibrio vulnificus, Journal of Structural Biology166(1) (2009), 8–15.
138.
SpinelliS.DesmyterA. et al., Lactococcal bacteriophage p2 receptor-binding protein structure suggests a common ancestor gene with bacterial and mammalian viruses, Nature Structural Molecular Biology13(1) (2006), 85.
139.
SachsG.WeeksD.L. et al., The gastric biology of helicobacter pylori, Annual Review of Physiology65(1) (2003), 349–369.
140.
BlaserM.J., Who are we: indigenous microbes and the ecology of human diseases, EMBO Reports7(10) (2006), 956–960.
141.
KustersJ.G.van VlietA.H. et al., Pathogenesis of helicobacter pylori infection, Clinical Microbiology Reviews19(3) (2006), 449–490.
142.
ArdekaniL.S.GargariS.L.M. et al., A novel nanobody against urease activity of helicobacter pylori, International Journal of Infectious Diseases17(9) (2013), e723–e8.
143.
AthertonJ.C., H. pylori virulence factors, British Medical Bulletin54(1) (1998), 105–120.
144.
PrinzC.HafsiN. et al., Helicobacter pylori virulence factors and the host immune response: implications for therapeutic vaccination, Trends in Microbiology11(3) (2003), 134–138.
145.
MalekshahiZ.V.GargariS.L.M. et al., Treatment of helicobacter pylori infection in mice with oral administration of egg yolk-driven anti-UreC immunoglobulin, Microbial Pathogenesis51(5) (2011), 366–372.
146.
RahbarizadehF.RasaeeM.J. et al., Over expression of anti-MUC1 single-domain antibody fragments in the yeast pichia pastoris, Molecular Immunology43(5) (2006), 426–435.
147.
PourasadiS.GargariS.L.M. et al., Efficient production of nanobodies against urease activity of helicobacter pylori in pichia pastoris, Turkish Journal of Medical Sciences47(2) (2017), 695–701.
148.
StijlemansB.ConrathK. et al., Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies african trypanosomes as paradigm, Journal of Biological Chemistry279(2) (2004), 1256–1261.
149.
BaralT.N.MagezS. et al., Experimental therapy of african trypanosomiasis with a nanobody-conjugated human trypanolytic factor, Nature Medicine12(5) (2006), 580.
150.
SaerensD.StijlemansB. et al., Parallel selection of multiple anti-infectome nanobodies without access to purified antigens, Journal of Immunological Methods329(1–2) (2008), 138–150.
151.
RatierL.UrrutiaM. et al., Relevance of the diversity among members of the trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies, PLoS One3(10) (2008), e3524.
152.
DeckersN.SaerensD. et al., Nanobodies, a promising tool for species-specific diagnosis of taenia solium cysticercosis, International Journal for Parasitology39(5) (2009), 625–633.
153.
DolkE.Van Der VaartM. et al., Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo, Appl Environ Microbiol71(1) (2005), 442–450.
154.
LeeV.HuangJ.L. et al., The evolution of multiple isotypic IgM heavy chain genes in the shark, The Journal of Immunology180(11) (2008), 7461–7470.
155.
LauneD.MolinaF. et al., Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins, Journal of Biological Chemistry272(49) (1997), 30937–30944.
156.
ZouX.SmithJ.A. et al., Expression of a dromedary heavy chain-only antibody and B cell development in the mouse, The Journal of Immunology175(6) (2005), 3769–3779.
157.
JanssensR.DekkerS. et al., Generation of heavy-chain-only antibodies in mice, Proceedings of the National Academy of Sciences103(41) (2006), 15130–15135.
158.
HammarströmS., ed. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues, Seminars in cancer biology, 1999: Elsevier.
159.
WilludaJ.HoneggerA. et al., High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment, Cancer Research59(22) (1999), 5758–5767.
160.
WhitlowM.BellB.A. et al., An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability, Protein Engineering, Design Selection6(8) (1993), 989–995.
161.
VuK.B.GhahroudiM.A. et al., Comparison of llama VH sequences from conventional and heavy chain antibodies, Molecular Immunology34(16–17) (1997), 1121–1131.
162.
BagshaweK., Antibody directed enzymes revive anti-cancer prodrugs concept, British Journal of Cancer56(5) (1987), 531.
163.
RoodinkI.FranssenM. et al., Isolation of targeting nanobodies against co-opted tumor vasculature, Laboratory Investigation90(1) (2010), 61.
164.
DoyleP.J.Arbabi-GhahroudiM. et al., Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol, Molecular Immunology45(14) (2008), 3703–3713.
165.
SpinelliS.FrenkenL.G. et al., Camelid heavy-chain variable domains provide efficient combining sites to haptens, Biochemistry39(6) (2000), 1217–1222.
166.
LadensonR.C.CrimminsD.L. et al., Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment, Analytical Chemistry78(13) (2006), 4501–4508.
167.
AndersonG.P. and GoldmanE.R., TNT detection using llama antibodies and a two-step competitive fluid array immunoassay, Journal of Immunological Methods339(1) (2008), 47–54.
168.
Alvarez-RuedaN.BeharG. et al., Generation of llama single-domain antibodies against methotrexate, a prototypical hapten, Molecular Immunology44(7) (2007), 1680–1690.
169.
GoldenbergD.M.SharkeyR.M. et al., Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy, Journal of Clinical Oncology24(5) (2006), 816.
170.
HuY.LiuC. et al., Nanobody-based delivery systems for diagnosis and targeted tumor therapy, Frontiers in Immunology8 (2017), 1442.
171.
GomesC.M.AbrunhosaA.J. et al., Molecular imaging with SPECT as a tool for drug development, Advanced Drug Delivery Reviews63(7) (2011), 547–554.
172.
AmetameyS.M.HonerM. et al., Molecular imaging with PET, Chemical Reviews108(5) (2008), 1501–1516.
173.
ChakravartyR.ShuklaR. et al., Nanoceria-PAN composite-based advanced sorbent material: a major step forward in the field of clinical-grade 68Ge/68Ga generator, ACS Applied Materials Interfaces2(7) (2010), 2069–2075.
174.
AlauddinM.M., Positron emission tomography (PET) imaging with 18F-based radiotracers, American Journal of Nuclear Medicine Molecular Imaging2(1) (2012), 55.
175.
HollandJ.P.CummingP. et al., PET radiopharmaceuticals for probing enzymes in the brain, American Journal of Nuclear Medicine Molecular Imaging3(3) (2013), 194.
176.
EckelmanW.C., Unparalleled contribution of technetium-99m to medicine over 5 decades, JACC: Cardiovascular Imaging2(3) (2009), 364–368.
177.
HuangL.GainkamL.O.T. et al., SPECT imaging with 99m Tc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression, Molecular Imaging Biology10(3) (2008), 167–175.
178.
XavierC.VaneyckenI. et al., Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 nanobodies for iPET imaging of HER2 receptor expression in cancer, Journal of Nuclear Medicine54(5) (2013), 776–784.
179.
SiontorouC.G. and BatziasF.A., Innovation in biotechnology: moving from academic research to product development – the case of biosensors, Critical Reviews in Biotechnology30(2) (2010), 79–98.
180.
BackmannN.ZahndC. et al., A label-free immunosensor array using single-chain antibody fragments, Proceedings of the National Academy of Sciences102(41) (2005), 14587–14592.
181.
SteinhauerC.WingrenC. et al., Single framework recombinant antibody fragments designed for protein chip applications, Biotechniques33(S6) (2002), S38–S45.
