Medical Microbiology and Immunology

  Previous Article | Back to Volume | Next Article
  Abstract | References | Citation | Download | Preview | Statistics
Sample volume 2
Title Single domain antibodies: promising experimental and therapeutic tools in infection and immunity
Author Janusz Wesolowski, Vanina Alzogaray, Jan Reyelt, Mandy Unger, Karla Juarez
Abstract Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.
Citation
References
1. Brekke OH, Sandlie I (2003) Therapeutic antibodies for human
diseases at the dawn of the twenty-Wrst century. Nat Rev Drug
Discov 2(1):52–62. doi:10.1038/nrd984
2. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson
G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature
363(6428):446–448. doi:10.1038/363446a0
3. Muyldermans S (2001) Single domain camel antibodies: current
status. J Biotechnol 74(4):277–302
4. Holliger P, Hudson PJ (2005) Engineered antibody fragments
and the rise of single domains. Nat Biotechnol 23(9):1126–1136.
doi:10.1038/nbt1142
5. Desmyter A, Transue TR, Ghahroudi MA, Thi MH, Poortmans F,
Hamers R, Muyldermans S, Wyns L (1996) Crystal structure of
a camel single-domain VH antibody fragment in complex with
lysozyme. Nat Struct Biol 3(9):803–811. doi:10.1038/nsb0996-
803
6. De Genst E, Silence K, Decanniere K, Conrath K, Loris R, Kinne
J, Muyldermans S, Wyns L (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies.
Proc Natl Acad Sci USA 103(12):4586–4591. doi:10.1073/
pnas.0505379103
7. Wu TT, Johnson G, Kabat EA (1993) Length distribution of
CDRH3 in antibodies. Proteins 16(1):1–7. doi:10.1002/prot.
340160102
8. Harmsen MM, De Haard HJ (2007) Properties, production, and
applications of camelid single-domain antibody fragments. Appl
Microbiol Biotechnol 77(1):13–22. doi:10.1007/s00253-007-
1142-2
9. Pini A, Bracci L (2000) Phage display of antibody fragments.
Curr Protein Pept Sci 1(2):155–169. doi:10.2174/138920300
3381397
10. Hoogenboom HR (2005) Selecting and screening recombinant
antibody libraries. Nat Biotechnol 23(9):1105–1116. doi:10.
1038/nbt1126
11. Cortez-Retamozo V, Lauwereys M, Hassanzadeh Gh G, Gobert
M, Conrath K, Muyldermans S, De Baetselier P, Revets H (2002)
EYcient tumor targeting by single-domain antibody fragments of
camels. Int J Cancer 98(3):456–462. doi:10.1002/ijc.10212
12. Dumoulin M, Last AM, Desmyter A, Decanniere K, Canet D,
Larsson G, Spencer A, Archer DB, Sasse J, Muyldermans S,
Wyns L, RedWeld C, Matagne A, Robinson CV, Dobson CM
(2003) A camelid antibody fragment inhibits the formation of
amyloid Wbrils by human lysozyme. Nature 424(6950):783–788.
doi:10.1038/nature01870
13. Conrath EK, Lauwereys M, Wyns L, Muyldermans S (2001)
Camel single-domain antibodies as modular building units in bispeciWc and bivalent antibody constructs. J Biol Chem
276(10):7346–7350. doi:10.1074/jbc.M007734200
14. Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L,
Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC,
Leonhardt H (2006) Targeting and tracing antigens in live cells
with  Xuorescent nanobodies. Nat Methods 3(11):887–889.
doi:10.1038/nmeth953
15. Olichon A, Surrey T (2007) Selection of genetically encoded
Xuorescent single domain antibodies engineered for eYcient
expression in  Escherichia coli. J Biol Chem 282(50):36314–
36320. doi:10.1074/jbc.M704908200
16. Coppieters K, Dreier T, Silence K, De Haard H, Lauwereys M,
Casteels P, Beirnaert E, Jonckheere H, Van de Wiele C, Staelens
L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P (2006)
Formatted anti-tumor necrosis factor alpha VHH proteins derived
from camelids show superior potency and targeting to inXamed
joints in a murine model of collagen-induced arthritis. Arthritis
Rheum 54(6):1856–1866. doi:10.1002/art.21827
17. Pant N, Hultberg A, Zhao Y, Svensson L, Pan-Hammarstrom Q,
Johansen K, Pouwels PH, Ruggeri FM, Hermans P, Frenken L,
Boren T, Marcotte H, Hammarstrom L (2006) Lactobacilli
expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced
diarrhea. J Infect Dis 194(11):1580–1588. doi:10.1086/508747
18. Gueorguieva D, Li S, Walsh N, Mukerji A, Tanha J, Pandey S
(2006) IdentiWcation of single-domain, Bax-speciWc intrabodies
that confer resistance to mammalian cells against oxidativestress-induced apoptosis. FASEB J 20(14):2636–2638. doi:10.
1096/fj.06-6306fje
19. Jobling SA, Jarman C, Teh MM, Holmberg N, Blake C, Verhoeyen ME (2003) Immunomodulation of enzyme function in
plants by single-domain antibody fragments. Nat Biotechnol
21(1):77–80. doi:10.1038/nbt772
20. Serruys B, Van Houtte F, Verbrugghe P, Leroux-Roels G, Vanlandschoot P (2009) Llama-derived single-domain intrabodies
inhibit secretion of hepatitis B virions in mice. Hepatology
49(1):39–49. doi:10.1002/hep.22609
21. Feldmann M (2002) Development of anti-TNF therapy for rheumatoid arthritis. Nat Rev Immunol 2(5):364–371. doi:10.1038/
nri802
22. Lonberg N (2008) Fully human antibodies from transgenic
mouse and phage display platforms. Curr Opin Immunol
20(4):450–459. doi:10.1016/j.coi.2008.06.004
23. Drucker A, Skedgel C, Virik K, Rayson D, Sellon M, Younis T
(2008) The cost burden of trastuzumab and bevacizumab therapy
for solid tumours in Canada. Curr Oncol 15(3):136–142.
doi:10.3747/co.v15i3.249
24. Goding JW, Howard MC (1998) Ecto-enzymes of lymphoid
cells. Immunol Rev 161:5–10. doi:10.1111/j.1600-065X.1998.
tb01567.x
25. Salmi M, Jalkanen S (2005) Cell-surface enzymes in control of
leukocyte traYcking. Nat Rev Immunol 5(10):760–771. doi:10.
1038/nri1705
26. Corda D, Di Girolamo M (2003) Functional aspects of protein
mono-ADP-ribosylation. EMBO J 22(9):1953–1958. doi:10.
1093/emboj/cdg209
27. Zolkiewska A (2005) Ecto-ADP-ribose transferases: cell-surface
response to local tissue injury. Physiology (Bethesda) 20:374–
381. doi:10.1152/physiol.00028.2005
28. Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli
A, Torboli M, Bolognesi G, Baricordi OR (2001) Nucleotide
receptors: an emerging family of regulatory molecules in blood
cells. Blood 97(3):587–600. doi:10.1182/blood.V97.3.58729. Koch-Nolte F, Reyelt J, Schössow B, Schwarz N, Scheuplein F,
Rothenburg S, Haag F, Alzogaray V, CauerhV A, Goldbaum FA
(2007) Single domain antibodies from llama eVectively and
speciWcally block T cell ecto-ADP-ribosyltransferase ART2.2
in vivo. FASEB J 21(13):3490–3498. doi:10.1096/fj.07-8661com
30. Adriouch S, Hubert S, Pechberty S, Koch-Nolte F, Haag F,
Seman M (2007) NAD released during inXammation participates
in T cell homeostasis by inducing ART2-mediated death of naive
T cells in vivo. J Immunol 179(1):186–194
31. Koch-Nolte F, Adriouch S, Bannas P, Krebs C, Scheuplein F,
Seman M, Haag F (2006) ADP-ribosylation of membrane proteins:
unveiling the secrets of a crucial regulatory mechanism in mammalian cells. Ann Med 38(3):188–199. doi:10.1080/07853
890600655499
32. Scheuplein F, Schwarz N, Adriouch S, Krebs C, Bannas P,
Rissiek B, Seman M, Haag F, Koch-Nolte F (2009) NAD and
ATP released from injured cells induce P2X7-dependent
shedding of CD62L and externalization of phosphatidylserine by
murine T cells. J Immunol 182(5):2898–2908. doi:10.4049/
jimmunol.0801711
33. Adriouch S, Bannas P, Schwarz N, Fliegert R, Guse AH, Seman
M, Haag F, Koch-Nolte F (2008) ADP-ribosylation at R125 gates
the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site. FASEB J 22(3):861–869. doi:10.1096/fj.07-
9294com
34. Seman M, Adriouch S, Scheuplein F, Krebs C, Freese D,
Glowacki G, Deterre P, Haag F, Koch-Nolte F (2003) NADinduced T cell death: ADP-ribosylation of cell surface proteins
by ART2 activates the cytolytic P2X7 purinoceptor. Immunity
19(4):571–582. doi:10.1016/S1074-7613(03)00266-8
35. Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C,
Ober RJ, Ward ES (1997) Increasing the serum persistence of an
IgG fragment by random mutagenesis. Nat Biotechnol
15(7):637–640. doi:10.1038/nbt0797-637
36. Behar G, Sibéril S, Groulet A, Chames P, Pugnière M, Boix C,
Sautès-Fridman C, Teillaud JL, Baty D (2008) Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain
antibodies that activate natural killer cells. Protein Eng Des Sel
21(1):1–10. doi:10.1093/protein/gzm064
37. Groot AJ, Khattabi ME, Sachs N, van der Groep P, van der Wall
E, van Diest PJ, Sonnenberg A, Verrips CT, Vooijs M (2009)
Reverse proteomic antibody screening identiWes anti adhesive
VHH targeting VLA-3. Mol Immunol 46(10):2022–2028
38. Muruganandam A, Tanha J, Narang S, Stanimirovic D (2002)
Selection of phage-displayed llama single-domain antibodies that
transmigrate across human blood-brain barrier endothelium.
FASEB J 16(2):240–242
39. Abulrob A, Sprong H, Van Bergen en Henegouwen P, Stanimirovic D (2005) The blood-brain barrier transmigrating single
domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells. J Neurochem 95(4):1201–
1214. doi:10.1111/j.1471-4159.2005.03463.x
40. Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De
Baetselier P, Muyldermans S, Revets H (2004) EYcient cancer
therapy with a nanobody-based conjugate. Cancer Res
64(8):2853–2857. doi:10.1158/0008-5472.CAN-03-3935
41. Omidfar K, Rasaee MJ, Modjtahedi H, Forouzandeh M,
Taghikhani M, Golmakani N (2004) Production of a novel
camel single-domain antibody speciWc for the type III mutant
EGFR. Tumour Biol 25(5–6):296–305. doi:10.1159/00008
1395
42. Roovers R, Laeremans T, Huang L, De Taeye S, Verkleij A,
Revets H, de Haard HJ, van Bergen en Henegouwen PM (2007)
EYcient inhibition of EGFR signaling and of tumour growth by
antagonistic anti-EFGR nanobodies. Cancer Immunol Immunother 56(3):303–317. doi:10.1007/s00262-006-0180-4
43. Huang L, Gainkam LO, Caveliers V, Vanhove C, Keyaerts M, De
Baetselier P, Bossuyt A, Revets H, Lahoutte T (2008) SPECT
imaging with 99mTc-labeled EGFR-speciWc nanobody for
in vivo monitoring of EGFR expression. Mol Imaging Biol
10(3):167–175
44. Tijink BM, Laeremans T, Budde M, Stigter-van Walsum M,
Dreier T, de Haard HJ, Leemans CR, van Dongen GA (2008)
Improved tumor targeting of anti-epidermal growth factor receptor
Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Mol Cancer Ther 7(8):2288–2297.
doi:10.1158/1535-7163.MCT-07-2384
45. Rahbarizadeh F, Rasaee MJ, Forouzandeh M, Allameh A,
Sarrami R, Nasiry H, Sadeghizadeh M (2005) The production
and characterization of novel heavy-chain antibodies against the
tandem repeat region of MUC1 mucin. Immunol Invest
34(4):431–452. doi:10.1080/08820130500265356
46. Ahmadvand D, Rasaee MJ, Rahbarizadeh F, Mohammadi M
(2008) Production and characterization of a high-aYnity nanobody against human endoglin. Hybridoma (Larchmt) 27(5):353–
360. doi:10.1089/hyb.2008.0014
47. Palladino MA, Bahjat FR, Theodorakis EA, Moldawer LL (2003)
Anti-TNF-alpha therapies: the next generation. Nat Rev Drug
Discov 2(9):736–746. doi:10.1038/nrd1175
48. Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB (2007)
Alpaca (Lama pacos) as a convenient source of recombinant
camelid heavy chain antibodies (VHHs). J Immunol Methods
324(1–2):13–25. doi:10.1016/j.jim.2007.04.008
49. Wolfson W (2006) Ablynx makes nanobodies from llama bodies.
Chem Biol 13(12):1243–1244. doi:10.1016/j.chembiol.2006.12.003
50. Hulstein JJ, de Groot PG, Silence K, Veyradier A, Fijnheer R,
Lenting PJ (2005) A novel nanobody that detects the gain-offunction phenotype of von Willebrand factor in ADAMTS13
deWciency and von Willebrand disease type 2B. Blood
106(9):3035–3042. doi:10.1182/blood-2005-03-1153
51. Schenk D (2002) Amyloid-beta immunotherapy for Alzheimer’s
disease: the end of the beginning. Nat Rev Neurosci 3(10):824–
828. doi:10.1038/nrn938
52. Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, Muyldermans S, Wyns L, Matagne A
(2002) Single-domain antibody fragments with high conformational stability. Protein Sci 11(3):500–515. doi:10.1110/ps.34602
53. Lafaye P, Achour I, England P, Duyckaerts C, Rougeon F (2009)
Single-domain antibodies recognize selectively small oligomeric
forms of amyloid beta, prevent Abeta-induced neurotoxicity and
inhibit  Wbril formation. Mol Immunol 46(4):695–704.
doi:10.1016/j.molimm.2008.09.008
54. Habicht G, Haupt C, Friedrich RP, Hortschansky P, Sachse C,
Meinhardt J, Wieligmann K, Gellermann GP, Brodhun M, Götz
J, Halbhuber KJ, Röcken C, Horn U, Fändrich M (2007) Directed
selection of a conformational antibody domain that prevents
mature amyloid Wbril formation by stabilizing Abeta protoWbrils.
Proc Natl Acad Sci USA 104(49):19232–19237. doi:10.1073/
pnas.0703793104
55. Klooster R, Maassen BT, Stam JC, Hermans PW, Ten Haaft MR,
Detmers FJ, de Haard HJ, Post JA, Theo Verrips C (2007)
Improved anti-IgG and HSA aYnity ligands: clinical application
of VHH antibody technology. J Immunol Methods 324(1–2):1–12.
doi:10.1016/j.jim.2007.04.005
56. Cohen-Saidon C, Nechushtan H, Kahlon S, Livni N, Nissim A,
Razin E (2003) A novel strategy using single-chain antibody to
show the importance of Bcl-2 in mast cell survival. Blood
102(7):2506–2512. doi:10.1182/blood-2002-12-3921
57. Avignolo C, Bagnasco L, Biasotti B, Melchiori A, Tomati V,
Bauer I, Salis A, Chiossone L, Mingari MC, Orecchia P, Carnemolla B, Neri D, Zardi L, Parodi S (2008) Internalization via
Antennapedia protein transduction domain of an scFv antibodytoward c-Myc protein. FASEB J 22(4):1237–1245. doi:10.1096/
fj.07-8865com
58. Groot AJ, Verheesen P, Westerlaken EJ, Gort EH, van der Groep
P, Bovenschen N, van der Wall E, van Diest PJ, Shvarts A (2006)
IdentiWcation by phage display of single-domain antibody fragments speciWc for the ODD domain in hypoxia-inducible factor
1alpha. Lab Invest 86(4):345–356. doi:10.1038/labinvest.3700
395
59. Groot AJ, Gort EH, van der Wall E, van Diest PJ, Vooijs M
(2008) Conditional inactivation of HIF-1 using intrabodies. Cell
Oncol 30(5):397–409
60. Verheesen P, de Kluijver A, van Koningsbruggen S, de Brij M,
de Haard HJ, van Ommen GJ, van der Maarel SM, Verrips CT
(2006) Prevention of oculopharyngeal muscular dystrophyassociated aggregation of nuclear polyA-binding protein with a
single-domain intracellular antibody. Hum Mol Genet
15(1):105–111. doi:10.1093/hmg/ddi432
61. Laune D, Molina F, Ferrieres G, Mani JC, Cohen P, Simon D,
Bernardi T, Piechaczyk M, Pau B, Granier C (1997) Systematic
exploration of the antigen binding activity of synthetic peptides
isolated from the variable regions of immunoglobulins. J Biol
Chem 272(49):30937–30944. doi:10.1074/jbc.272.49.30937
62. Marquardt A, Muyldermans S, Przybylski M (2006) A synthetic
camel anti-lysozyme peptide antibody (peptibody) with Xexible
loop structure identiWed by high-resolution aYnity mass spectrometry. Chem Eur J 12(7):1915–1923. doi:10.1002/chem.
200500785
63. Laplagne DA, Zylberman V, Ainciart N, Steward MW, Sciutto E,
Fossati CA, Goldbaum FA (2004) Engineering of a polymeric
bacterial protein as a scaVold for the multiple display of peptides.
Proteins 57(4):820–828. doi:10.1002/prot.20248
64. Zarebski LM, Urrutia M, Goldbaum FA (2005) Llama single
domain antibodies as a tool for molecular mimicry. J Mol Biol
349(4):814–824. doi:10.1016/j.jmb.2005.03.072
65. Simmons DP, Streltsov VA, Dolezal O, Hudson PJ, Coley AM,
Foley M, Proll DF, Nuttall SD (2008) Shark IgNAR antibody
mimotopes target a murine immunoglobulin through extended
CDR3 loop structures. Proteins 71(1):119–130. doi:10.1002/
prot.21663
66. Gura T (2002) Therapeutic antibodies: magic bullets hit the
target. Nature 417(6889):584–586. doi:10.1038/417584a
67. Strebhardt K, Ullrich A (2008) Paul Ehrlich’s magic bullet
concept: 100 years of progress. Nat Rev Cancer 8(6):473–480.
doi:10.1038/nrc2394
68. Aktories K, Just I (2000) Bacterial protein toxins. Springer
Verlag, Berlin
69. Goldman ER, Anderson GP, Liu JL, Delehanty JB, Sherwood LJ,
Osborn LE, Cummins LB, Hayhurst A (2006) Facile generation
of heat-stable antiviral and antitoxin single domain antibodies
from a semisynthetic llama library. Anal Chem 78(24):8245–
8255. doi:10.1021/ac0610053
70. Liu JL, Anderson GP, Delehanty JB, Baumann R, Hayhurst A,
Goldman ER (2007) Selection of cholera toxin speciWc IgNAR
single-domain antibodies from a naïve shark library. Mol Immunol 44(7):1775–1783. doi:10.1016/j.molimm.2006.07.299
71. Goldman ER, Anderson GP, Conway J, Sherwood LJ, Fech M,
Vo B, Liu JL, Hayhurst A (2008) Thermostable llama single
domain antibodies for detection of botulinum A neurotoxin
complex. Anal Chem 80(22):8583–8591. doi:10.1021/ac801
4774
72. El Khattabi M, Adams H, Heezius E, Hermans P, Detmers F,
Maassen B, van der Ley P, Tommassen J, Verrips T, Stam J
(2006) Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: prospects for therapeutic applications.
Clin Vaccine Immunol 13(10):1079–1086. doi:10.1128/
CVI.00107-06
73. Harrison RA, Wernery U (2007) The unique properties of camelid IgG have potential to improve the treatment of snake bite. J
Camel Pract Res 14(1):15–16
74. Meddeb-Mouelhi F, Bouhaouala-Zahar B, Benlasfar Z, Hammadi M, Mejri T, Moslah M, Karoui H, Khorchani T, El Ayeb M
(2003) Immunized camel sera and derived immunoglobulin subclasses neutralizing Androctonus australis hector scorpion toxins. Toxicon 42(7):785–791. doi:10.1016/j.toxicon.2003.10.021
75. Harrison RA, Hasson SS, Harmsen M, Laing GD, Conrath K,
Theakston RD (2006) 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. Toxicon 47(3):364–368. doi:10.1016/j.toxicon.2005.10.017
76. Stewart CS, MacKenzie CR, Hall JC (2007) Isolation, characterization and pentamerization of alpha-cobrotoxin speciWc singledomain antibodies from a naïve phage display library: preliminary
Wndings for antivenom development. Toxicon 49(5):699–709.
doi:10.1016/j.toxicon.2006.11.023
77. Hmila I, Abdallah RBA, Saerens D, Benlasfar Z, Conrath K,
Ayeb ME, Muyldermans S, Bouhaouala-Zahar B (2008) VHH,
bivalent domains and chimeric heavy chain-only antibodies with
high neutralizing eYcacy for scorpion toxin AahI. Mol Immunol
45(14):3847–3856. doi:10.1016/j.molimm.2008.04.011
78. Doyle PJ, Arbabi-Ghahroudi M, Gaudette N, Furzer G, Savard
ME, Gleddie S, McLean MD, MacKenzie CR, Hall JC (2008)
Cloning, expression, and characterization of a single-domain
antibody fragment with aYnity for 15-acetyl-deoxynivalenol.
Mol Immunol 45(14):3703–3713. doi:10.1016/j.molimm.2008.
06.005
79. Spinelli S, Frenken LG, Hermans P, Verrips T, Brown K, Tegoni
M, Cambillau C (2000) Camelid heavy-chain variable domains
provide eYcient combining sites to haptens. Biochemistry
39(6):1217–1222. doi:10.1021/bi991830w
80. Ladenson RC, Crimmins DL, Landt Y, Ladenson JH (2006) Isolation and characterization of a thermally stable recombinant
anti-caVeine heavy-chain antibody fragment. Anal Chem
78(13):4501–4508. doi:10.1021/ac058044j
81. Anderson GP, Goldman ER (2008) TNT detection using llama
antibodies and a two-step competitive Xuid array immunoassay.
J Immunol Methods 339(1):47–54. doi:10.1016/j.jim.2008.
08.001
82. Alvarez-Rueda N, Behar G, Ferré V, Pugnière M, Roquet F, Gastinel L, Jacquot C, Aubry J, Baty D, Barbet J, Birklé S (2007)
Generation of llama single-domain antibodies against methotrexate, a prototypical hapten. Mol Immunol 44(7):1680–1690.
doi:10.1016/j.molimm.2006.08.007
83. Goldenberg DM, Sharkey RM, Paganelli G, Barbet J, Chatal JF
(2006) Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy. J Clin Oncol 24(5):823–834.
doi:10.1200/JCO.2005.03.8471
84. Forsman A, Beirnaert E, Aasa-Chapman MM, Hoorelbeke B,
Hijazi K, Koh W, Tack V, Szynol A, Kelly C, McKnight A, Verrips T, De Haard H, Weiss RA (2008) Llama antibody fragments
with cross-subtype human immunodeWciency virus type 1 (HIV-1)-
neutralizing properties and high aYnity for HIV-1 gp120. J Virol
82(24):12069–12081. doi:10.1128/JVI.01379-08
85. Garaicoechea L, Olichon A, Marcoppido G, Wigdorovitz A,
Mozgovoj M, Saif L, Surrey T, Parreño V (2008) Llama-derived
single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice. J Virol 82(19):9753–9764.
doi:10.1128/JVI.00436-08
86. Harmsen MM, van Solt CB, Fijten HP, van Keulen L, Rosalia
RA, Weerdmeester K, Cornelissen AH, De Bruin MG, Eble PL,
Dekker A (2007) Passive immunization of guinea pigs with llama
single-domain antibody fragments against foot-and-mouthdisease. Vet Microbiol 120(3–4):193–206. doi:10.1016/j.vetmic.
2006.10.029
87. Harmsen MM, Fijten HP, Dekker A, Eblé PL (2008) Passive
immunization of pigs with bispeciWc llama single-domain antibody fragments against foot-and-mouth disease and porcine
immunoglobulin. Vet Microbiol 132(1–2):56–64. doi:10.1016/
j.vetmic.2008.04.030
88. Hultberg A, Tremblay D, De Haard H, Verrips T, Moineau S,
Hammarström L, Marcotte H (2007) Lactobacilli expressing
llama VHH fragments neutralise Lactococcus phages. BMC Biotechnol 7:58. doi:10.1186/1472-6750-7-58
89. de Haard HJ, Bezemer S, Ledeboer AM, Müller WH, Boender
PJ, Moineau S, Coppelmans MC, Verkleij A, Frenken LG, Verrips CT (2005) Llama antibodies against a lactococcal protein
located at the tip of the phage tail prevent phage infection. J
Bacteriol 187(13):4531–4541. doi:10.1128/JB.187.13.4531-
4541.2005
90. Ledeboer AM, Bezemer S, de Hiaard JJ, SchaVers IM, Verrips
CT, van Vliet C, Düsterhöft EM, Zoon P, Moineau S, Frenken
LG (2002) Preventing phage lysis of Lactococcus lactis in cheese
production using a neutralizing heavy-chain antibody fragment
from llama. J Dairy Sci 85(6):1376–1382
91. Dekker S, Toussaint W, Panayotou G, de Wit T, Visser P, Grosveld F, Drabek D (2003) Intracellularly expressed single-domain
antibody against p15 matrix protein prevents the production of
porcine retroviruses. J Virol 77(22):12132–12139. doi:10.1128/
JVI.77.22.12132-12139.2003
92. Sherwood LJ, Osborn LE, Carrion R, Patterson JL, Hayhurst A
(2007) Rapid assembly of sensitive antigen-capture assays for
Marburg virus, using in vitro selection of llama single-domain
antibodies, at biosafety level 4. J Infect Dis 196(Suppl 2):S213–
S219. doi:10.1086/520586
93. Harmsen MM, van Solt CB, van Zijderveld-van Bemmel AM,
Niewold TA, van Zijderveld FG (2006) Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Appl Microbiol Biotechnol
72(3):544–551. doi:10.1007/s00253-005-0300-7
94. Szynol A, de Soet JJ, Sieben-van Tuyl E, Bos JW, Frenken LG
(2004) Bactericidal eVects of a fusion protein of llama heavychain antibodies coupled to glucose oxidase on oral bacteria.
Antimicrob Agents Chemother 48(9):3390–3395. doi:10.1128/
AAC.48.9.3390-3395.2004
95. Krüger C, Hultberg A, Marcotte H, Hermans P, Bezemer S,
Frenken LG, Hammarström L (2006) Therapeutic eVect of llama
derived VHH fragments against  Streptococcus mutans on the
development of dental caries. Appl Microbiol Biotechnol
72(4):732–737. doi:10.1007/s00253-006-0347-0
96. Korotkov KV, Pardon E, Steyaert J, Hol WG (2009) Crystal
structure of the N-terminal domain of the secretin GspD from
ETEC determined with the assistance of a nanobody.
Structure 17(2):255–265. doi:10.1016/j.str.2008.11.011
97. Lam AY, Pardon E, Korotkov KV, Hol WG, Steyaert J (2009)
Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from  Vibrio vulniWcus. J Struct Biol
166(1):8–15. doi:10.1016/j.jsb.2008.11.008
98. Spinelli S, Desmyter A, Verrips CT, de Haard HJ, Moineau S,
Cambillau C (2006) Lactococcal bacteriophage p2 receptor-binding protein structure suggests a common ancestor gene with bacterial and mammalian viruses. Nat Struct Mol Biol 13(1):85–89.
doi:10.1038/nsmb1029
99. Conrath KE, Lauwereys M, Galleni M, Matagne A, Frère JM,
Kinne J, Wyns L, Muyldermans S (2001) Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in
the camelidae. Antimicrob Agents Chemother 45(10):2807–
2812. doi:10.1128/AAC.45.10.2807-2812.2001
100. Stijlemans B, Conrath K, Cortez-Retamozo V, Van Xong H,
Wyns  L,  Senter  P,  Revets  H,  De  Baetselier  P,  Muyldermans  S,
Magez S (2004) EYcient targeting of conserved cryptic epitopes
of infectious agents by single domain antibodies. African trypanosomes as paradigm. J Biol Chem 279(2):1256–1261.
doi:10.1074/jbc.M307341200
101. Baral TN, Magez S, Stijlemans B, Conrath K, Vanhollebeke B,
Pays E, Muyldermans S, De Baetselier P (2006) Experimental
therapy of African trypanosomiasis with a nanobody-conjugated
human trypanolytic factor. Nat Med 12(5):580–584.
doi:10.1038/nm1395
102. Saerens D, Stijlemans B, Baral T, Nguyen Thi GT, Wernery U,
Magez S, De Baetselier P, Muyldermans S, Conrath K (2008)
Parallel selection of multiple anti-infectome Nanobodies without
access to puriWed antigens. J Immunol Methods 329(1–2):138–
150. doi:10.1016/j.jim.2007.10.005
103. Ratier L, Urrutia M, Paris G, Zarebski L, Frasch AC, Goldbaum
FA (2008) Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids
single-domain antibodies. PLoS One 3(10):e3524. doi:10.1371/
journal.pone.0003524
104. Deckers N, Saerens D, Kanobana K, Conrath K, Victor B, Wernery U, Vercruysse J, Muyldermans S, Dorny P (2009) Nanobodies, a promising tool for species-speciWc diagnosis of  Taenia
solium cysticercosis. Int J Parasitol 39(5):625–633. doi:10.1016/
j.ijpara.2008.10.012
105. Dolk E, van der Vaart M, Lutje Hulsik D, Vriend G, De Haard H,
Spinelli S, Cambillau C, Frenken L, Verrips T (2005) Isolation of
llama antibody fragments for prevention of dandruV by phage
display in shampoo. Appl Environ Microbiol 71(1):442–450.
doi:10.1128/AEM.71.1.442-450.2005
106. Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC,
Flajnik MF (1995) A new antigen receptor gene family that
undergoes rearrangement and extensive somatic diversiWcation
in sharks. Nature 374(6518):168–173. doi:10.1038/374168a0
107. Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K (2009) General strategy to humanize a camelid single-domain antibody and identiWcation of a universal
humanized nanobody scaVold. J Biol Chem 284(5):3273–3284.
doi:10.1074/jbc.M806889200
108. Zou X, Smith JA, Nguyen VK, Ren L, Luyten K, Muyldermans
S, Bruggemann M (2005) Expression of a dromedary heavy
chain-only antibody and B cell development in the mouse. J
Immunol 175(6):3769–3779
109. Janssens R, Dekker S, Hendriks RW, Panayotou G, van Remoortere A, San JK, Grosveld F, Drabek D (2006) Generation of
heavy-chain-only antibodies in mice. Proc Natl Acad Sci USA
103(41):15130–15135. doi:10.1073/pnas.0601108103
110. Hofman EG, Ruonala MO, Bader AN, van den Heuvel D, Voortman J, Roovers R, Verkleij A, Gerritsen HC, van Bergen en
Henegouwen PM (2008) EGF induces coalescence of diVerent
lipid rafts. J Cell Sci 121(Pt 15):2519–2528. doi:10.1242/
jcs.028753
111. Gainkam LO, Huang L, Caveliers V, Keyaerts M, Hernot S,
Vaneycken I, Vanhove C, Revets H, De Baetselier P, Lahoutte T
(2008) Comparison of the biodistribution and tumor targeting of
two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CT. J Nucl Med 49(5):788–795. doi:10.2967/
jnumed.107.048538
112. Rahbarizadeh F, Rasaee MJ, Forouzandeh M, Allameh AA
(2006) Over expression of anti-MUC1 single-domain antibody
fragments in the yeast Pichia pastoris. Mol Immunol 43(5):426–
435. doi:10.1016/j.molimm.2005.03.003
113. Winichayakul S, Pernthaner A, Scott R, Vlaming R, Roberts N
(2008) Head-to-tail fusions of camelid antibodies can beexpressed in planta and bind in rumen  Xuid. Biotechnol Appl
Biochem 53(Pt 2):111–122
114. Saerens D, Frederix F, Reekmans G, Conrath K, Jans K, Brys L,
Huang L, Bosmans E, Maes G, Borghs G, Muyldermans S (2005)
Engineering camel single-domain antibodies and immobilization
chemistry for human prostate-speciWc antigen sensing. Anal
Chem 77(23):7547–7555. doi:10.1021/ac051092j
115. Saerens D, Kinne J, Bosmans E, Wernery U, Muyldermans S,
Conrath K (2004) Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing
conformational variants of prostate-speciWc antigen. J Biol Chem
279(50):51965–51972. doi:10.1074/jbc.M409292200
116. Chan PH, Pardon E, Menzer L, De Genst E, Kumita JR, Christodoulou J, Saerens D, Brans A, Bouillenne F, Archer DB, Robinson CV, Muyldermans S, Matagne A, RedWeld C, Wyns L,
Dobson CM, Dumoulin M (2008) Engineering a camelid antibody fragment that binds to the active site of human lysozyme
and inhibits its conversion into amyloid  Wbrils. Biochemistry
47(42):11041–11054. doi:10.1021/bi8005797
117. Chartier A, Raz V, Sterrenburg E, Verrips CT, van der Maarel
SM, Simonelig M (2009) Prevention of oculopharyngeal muscular dystrophy by muscular expression of Llama single-chain
intrabodies in vivo. Hum Mol Genet 18(10):1849–1859.
doi:10.1093/hmg/ddp101
118. van Koningsbruggen S, De Haard H, de Kievit P, Dirks RW, van
Remoortere A, Groot AJ, van Engelen BG, den Dunnen JT, Verrips CT, Frants RR, van der Maarel SM (2003) Llama-derived
phage display antibodies in the dissection of the human disease
oculopharyngeal muscular dystrophy. J Immunol Methods
279(1–2):149–161. doi:10.1016/S0022-1759(03)00232-1
119. Liu JL, Anderson GP, Goldman ER (2007) Isolation of anti-toxin
single domain antibodies from a semi-synthetic spiny dogWsh
shark display library. BMC Biotechnol 7:78. doi:10.1186/1472-
6750-7-78
120. van der Vaart JM, Pant N, Wolvers D, Bezemer S, Hermans PW,
Bellamy K, Sarker SA, van der Logt CP, Svensson L, Verrips CT,
Hammarstrom L, van Klinken BJ (2006) Reduction in morbidity
of rotavirus induced diarrhoea in mice by yeast produced
monovalent llama-derived antibody fragments. Vaccine
24(19):4130–4137. doi:10.1016/j.vaccine.2006.02.045
121. Harmsen MM, van Solt CB, Hoogendoorn A, van Zijderveld FG,
Niewold TA, van der Meulen J (2005) Escherichia coli F4 Wmbriae speciWc llama single-domain antibody fragments eVectively
inhibit bacterial adhesion in vitro but poorly protect against
diarrhoea. Vet Microbiol 111(1–2):89–98. doi:10.1016/j.vetmic.
2005.09.005
122. Henderson KA, Streltsov V, Coley A, Dolezal O, Hudson PJ,
Batchelor AH, Gupta A, Bai T, Murphy VJ, Anders RF, Foley M,
Nuttall S (2007) Structure of an IgNAR-AMA1 complex: targeting
a conserved hydrophobic cleft broadens malarial strain recognition. Structure 15(11):1452–1466. doi:10.1016/j.str.2007.09.011
123. DeLano W (2002) The PyMOL user’s manual. DeLano ScientiWc, San Carlos, CA
124. StanWeld RL, Dooley H, Flajnik MF, Wilson IA (2004) Crystal
structure of a shark single-domain antibody V region in complex
with lysozyme. Science 305(5691):1770–1773. doi:10.1126/science.1101148
125. HadWeld AT, Harvey DJ, Archer DB, MacKenzie DA, Jeenes DJ,
Radford SE, Lowe G, Dobson CM, Johnson LN (1994) Crystal
structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product. J Mol Biol 243(5):856–872. doi:10.1006/
jmbi.1994.1688
126. Braden BC, Souchon H, Eisele JL, Bentley GA, Bhat TN, Navaza
J, Poljak RJ (1994) Three-dimensional structures of the free and
the antigen-complexed Fab from monoclonal anti-lysozyme antibody D44.1. J Mol Biol 243(4):767–781. doi:10.1016/0022-
2836(94)90046-9
127. Bazl MR, Rasaee MJ, Foruzandeh M, Rahimpour A, Kiani J,
Rahbarizadeh F, Alirezapour B, Mohammadi M (2007) Production of chimeric recombinant single domain antibody-green Xuorescent fusion protein in Chinese hamster ovary cells. Hybridoma
(Larchmt) 26(1):1–9. doi:10.1089/hyb.2006.037
128. Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso
MC, Leonhardt H (2008) A versatile nanotrap for biochemical
and functional studies with Xuorescent fusion proteins. Mol Cell
Proteomics 7(2):282–289. doi:10.1074/mcp.M700342-MCP200
Keywords Single domain antibodies - Recombinant antibodies - VHH - Nanobody - Enzyme inhibitors - Virus neutralization
Download Full PDF Download
  Previous Article | Back to Volume | Next Article
Share
Search in articles
Statistics
Journal Published articles
MMI 51
Journal Hits
MMI 121898
Journal Downloads
MMI 585
Total users online -