Properties, production, and applications of camelid single-domain antibody fragments

Harmsen, M. M.; De Haard, H. J.

doi:10.1007/s00253-007-1142-2

Cited by 809Monoclonal and Polyclonal Antibodies ResearchProtein purification and stabilityAnimal Diversity and Health Studies

Properties, production, and applications of camelid single-domain antibody fragments

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Harmsen, M. M., De Haard, H. J.·2007·Applied Microbiology and Biotechnology

DOI 10.1007/s00253-007-1142-2PMC PMC2039825

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Abstract

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.

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Camelid VHH and shark V-NAR domains are used as templates to generate highly efficient and stable nanobodies.
“Given their high-affinity, solubility and stability also in absence of the partner VL domain, the camelid VH domains (VHH) [4] and the shark VH domains called V-NAR (New Antigen Receptor) [5], are currently used as templates to generate highly efficient and stable new antibody fragments collectively termed nanobodies [6,7,8].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [4]

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Naturally occurring antibodies devoid of light chains
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“As compared to human VH domains, the VHH FRs show a high sequence homology of more than 80%, and their 3D structures can be superimposed (Muyldermans et al. 2001; Holliger and Hudson 2005).”
Background
Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli
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Background
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Background
Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies
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Background
Targeting and tracing antigens in live cells with fluorescent nanobodies
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Background
Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies
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“Occasionally, VHHs with affinity constants (KD) as low as 100 pM are isolated (Saerens et al. 2004; De Genst et al. 2006; Harmsen et al. 2006), which equals the affinity ceiling proposed for natural antibodies (Sundberg and Mariuzza 2002).”
BackgroundInfluential
Single‐domain antibody fragments with high conformational stability
Dumoulin, M., Conrath, K., Meirhaeghe, A. V., Meersman, F., Heremans, K., Frenken, L. G. J., Muyldermans, S., Wyns, L., & Matagne, A. (2002) · Protein Science · doi:10.1110/ps.34602 · Cited by 641 · cited 4× · see citing works
“VHHs are especially suited for oral immunotherapy because of their resistance against extremes of pH and the capacity to bind to the target at high concentrations of chaotropic agents (Dumoulin et al. 2002, 2003).”
BackgroundInfluential
Structure-Immunogenicity Relationships of Therapeutic Proteins
Hermeling, S., Crommelin, D. J. A., Schellekens, H., & Jiskoot, W. (2004) · Pharmaceutical Research · doi:10.1023/b:pham.0000029275.41323.a6 · Cited by 617 · cited 1× · see citing works
“This could rely on their high sequence homology to conventional VH domains and on their high stability because aggregation of proteins is known to increase immunogenicity (Hermeling et al. 2004).”
Background
Single domain camel antibodies: current status
Muyldermans, S. (2001) · Reviews in Molecular Biotechnology · doi:10.1016/s1389-0352(01)00021-6 · Cited by 553 · cited 1× · see citing works
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Method
Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme
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BackgroundInfluential
Humanization of yeast to produce complex terminally sialylated glycoproteins
Hamilton, S. R., Davidson, R. C., Sethuraman, N., Nett, J. H., Jiang, Y., Rios, S., Bobrowicz, P., Stadheim, T. A., Li, H., Choi, B.-K., et al. (2006) · Science · doi:10.1126/science.1130256 · Cited by 511 · cited 2× · see citing works
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Background
Sequence and structure of V H domain from naturally occurring camel heavy chain immunoglobulins lacking light chains
Muyldermans, S., Atarhouch, T., Saldanha, J. W., Barbosa, J. A. R. G., & HAMERS, R. (1994) · Protein Engineering Design and Selection · doi:10.1093/protein/7.9.1129 · Cited by 495 · cited 7× · see citing works
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BackgroundInfluential
Potent enzyme inhibitors derived from dromedary heavy‐chain antibodies
Lauwereys, M., Ghahroudi, M. A., Desmyter, A., Kinne, J., Hölzer, W., Genst, E. D., Wyns, L., & Muyldermans, S. (1998) · The EMBO Journal · doi:10.1093/emboj/17.13.3512 · Cited by 492 · cited 2× · see citing works
“VHHs can also recognize antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites (Lauwereys et al. 1998; De Genst et al. 2006) and conserved cryptic epitopes (Stijlemans et al. 2004).”
Background
A comparison of strategies to stabilize immunoglobulin Fv-fragments
Glockshuber, R., Malia, M., Pfitzinger, I., & Plückthun, A. (1990) · Biochemistry · doi:10.1021/bi00458a002 · Cited by 473 · cited 2× · see citing works
“Such molecules (diabodies, Fig. 1a) can be produced using conventional recombinant antibodies using linkers between the VH and VL domains of a specific length, although this often results in aggregation and reduced affinity because of mispairing of VH and VL domains ( Glockshuber et al. 1990; Whitlow et al. 1993).”
Background
Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies
van der Linden, R. H. J., Frenken, L. G. J., de Geus, B., Harmsen, M. M., Ruuls, R. C., Stok, W., de Ron, L., Wilson, S., Davis, P., & Verrips, C. T. (1999) · Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology · doi:10.1016/s0167-4838(99)00030-8 · Cited by 471 · cited 3× · see citing works
“Contrary to conventional antibodies, VHHs have been shown to remain functional at 90°C (Van der Linden et al. 1999) or after incubation at high temperatures (Van der Linden et al. 1999; Perez et al. 2001).”
BackgroundSelf-citation
Advances in the production of human therapeutic proteins in yeasts and filamentous fungi
Gerngross, T. U. (2004) · Nature Biotechnology · doi:10.1038/nbt1028 · Cited by 409 · see citing works
Development trends for monoclonal antibody cancer therapeutics
Reichert, J. M., & Valge-archer, V. E. (2007) · Nature Reviews Drug Discovery · doi:10.1038/nrd2241 · Cited by 392 · cited 3× · see citing works
“They contain an N-linked oligosaccharide attached to the second heavy-chain constant domain (CH2) that is essential for antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC), and for retaining a long serum half-life.”
BackgroundInfluential
Efficient Cancer Therapy with a Nanobody-Based Conjugate
Cortez‐Retamozo, V., Backmann, N., Senter, P. D., Wernery, U., BAETSELIER, P. D., Muyldermans, S., & Revets, H. (2004) · Cancer Research · doi:10.1158/0008-5472.can-03-3935 · Cited by 383 · cited 3× · see citing works
“…FMD virus VP1 Monovalent PEG Harmsen et al. 2007 Sepsis N. meningitidis LPS Monovalent None El Khattabi et al. 2006 Cancer – CEA Monovalent β-Lactamase Cortez-Retamozo et al. 2004 Cancer – EGF receptor Bivalent Anti-albumin VHH Roovers et al. 2007 Rheumatoid arthritis – TNFα Bivalent…”
Background
β-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the Camelidae
Conrath, K., Lauwereys, M., Galleni, M., Matagne, A., Frère, J., Kinne, J., Wyns, L., & Muyldermans, S. (2001) · Antimicrobial Agents and Chemotherapy · doi:10.1128/aac.45.10.2807-2812.2001 · Cited by 348 · cited 3× · see citing works
“However, antigen binding by the C-terminal VHH in such fusions can be compromised (Conrath et al. 2001b) presumably because of steric hindrance by the N-terminal VHH.”
Background
Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR Nanobodies
Roovers, R. C., Laeremans, T., Huang, L., Taeye, S. D., Verkleij, A. J., Revets, H., de Haard, H., & Henegouwen, P. M. P. V. B. E. (2006) · Cancer Immunology Immunotherapy · doi:10.1007/s00262-006-0180-4 · Cited by 348 · cited 7× · see citing works
“Therefore, VHHs have been targeted to normally long-lived serum proteins such as albumin (Coppieters et al. 2006; Roovers et al. 2007) or immunoglobulin (Harmsen et al. 2005a) using bispecific VHHs recognizing these serum proteins in addition to the therapeutic target, resulting in half-lives that…”
BackgroundInfluential
Comparison of llama VH sequences from conventional and heavy chain antibodies
Bang, V. K., Ghahroudi, M. A., Wyns, L., & Muyldermans, S. (1997) · Molecular Immunology · doi:10.1016/s0161-5890(97)00146-6 · Cited by 344 · cited 3× · see citing works
“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al.”
Background
Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody
Nowakowski, A., Wang, C., Powers, D. B., Amersdorfer, P., Smith, T. J., Montgomery, V. A., Sheridan, R., Blake, R., Smith, L. A., & Marks, J. D. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.172229899 · Cited by 331 · cited 1× · see citing works
“The use of mixtures of a limited number of monoclonal antibodies (oligoclonal antibodies) is advantageous over single monoclonal antibodies for particular applications, such as toxin neutralization (Nowakowski et al. 2002).”
Background
Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains
Muyldermans, S., Cambillau, C., & Wyns, L. (2001) · Trends in Biochemical Sciences · doi:10.1016/s0968-0004(01)01790-x · Cited by 322 · cited 2× · see citing works
“As compared to human VH domains, the VHH FRs show a high sequence homology of more than 80%, and their 3D structures can be superimposed (Muyldermans et al. 2001; Holliger and Hudson 2005).”
Background
An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability
Whitlow, M., Bell, B. A., Feng, S.-L. Y., Filpula, D. R., Hardman, K. D., Hubert, S. L., Rollence, M. L., Wood, J. F., Schott, M. E., Milenic, D. E., et al. (1993) · Protein Engineering Design and Selection · doi:10.1093/protein/6.8.989 · Cited by 318 · cited 1× · see citing works
“1a) can be produced using conventional recombinant antibodies using linkers between the VH and VL domains of a specific length, although this often results in aggregation and reduced affinity because of mispairing of VH and VL domains (Glockshuber et al. 1990; Whitlow et al. 1993).”
Background
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
Coppieters, K. T., Dreier, T., Silence, K., de Haard, H., Lauwereys, M., Casteels, P., Beirnaert, E., Jonckheere, H., de Wiele, C. V., Staelens, L., et al. (2006) · Arthritis & Rheumatism · doi:10.1002/art.21827 · Cited by 303 · see citing works
Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs
Conrath, K., Lauwereys, M., Wyns, L., & Muyldermans, S. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m007734200 · Cited by 301 · cited 3× · see citing works
“However, antigen binding by the C-terminal VHH in such fusions can be compromised (Conrath et al. 2001b) presumably because of steric hindrance by the N-terminal VHH.”
Background
Camel heavy‐chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen‐binding repertoire
Nguyen, V. K., HAMERS, R., Wyns, L., & Muyldermans, S. (2000) · The EMBO Journal · doi:10.1093/emboj/19.5.921 · Cited by 301 · cited 1× · see citing works
“Firstly, the N-terminal part of CDR1 is more variable (Vu et al. 1997; Harmsen et al. 2000; Nguyen et al. 2000).”
Background
Isolation of antigen specific Llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae
Frenken, L. G. J., van der Linden, R. H. J., Hermans, P., Bos, J. W., Ruuls, R. C., de Geus, B., & Verrips, C. T. (2000) · Journal of Biotechnology · doi:10.1016/s0168-1656(99)00228-x · Cited by 287 · cited 6× · see citing works
“As a result, high-affinity antigenbinding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b).”
BackgroundInfluential
Efficient Targeting of Conserved Cryptic Epitopes of Infectious Agents by Single Domain Antibodies
Stijlemans, B., Conrath, K., Cortez‐Retamozo, V., Xong, H. V., Wyns, L., Senter, P. D., Revets, H., BAETSELIER, P. D., Muyldermans, S., & Magez, S. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m307341200 · Cited by 277 · cited 2× · see citing works
“VHHs can also recognize antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites (Lauwereys et al. 1998; De Genst et al. 2006) and conserved cryptic epitopes ( Stijlemans et al. 2004 ).”
Background
Aggregation-resistant domain antibodies selected on phage by heat denaturation
Jespers, L. S., Schön, O., Famm, K., & Winter, G. P. (2004) · Nature Biotechnology · doi:10.1038/nbt1000 · Cited by 266 · cited 2× · see citing works
“For further reading on these topics, we refer to recently published reviews (Muyldermans 2001; Dufner et al. 2006).”
BackgroundMethod
Selection of phage‐displayed llama single‐domain antibodies that transmigrate across human blood‐brain barrier endothelium
Muruganandam, A., Tanha, J., Narang, S. A., & Stanimirovic, D. B. (2001) · The FASEB Journal · doi:10.1096/fj.01-0343fje · Cited by 261 · cited 2× · see citing works
“For their use in targeting drugs across the blood–brain barrier (BBB) into the brain, VHHs were selected that transmigrate the human BBB in an in vitro model and accumulate in the brain after intravenous injection into mice (Muruganandam et al. 2002).”
Background
Efficient tumor targeting by single‐domain antibody fragments of camels
Cortez‐Retamozo, V., Lauwereys, M., Gh., G. H., Gobert, M., Conrath, K., Muyldermans, S., BAETSELIER, P. D., & Revets, H. (2002) · International Journal of Cancer · doi:10.1002/ijc.10212 · Cited by 260 · cited 2× · see citing works
“Until now, multiple injections of VHHs have not shown any immunogenicity in mice, as assessed by the presence of specific antibodies, T cell proliferation, or cytokine levels (Cortez-Retamozo et al. 2002; Coppieters et al. 2006).”
Background
Molecular recognition in antibody-antigen complexes
Sundberg, E. J., & Mariuzza, R. A. (2002) · Advances in protein chemistry · doi:10.1016/s0065-3233(02)61004-6 · Cited by 255 · cited 6× · see citing works
“However, the paired N-terminal variable domains of heavy (VH) and light (VL) chains are sufficient for antigen binding (Sundberg and Mariuzza 2002).”
BackgroundInfluential
Nanobodies as novel agents for cancer therapy
Revets, H., BAETSELIER, P. D., & Muyldermans, S. (2005) · Expert Opinion on Biological Therapy · doi:10.1517/14712598.5.1.111 · Cited by 254 · cited 2× · see citing works
“Similar to conventional VH domains, VHHs contain four framework regions (FRs) that form the core structure of the immunoglobulin domain and three complementarity-determining regions (CDRs) that are involved in antigen binding.”
Background
Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment
Nieba, L., Honegger, A., Krebber, C., & Plückthun, A. (1997) · Protein Engineering Design and Selection · doi:10.1093/protein/10.4.435 · Cited by 248 · cited 2× · see citing works
“Fourth, replacement of hydrophobic residues of conventional VH domains normally interacting with CH1 increased scFv production in E. coli (Nieba et al. 1997), suggesting that the hydrophilic mutations that naturally occur at these positions in VHHs also contributes to their high expression level.”
BackgroundMethod
Challenges in therapeutic glycoprotein production
Sethuraman, N., & Stadheim, T. A. (2006) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2006.06.010 · Cited by 247 · cited 2× · see citing works
“Furthermore, it could complicate their therapeutic use because the addition of yeast-specific high-mannose oligosaccharides results in a high immunogenicity and decreased serum half-life because of binding to specific mannose receptors on cells of the reticulo-endothelial system (Sethuraman and Stadheim 2006).”
Background
Engineering of therapeutic antibodies to minimize immunogenicity and optimize function
Presta, L. G. (2006) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2006.01.026 · Cited by 247 · cited 2× · see citing works
“If necessary, technologies developed to decrease immunogenicity of mouse monoclonal antibodies (Presta 2006) could also be applied to VHHs.”
Background
A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme
Dumoulin, M., Last, A. M., Desmyter, A., Decanniere, K., Canet, D., Larsson, G., Spencer, A. G., Archer, D. B., Sasse, J., Muyldermans, S., et al. (2003) · Nature · doi:10.1038/nature01870 · Cited by 245 · cited 2× · see citing works
“VHHs are especially suited for oral immunotherapy because of their resistance against extremes of pH and the capacity to bind to the target at high concentrations of chaotropic agents (Dumoulin et al. 2002, 2003).”
Background
Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features
Harmsen, M. M., Ruuls, R. C., Nijman, I. J., Niewold, T., Frenken, L. G. J., & de Geus, B. (2000) · Molecular Immunology · doi:10.1016/s0161-5890(00)00081-x · Cited by 218 · cited 5× · see citing works
“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”
BackgroundInfluentialSelf-citation
Thermal Unfolding of a Llama Antibody Fragment: A Two-State Reversible Process
Pérez, J. M. J., Renisio, J., Prompers, J. J., van Platerink, C. J., Cambillau, C., Darbon, H., & Frenken, L. G. J. (2000) · Biochemistry · doi:10.1021/bi0009082 · Cited by 200 · cited 3× · see citing works
“Contrary to conventional antibodies, VHHs have been shown to remain functional at 90°C (Van der Linden et al. 1999) or after incubation at high temperatures (Van der Linden et al. 1999; Perez et al. 2001).”
Background
Prokaryotic expression of antibodies
Arbabi‐Ghahroudi, M., Tanha, J., & MacKenzie, R. (2005) · Cancer and Metastasis Reviews · doi:10.1007/s10555-005-6193-1 · Cited by 191 · cited 2× · see citing works
“However, antibody fragments that lack the Fc with its N-linked oligosaccharide are preferably produced in microbial systems (Arbabi-Ghahroudi et al. 2005).”
Background
Biophysical Properties of Camelid VHH Domains Compared to Those of Human VH3 Domains
Ewert, S., Cambillau, C., Conrath, K., & Plückthun, A. (2002) · Biochemistry · doi:10.1021/bi011239a · Cited by 184 · cited 2× · see citing works
“This high apparent stability is mainly attributed to their efficient refolding after chemical or thermal denaturation and to a lesser extent because of an increased resistance against denaturation (Perez et al. 2001; Dumoulin et al. 2002; Ewert et al. 2002).”
Background
Facile Generation of Heat-Stable Antiviral and Antitoxin Single Domain Antibodies from a Semisynthetic Llama Library
Goldman, E. R., Anderson, G. P., Liu, J. L., Delehanty, J. B., Sherwood, L. J., Osborn, L. E., Cummins, L. B., & Hayhurst, A. (2006) · Analytical Chemistry · doi:10.1021/ac0610053 · Cited by 183 · cited 2× · see citing works
“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”
Background
Pentamerization of Single-domain Antibodies from Phage Libraries: A Novel Strategy for the Rapid Generation of High-avidity Antibody Reagents
Zhang, J., Tanha, J., Hirama, T., Khieu, N. H., To, R., Tong-Sevinc, H., Stone, E., Brisson, J., & MacKenzie, C. R. (2003) · Journal of Molecular Biology · doi:10.1016/j.jmb.2003.09.034 · Cited by 171 · cited 2× · see citing works
“The avidity of VHHs has also been strongly increased using genetic fusions to the B-subunits of an E. coli toxin that selfassembles into a homopentamer (Zhang et al. 2004), resulting in pentameric recombinant antibodies (“pentabodies,” Fig.”
Background
Antigen Specificity and High Affinity Binding Provided by One Single Loop of a Camel Single-domain Antibody
Desmyter, A., Decanniere, K., Muyldermans, S., & Wyns, L. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m102107200 · Cited by 168 · cited 3× · see citing works
“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”
Background
Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor
Baral, T. N., Magez, S., Stijlemans, B., Conrath, K., Vanhollebeke, B., Pays, É., Muyldermans, S., & BAETSELIER, P. D. (2006) · Nature Medicine · doi:10.1038/nm1395 · Cited by 166 · cited 3× · see citing works
“For example, sleeping sickness was successfully treated with VHHs that bind to a trypanosome coat protein and were fused to the apolipoprotein L-1 enzyme, resulting in trypanosome lysis (Baral et al. 2006).”
BackgroundInfluential
Camelid Heavy-Chain Variable Domains Provide Efficient Combining Sites to Haptens
Spinelli, S., Frenken, L. G. J., Hermans, P., Verrips, T. C., Brown, K., Tegoni, M., & Cambillau, C. (2000) · Biochemistry · doi:10.1021/bi991830w · Cited by 163 · see citing works
The Subunits of Purified Rabbit Antibody*
Utsumi, S., & Karush, F. (1964) · Biochemistry · doi:10.1021/bi00897a024 · Cited by 158 · cited 2× · see citing works
“Although isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al.”
Background
Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability
Davies, J., & Riechmann, L. (1996) · Protein Engineering Design and Selection · doi:10.1093/protein/9.6.531 · Cited by 155 · cited 1× · see citing works
“…human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005).”
Background
Elbow motion in the immunoglobulins involves a molecular ball-and-socket joint
Lesk, A. M., & Chothia, C. (1988) · Nature · doi:10.1038/335188a0 · Cited by 152 · cited 7× · see citing works
“Fourth, replacement of hydrophobic residues of conventional VH domains normally interacting with CH1 increased scFv production in E. coli (Nieba et al. 1997), suggesting that the hydrophilic mutations that naturally occur at these positions in VHHs also contributes to their high expression level.”
BackgroundInfluential
Lactobacilli Expressing Variable Domain of Llama Heavy‐Chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus‐Induced Diarrhea
Pant, N., Hultberg, A., Zhao, Y., Svensson, L., Pan‐Hammarström, Q., Johansen, K., Pouwels, P. H., Ruggeri, F. M., Hermans, P., Frenken, L. G. J., et al. (2006) · The Journal of Infectious Diseases · doi:10.1086/508747 · Cited by 152 · cited 3× · see citing works
“Thus, diarrhea could also be prevented by lactobacilli that produce rotavirus-neutralizing VHHs fused to a cell surface anchor (Pant et al. 2006).”
BackgroundInfluential
The crystal structure of a llama heavy chain variable domain
Spinelli, S., Frenken, L. A. M., Bourgeois, D., de Ron, L., Bos, W., Verrips, T. C., Anguille, C., Cambillau, C., & TEGONI, M. (1996) · Nature Structural & Molecular Biology · doi:10.1038/nsb0996-752 · Cited by 148 · cited 2× · see citing works
“Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains.”
Background
Single Domain Antibodies Derived from Dromedary Lymph Node and Peripheral Blood Lymphocytes Sensing Conformational Variants of Prostate-specific Antigen
Saerens, D., Kinne, J., Bosmans, E., Wernery, U., Muyldermans, S., & Conrath, K. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m409292200 · Cited by 148 · cited 4× · see citing works
“Occasionally, VHHs with affinity constants (KD) as low as 100 pM are isolated (Saerens et al. 2004; De Genst et al. 2006; Harmsen et al. 2006), which equals the affinity ceiling proposed for natural antibodies (Sundberg and Mariuzza 2002).”
BackgroundInfluential
Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor
Streltsov, V. A., Varghese, J. N., Carmichael, J. A., Irving, R. A., Hudson, P. J., & Nuttall, S. D. (2004) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0403509101 · Cited by 141 · cited 2× · see citing works
“This contrasts with shark single-domain antibodies that have a vestigial CDR2 that does not contribute to antigen binding (Streltsov et al. 2004).”
Background
Antibody VH Domains as Small Recognition Units
Davies, J., & Riechmann, L. (1995) · Nature Biotechnology · doi:10.1038/nbt0595-475 · Cited by 141 · cited 2× · see citing works
“…human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005).”
BackgroundResult
Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama
van der Linden, R. H. J., de Geus, B., Stok, W., Bos, W., van Wassenaar, D., Verrips, T. C., & Frenken, L. A. M. (2000) · Journal of Immunological Methods · doi:10.1016/s0022-1759(00)00188-5 · Cited by 140 · cited 3× · see citing works
“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”
Background
Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo
Dolk, E., van der Vaart, M., Hulsik, D. L., Vriend, G., de Haard, H., Spinelli, S., Cambillau, C., Frenken, L. G. J., & Verrips, T. C. (2005) · Applied and Environmental Microbiology · doi:10.1128/aem.71.1.442-450.2005 · Cited by 130 · cited 2× · see citing works
“Although VHHs are highly suited for applications that require a high stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to…”
Background
Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy
Harmsen, M. M., van Solt, C. B., Bemmel, A. M. V. Z.-V., Niewold, T., & van Zijderveld, F. G. (2006) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-005-0300-7 · Cited by 123 · cited 5× · see citing works
“However, by selection for proteolytic stability, a VHH could be isolated from the original library that was not degraded in vivo (Harmsen et al. 2006).”
BackgroundInfluentialSelf-citation
Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH properties
Tanha, J., Dubuc, G., Hirama, T., Narang, S. A., & MacKenzie, C. R. (2002) · Journal of Immunological Methods · doi:10.1016/s0022-1759(02)00027-3 · Cited by 123 · cited 8× · see citing works
“Methods to isolate antigen-specific VHHs from immune (Arbabi-Ghahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune ( Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”
BackgroundResultInfluential
Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library
Yau, K. Y. F., Groves, M. A. T., Li, S., Sheedy, C., Lee, H., Tanha, J., MacKenzie, C. R., Jermutus, L., & Hall, J. C. (2003) · Journal of Immunological Methods · doi:10.1016/j.jim.2003.07.011 · Cited by 119 · cited 3× · see citing works
“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”
Background
Antigen Binding and Solubility Effects upon the Veneering of a Camel VHH in Framework-2 to Mimic a VH
Conrath, K., Vincke, C., Stijlemans, B., Schymkowitz, J. W. H., Decanniere, K., Wyns, L., Muyldermans, S., & Loris, R. (2005) · Journal of Molecular Biology · doi:10.1016/j.jmb.2005.04.050 · Cited by 117 · cited 2× · see citing works
“…human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005).”
Background
Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments
Vandervaart, J. P., Pant, N., Wolvers, D. A. W., Bezemer, S., Hermans, P., Bellamy, K., Sarker, S. A., VANDERLOGT, C., Svensson, L., & Verrips, C. T. (2006) · Vaccine · doi:10.1016/j.vaccine.2006.02.045 · Cited by 112 · cited 5× · see citing works
“VHHs that successfully prevented diarrhea caused by rotavirus in a mouse model were similarly selected for resistance against the acidic environment of the stomach ( Van der Vaart et al. 2006 ).”
BackgroundInfluential
Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120
Zwick, M. B., Parren, P. W. H. I., Saphire, E. O., Church, S., Wang, M., Scott, J. K., Dawson, P. E., Wilson, I. A., & Burton, D. R. (2003) · Journal of Virology · doi:10.1128/jvi.77.10.5863-5876.2003 · Cited by 108 · see citing works
An S-Layer Heavy Chain Camel Antibody Fusion Protein for Generation of a Nanopatterned Sensing Layer To Detect the Prostate-Specific Antigen by Surface Plasmon Resonance Technology
Pleschberger, M., Saerens, D., Weigert, S., Sleytr, U. B., Muyldermans, S., Sára, M., & Egelseer, E. M. (2004) · Bioconjugate Chemistry · doi:10.1021/bc049964w · Cited by 105 · cited 2× · see citing works
“…stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to focus on their therapeutic applications, which are more challenging.”
Background
Contributions of CDR3 to VHH Domain Stability and the Design of Monobody Scaffolds for Naive Antibody Libraries
Bond, C. J., Marsters, J. C., & Sidhu, S. S. (2003) · Journal of Molecular Biology · doi:10.1016/s0022-2836(03)00967-7 · Cited by 100 · cited 1× · see citing works
“In addition to these specific mutations, the packing of extended CDR3 loops against this former VL interface contributes to domain stability (Bond et al. 2003).”
Background
Generation of llama single-domain antibodies against methotrexate, a prototypical hapten
Alvarez-Rueda, N., Béhar, G., Ferré, V., Pugnière, M., Roquet, F., Gastinel, L. N., Jacquot, C., Aubry, J., Baty, D., Barbet, J., et al. (2006) · Molecular Immunology · doi:10.1016/j.molimm.2006.08.007 · Cited by 99 · cited 1× · see citing works
“However, hapten- and peptide-binding VHHs have been successfully isolated using strong selection systems (Spinelli et al. 2000; Yau et al. 2003; Alvarez-Rueda et al. 2007; Harmsen et al. 2007).”
Background
Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts
Sagt, C. M. J., Kleizen, B., Verwaal, R., de Jong, M. D. M., Müller, W. H., Smits, A., Visser, C. J., Boonstra, J., Verkleij, A. J., & Verrips, C. T. (2000) · Applied and Environmental Microbiology · doi:10.1128/aem.66.11.4940-4944.2000 · Cited by 94 · cited 2× · see citing works
“First, the presence of a potential N-linked glycosylation site often increases production levels in yeast (Sagt et al. 2000).”
Background
Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris
Rahbarizadeh, F., Rasaee, M. J., Forouzandeh, M., & Allameh, A. (2005) · Molecular Immunology · doi:10.1016/j.molimm.2005.03.003 · Cited by 90 · cited 2× · see citing works
“VHH production by the favored yeast expression host Pichia pastoris was only recently described (Rahbarizadeh et al. 2006).”
Background
Generation of heavy-chain-only antibodies in mice
Janssens, R., Dekker, S., Hendriks, R. W., Panayotou, G., van Remoortere, A., San, J. K. A., Grosveld, F. G., & Drabek, D. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0601108103 · Cited by 88 · cited 2× · see citing works
“Furthermore, transgenic mice containing hybrid llama/ human antibody loci that contain llama V regions and human D, J, and C regions have recently been used to generate human heavy-chain antibodies in mice (Janssens et al. 2006).”
Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins
Harmsen, M. M., van Solt, C. B., Fijten, H., & Setten, M. C. V. (2005) · Vaccine · doi:10.1016/j.vaccine.2005.05.017 · Cited by 85 · cited 9× · see citing works
“As a result, high-affinity antigenbinding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b).”
BackgroundInfluentialSelf-citation
Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease
Harmsen, M. M., van Solt, C. B., Fijten, H., van Keulen, L. J. M., Rosalia, R. A., Weerdmeester, K., Cornelissen, A. H. M., de Bruin, M. G. M., Eblé, P. L., & Dekker, A. (2006) · Veterinary Microbiology · doi:10.1016/j.vetmic.2006.10.029 · Cited by 85 · cited 5× · see citing works
“However, in contrast to the full protection afforded by the hyperimmune serum, these VHHs poorly protected guinea pigs from FMD viral challenge infection, suggesting that Fc-mediated effector functions are required for efficient in vivo protection (Harmsen et al. 2007).”
BackgroundInfluentialSelf-citation
Harnessing phage and ribosome display for antibody optimisation
Dufner, P., Jermutus, L., & Minter, R. (2006) · Trends in biotechnology · doi:10.1016/j.tibtech.2006.09.004 · Cited by 81 · cited 3× · see citing works
“2000b), as has often been done for conventional antibody fragments (Dufner et al. 2006).”
BackgroundMethod
Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains
Olichon, A., Schweizer, D., Muyldermans, S., & de Marco, A. (2007) · BMC Biotechnology · doi:10.1186/1472-6750-7-7 · Cited by 81 · see citing works
Retargeting serum immunoglobulin with bispecific diabodies
Holliger, P., Wing, M. G., Pound, J. D., Bohlen, H., & Winter, G. P. (1997) · Nature Biotechnology · doi:10.1038/nbt0797-632 · Cited by 79 · cited 1× · see citing works
“Although such effector functions can be indirectly recruited using bispecific (conventional) antibody fragments binding to host immunoglobulin (Holliger et al. 1997), it may be more efficient to recruit these functions by fusing VHHs to host Fc domains.”
Background
Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography
Verheesen, P., ten Haaft, M. R., Lindner, N. M., Verrips, C. T., & de Haard, J. J. W. (2003) · Biochimica et Biophysica Acta (BBA) - General Subjects · doi:10.1016/j.bbagen.2003.09.006 · Cited by 76 · cited 1× · see citing works
“…a high stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to focus on their therapeutic applications, which are more…”
Background
Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation
Verheesen, P., Roussis, A., de Haard, H., Groot, A. J., Stam, J. C., den Dunnen, J. T., Frants, R. R., Verkleij, A. J., Verrips, C. T., & van der Maarel, S. M. (2006) · Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics · doi:10.1016/j.bbapap.2006.05.011 · Cited by 69 · cited 2× · see citing works
“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”
Background
Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea
Harmsen, M. M., van Solt, C. B., Hoogendoorn, A., van Zijderveld, F., Niewold, T., & van der Meulen, J. (2005) · Veterinary Microbiology · doi:10.1016/j.vetmic.2005.09.005 · Cited by 62 · cited 13× · see citing works
“As a result, high-affinity antigenbinding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b).”
BackgroundInfluentialSelf-citation
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
Harrison, R. A., Hasson, S. S., Harmsen, M. M., Laing, G. D., Conrath, K., & Theakston, R. D. G. (2005) · Toxicon · doi:10.1016/j.toxicon.2005.10.017 · Cited by 60 · cited 2× · see citing works
“2004), in vivo diagnosis using imaging, and treatment of snake bites (Harrison et al. 2006).”
BackgroundSelf-citation
Antibody phage display : methods and protocols
Aitken, R. (2002) · Cited by 58 · see citing works
Large-scale production of VHH antibody fragments by Saccharomyces cerevisiae
Thomassen, Y. E., Meijer, W., Sierkstra, L. N., & Verrips, C. T. (2002) · Enzyme and Microbial Technology · doi:10.1016/s0141-0229(01)00497-5 · Cited by 55 · cited 1× · see citing works
“VHHs have also often been produced in the yeast Saccharomyces cerevisiae (Frenken et al. 2000; Thomassen et al. 2002; Van der Vaart 2002).”
Background
Identification of single‐domain, Bax‐specific intrabodies that confer resistance to mammalian cells against oxidative‐stress‐induced apoptosis
Gueorguieva, D., Li, S., Walsh, N., Mukerji, A., Tanha, J., & Pandey, S. (2006) · The FASEB Journal · doi:10.1096/fj.06-6306fje · Cited by 53 · cited 5× · see citing works
“Finally, Bax-specific VHHs have been expressed in the cytoplasm, resulting in so-called intrabodies, to prevent oxidative-stress-induced apoptosis that is implicated in several neurodegenerative diseases (Gueorguieva et al. 2006).”
BackgroundInfluential
Specific Binding Activity of Isolated Light Chains of Antibodies
Yoo, T. J., Roholt, O. A., & Pressman, D. (1967) · Science · doi:10.1126/science.157.3789.707 · Cited by 52 · cited 2× · see citing works
“Although isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al. 1967) can retain antigenbinding specificity, their affinity and solubility is often reduced (Ward et al. 1989).”
Background
Dimerisation strategies for shark IgNAR single domain antibody fragments
Simmons, D. P., Abregu, F. A., Krishnan, U. V., Proll, D. F., Streltsov, V. A., Doughty, L., Hattarki, M. K., & Nuttall, S. D. (2006) · Journal of Immunological Methods · doi:10.1016/j.jim.2006.07.019 · Cited by 52 · cited 1× · see citing works
“Third, the presence of unpaired C-terminal cysteines reduces expression levels (Simmons et al. 2006).”
Background
Improved production and function of llama heavy chain antibody fragments by molecular evolution
van der Linden, R. H. J., de Geus, B., Frenken, L. G. J., Peters, H., & Verrips, C. T. (2000) · Journal of Biotechnology · doi:10.1016/s0168-1656(00)00274-1 · Cited by 52 · cited 3× · see citing works
“Methods to isolate antigen-specific VHHs from immune (ArbabiGhahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established.”
Background
Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source
van de Laar, T., Visser, C., Holster, M., López, C. G., Kreuning, D., Sierkstra, L. N., Lindner, N. M., & Verrips, T. C. (2006) · Biotechnology and Bioengineering · doi:10.1002/bit.21150 · Cited by 40 · see citing works
High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli
Rahbarizadeh, F., Rasaee, M. J., Forouzandeh‐Moghadam, M., & Allameh, A. (2005) · Protein Expression and Purification · doi:10.1016/j.pep.2005.04.008 · Cited by 38 · cited 2× · see citing works
“VHHs have often been produced in E. coli (ArbabiGhahroudi et al. 1997; Rahbarizadeh et al. 2005).”
Background
Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries
Krüger, C., Hultberg, A., Marcotte, H., Hermans, P., Bezemer, S., Frenken, L. G. J., & Hammarström, L. L. G. (2006) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-006-0347-0 · Cited by 37 · cited 4× · see citing works
“…Pant et al. 2006 Piglet diarrhea E. coli F4 fimbriae Monovalent None Harmsen et al. 2006 Caries S. mutans I/II adhesion Monovalent None Kruger et al. 2006 FMD FMD virus VP1 Monovalent PEG Harmsen et al. 2007 Sepsis N. meningitidis LPS Monovalent None El Khattabi et al. 2006 Cancer –…”
BackgroundInfluential
Llama Single-Chain Antibody That Blocks Lipopolysaccharide Binding and Signaling: Prospects for Therapeutic Applications
Khattabi, M. E., Adams, H., Heezius, E. C. J. M., Hermans, P., Detmers, F. J. M., Maassen, B., van der Ley, P., Tommassen, J., Verrips, T. C., & Stam, J. C. (2006) · Clinical and Vaccine Immunology · doi:10.1128/cvi.00107-06 · Cited by 35 · cited 4× · see citing works
“Piglet diarrhea E. coli F4 fimbriae Monovalent None Harmsen et al. 2006 Caries S. mutans I/II adhesion Monovalent None Kruger et al. 2006 FMD FMD virus VP1 Monovalent PEG Harmsen et al. 2007 Sepsis N. meningitidis LPS Monovalent None El Khattabi et al. 2006”
BackgroundInfluential
Heavy‐chain only antibodies derived from dromedary are secreted and displayed by mouse B cells
Nguyen, V. K., Zou, X., Lauwereys, M., Brys, L., Brüggemann, M., & Muyldermans, S. (2003) · Immunology · doi:10.1046/j.1365-2567.2003.01633.x · Cited by 33 · cited 1× · see citing works
“Production of such functional antibodies requires the correct glycosylation of the CH2 domain, which until recently could only be accomplished using higher eukaryotic cells (Nguyen et al. 2003) but not by microbial production.”
Background
Specific production rate of VHH antibody fragments by Saccharomyces cerevisiae is correlated with growth rate, independent of nutrient limitation
Thomassen, Y. E., Verkleij, A. J., Boonstra, J., & Verrips, C. T. (2005) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2005.05.010 · Cited by 28 · cited 2× · see citing works
“In baker’s yeast, the specific VHH production rate is correlated with growth rate (Thomassen et al. 2005) and can be up to fivefold increased by growing on ethanol as the carbon source (Van de Laar et al.”
Background
Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)
Joosten, V., Roelofs, M. S., van den Dries, N., Goosen, T., Verrips, C. T., van den Hondel, C. A. M. J. J., & Lokman, B. C. (2005) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2005.06.034 · Cited by 25 · cited 1× · see citing works
“There is only one example of VHH production in filamentous fungi, which resulted in limited proteolytic degradation of the secreted product (Joosten et al. 2005) because of the high levels of proteases secreted by filamentous fungi (Gerngross 2004).”
Background
“Baculophage:” A new tool for protein display
Davies, A. H. (1995) · Nature Biotechnology · doi:10.1038/nbt1095-1046 · Cited by 24 · see citing works
Studies on the antibody response of Lama glama — evaluation of the binding capacity of different IgG subtypes in ELISAs for clenbuterol and BSA
Lange, I. G., Daxenberger, A., & MEYER, H. H. D. (2001) · Veterinary Immunology and Immunopathology · doi:10.1016/s0165-2427(01)00376-2 · Cited by 22 · cited 1× · see citing works
“Indeed, llamas immunized with clenbuterol developed conventional but not heavychain antibodies against this hapten (Lange et al. 2001).”
Background
Expression of VHH Antibody Fragments in Saccharomyces cerevisiae
van der Vaart, J. M. (2003) · Humana Press eBooks · doi:10.1385/1-59259-240-6:359 · Cited by 18 · see citing works

Flagged first, then by citations

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Nanobodies: natural single-domain antibodies
Muyldermans, S. (2013) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-063011-092449 · Cited by 2,304 · see citing works
Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing
Yang, B., Treweek, J. B., Kulkarni, R. P., Deverman, B. E., Chen, C., Lubeck, E., Shah, S., Cai, L., & Gradinaru, V. (2014) · Cell · doi:10.1016/j.cell.2014.07.017 · Cited by 933 · see citing works
The therapeutic potential of nanobodies
Jovčevska, I., & Muyldermans, S. (2020) · BioDrugs · doi:10.1007/s40259-019-00392-z · Cited by 749 · see citing works
Overcoming the Blood–Brain Barrier: The Role of Nanomaterials in Treating Neurological Diseases
Furtado, D., Björnmalm, M., Ayton, S., Bush, A. I., Kempe, K., & Caruso, F. (2018) · Advanced Materials · doi:10.1002/adma.201801362 · Cited by 708 · see citing works
PEGylation of therapeutic proteins
Jevševar, S., Kunstelj, M., & Porekar, V. G. (2010) · Biotechnology Journal · doi:10.1002/biot.200900218 · Cited by 681 · see citing works
“An example of a successful application of PEGylation to nanobodies is the PEGylated nanobody neutralizing foot and mouth disease (FMD) virus [15].”
Background
General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold
Vincke, C., Loris, R., Saerens, D., Martínez‐Rodríguez, S., Muyldermans, S., & Conrath, K. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m806889200 · Cited by 577 · see citing works
Single domain antibodies: promising experimental and therapeutic tools in infection and immunity
Wesolowski, J., Alzogaray, V., Reyelt, J., Unger, M., Juárez‐Moreno, K., Urrutia, M., Cauerhff, A., Danquah, W., Rissiek, B., Scheuplein, F., et al. (2009) · Medical Microbiology and Immunology · doi:10.1007/s00430-009-0116-7 · Cited by 516 · see citing works
“sdAbs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells [8].”
Background
A robust pipeline for rapid production of versatile nanobody repertoires
Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., Oeffinger, M., Nussenzweig, M. C., Fenyö, D., Chait, B. T., et al. (2014) · Nature Methods · doi:10.1038/nmeth.3170 · Cited by 493 · see citing works
Emerging new therapeutic antibody derivatives for cancer treatment
Jin, S., Sun, Y., Xiao, L., Gu, X., Ning, J., Xu, Y., Chen, S., & Pan, L. (2022) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-021-00868-x · Cited by 461 · see citing works
Methods for converting cysteine to dehydroalanine on peptides and proteins
Chalker, J. M., Gunnoo, S. B., Boutureira, O., Gerstberger, S. C., Fernández‐González, M., Bernardes, G. J. L., Griffin, L., Hailu, H., Schofield, C. J., & Davis, B. G. (2011) · Chemical Science · doi:10.1039/c1sc00185j · Cited by 367 · see citing works
The state-of-play and future of antibody therapeutics
Elgundi, Z., Reslan, M., Cruz, E., Sifniotis, V., & Kayser, V. (2016) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2016.11.004 · Cited by 318 · see citing works
Preclinical screening of anti‐HER2 nanobodies for molecular imaging of breast cancer
Vaneycken, I., Devoogdt, N., Gassen, N. V., Vincke, C., Xavier, C., Wernery, U., Muyldermans, S., Lahoutte, T., & Caveliers, V. (2011) · The FASEB Journal · doi:10.1096/fj.10-180331 · Cited by 315 · see citing works
Development trends for therapeutic antibody fragments
Nelson, A. L., & Reichert, J. M. (2009) · Nature Biotechnology · doi:10.1038/nbt0409-331 · Cited by 305 · see citing works
Highly Sensitive and Multiplexed Protein Measurements
Cohen, L., & Walt, D. R. (2018) · Chemical Reviews · doi:10.1021/acs.chemrev.8b00257 · Cited by 294 · see citing works
Antibody fragments as nanoparticle targeting ligands: a step in the right direction
Richards, D. A., Maruani, A., & Chudasama, V. (2016) · Chemical Science · doi:10.1039/c6sc02403c · Cited by 294 · see citing works
Nanobody: The “Magic Bullet” for Molecular Imaging?
Chakravarty, R., Goel, S., & Cai, W. (2014) · Theranostics · doi:10.7150/thno.8006 · Cited by 288 · see citing works
“The detailed procedures for these processes have been well-documented in the literature and will not be discussed here [34, 37, 40].”
BackgroundInfluential
Phage Display Derived Monoclonal Antibodies: From Bench to Bedside
Alfaleh, M. A., Alsaab, H. O., Mahmoud, A. B., Alkayyal, A. A., Jones, M. L., Mahler, S. M., & Hashem, A. M. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.01986 · Cited by 280 · see citing works
Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies/VHH Domains
Schmitz, K. R., Bagchi, A., Roovers, R. C., Henegouwen, P. M. P. V. B. E., & Ferguson, K. M. (2013) · Structure · doi:10.1016/j.str.2013.05.008 · Cited by 251 · see citing works
CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells
Jähnichen, S., Blanchetot, C., Maussang, D., González-Pajuelo, M., Chow, K. Y. C., Bosch, L., Vrieze, S. D., Serruys, B., Ulrichts, H., Vandevelde, W., et al. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1012865107 · Cited by 251 · see citing works
“Nanobodies have several favorable biological and physicochemical characteristics, including low immunogenicity, increased tissue penetration, high solubility, and physical stability (3).”
Background
Techniques for the analysis of protein-protein interactions in vivo
Xing, S., Wallmeroth, N., Berendzen, K. W., & Grefen, C. (2016) · PLANT PHYSIOLOGY · doi:10.1104/pp.16.00470 · Cited by 246 · see citing works
David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments
Bates, A., & Power, C. A. (2019) · Antibodies · doi:10.3390/antib8020028 · Cited by 237 · see citing works
The roles of TNF in brain dysfunction and disease
Clark, I. A., Alleva, L. M., & Vissel, B. (2010) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2010.08.007 · Cited by 232 · see citing works
From seeing to believing: labelling strategies for in vivo cell-tracking experiments
Progatzky, F., Dallman, M. J., & Celso, C. L. (2013) · Interface Focus · doi:10.1098/rsfs.2013.0001 · Cited by 232 · see citing works
A comprehensive comparison between camelid nanobodies and single chain variable fragments
Asaadi, Y., Jouneghani, F. F., Janani, S., & Rahbarizadeh, F. (2021) · Biomarker Research · doi:10.1186/s40364-021-00332-6 · Cited by 209 · see citing works
“VL and VH exhibit only 53% and 51% sequence identity, respectively, with corresponding regions in humans [72], while nanobodies display high sequence similarity with human VH (VH3 gene family) with ~75–90% identity correlated with their Low immunogenicity in clinical applications [73, 74].”
Background
Targeting tumors with nanobodies for cancer imaging and therapy
Oliveira, S., Heukers, R., Sornkom, J., Kok, R. J., & Henegouwen, P. M. P. V. B. E. (2013) · Journal of Controlled Release · doi:10.1016/j.jconrel.2013.08.298 · Cited by 206 · see citing works
Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules
Hultberg, A., Temperton, N. J., Rosseels, V., Koenders, M., González-Pajuelo, M., Schepens, B., Ibañez, L. I., Vanlandschoot, P., Schillemans, J. P., Saunders, M. J., et al. (2011) · PLoS ONE · doi:10.1371/journal.pone.0017665 · Cited by 195 · see citing works
“VHHs are small (12– 15 kDa), stable molecules with improved solubility and similar affinities as conventional antibodies [10].”
Alternative affinity tools: more attractive than antibodies?
Ruigrok, V. J. B., Levisson, M., Eppink, M. H. M., Smidt, H., & John van der, O. O. S. T. (2011) · Biochemical Journal · doi:10.1042/bj20101860 · Cited by 193 · see citing works
Strategies for overcoming protein and peptide instability in biodegradable drug delivery systems
Shi, M., & McHugh, K. J. (2023) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2023.114904 · Cited by 194 · see citing works
Next generation matrix metalloproteinase inhibitors — Novel strategies bring new prospects
Levin, M., Udi, Y., Solomonov, I., & Sagi, I. (2017) · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research · doi:10.1016/j.bbamcr.2017.06.009 · Cited by 185 · see citing works
Single-domain antibodies as building blocks for novel therapeutics
Saerens, D., Ghassabeh, G. H., & Muyldermans, S. (2008) · Current Opinion in Pharmacology · doi:10.1016/j.coph.2008.07.006 · Cited by 186 · see citing works
“However, an in vitro affinity maturation step of random mutation and selection (such as an error-prone PCR and ribosome display selections) of the selected clones might be required to obtain more potent antigen binders [13–15] with affinities matching those from ‘immune’ libraries.”
Background
Insights into maleimide-thiol conjugation chemistry: Conditions for efficient surface functionalization of nanoparticles for receptor targeting
Martínez-Jothar, L., Doulkeridou, S., Schiffelers, R. M., Toraño, J. S., Oliveira, S., van Nostrum, C. F., & Hennink, W. E. (2018) · Journal of Controlled Release · doi:10.1016/j.jconrel.2018.03.002 · Cited by 186 · see citing works
Pros and cons of different therapeutic antibody formats for recombinant antivenom development
Laustsen, A. H., Gutiérrez, J. M., Knudsen, C., Johansen, K. H., Bermúdez-Méndez, E., Cerni, F. A., Jürgensen, J. A., Ledsgaard, L., Martos-Esteban, A., Øhlenschlæger, M., et al. (2018) · Toxicon · doi:10.1016/j.toxicon.2018.03.004 · Cited by 174 · see citing works
Nanobodies® † †Nanobody is a registered trademark of Ablynx NV. as inhaled biotherapeutics for lung diseases
Heeke, G. V., Allosery, K., Brabandere, V. D., Smedt, T. D., Detalle, L., & de Fougerolles, A. (2016) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2016.06.012 · Cited by 172 · see citing works
Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy
Hu, Y., Liu, C., & Muyldermans, S. (2017) · Frontiers in Immunology · doi:10.3389/fimmu.2017.01442 · Cited by 171 · see citing works
“Altogether, most Nbs exhibit a high stability against elevated temperature, high pressure, or chemical denaturants as demonstrated by thermo fluorescence or circular dichroism measurements (34, 51, 52).”
Background
Macrophage migration inhibitory factor (MIF) as a therapeutic target for rheumatoid arthritis and systemic lupus erythematosus
Bilsborrow, J. B., Doherty, E. H., Tilstam, P. V., & Bucala, R. (2019) · Expert Opinion on Therapeutic Targets · doi:10.1080/14728222.2019.1656718 · Cited by 169 · see citing works
Molecular and cellular analysis of human histamine receptor subtypes
Seifert, R., Straßer, A., Schneider, E. H., Neumann, D., Dove, S., & Buschauer, A. (2012) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2012.11.001 · Cited by 168 · see citing works
Immunological and molecular diagnosis of cysticercosis
Rodríguez, S., Wilkins, P. P., & Dorny, P. (2012) · Pathogens and Global Health · doi:10.1179/2047773212y.0000000048 · Cited by 157 · see citing works
Neutralization of Clostridium difficile Toxin A with Single-domain Antibodies Targeting the Cell Receptor Binding Domain
Hussack, G., Arbabi‐Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K.-S., MacKenzie, R., & Tanha, J. (2011) · Journal of Biological Chemistry · doi:10.1074/jbc.m110.198754 · Cited by 153 · see citing works
Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease
Rotman, M., Welling, M. M., Bunschoten, A., de Backer, M. E., Rip, J., Nabuurs, R. J. A., Gaillard, P. J., van Buchem, M. A., van der Maarel, S. M., & van der Weerd, L. (2015) · Journal of Controlled Release · doi:10.1016/j.jconrel.2015.02.012 · Cited by 152 · see citing works
Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications
Wang, Y., Fan, Z., Shao, L., Kong, X., Hou, X., Tian, D., Sun, Y., Xiao, Y., & Yu, L. (2016) · International Journal of Nanomedicine · doi:10.2147/ijn.s107194 · Cited by 151 · see citing works
“10, 34–37 research, bioimaging, clinical diagnosis, therapeutics, and agricultural and plant protection.”
Subcutaneous envafolimab monotherapy in patients with advanced defective mismatch repair/microsatellite instability high solid tumors
Li, J., Deng, Y., Zhang, W., Zhou, A., Guo, W., Yang, J., Yuan, Y., Zhu, L., Qin, S., Xiang, S., et al. (2021) · Journal of Hematology & Oncology · doi:10.1186/s13045-021-01095-1 · Cited by 151 · see citing works
“Although they tend to have a short half-life due to rapid renal clearance, they are more soluble and stable than full monoclonal antibodies and, importantly, more rapidly penetrate tissues; collectively, these properties enable subcutaneous administration [16].”
Background
Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo
Maussang, D., Mujić-Delić, A., Descamps, F. J., Stortelers, C., Vanlandschoot, P., van Walsum, M., Vischer, H. F., Roy, M. V., Vosjan, M. J. W. D., González-Pajuelo, M., et al. (2013) · Journal of Biological Chemistry · doi:10.1074/jbc.m113.498436 · Cited by 148 · see citing works
Nanobody-Based Apolipoprotein E Immunosensor for Point-of-Care Testing
Ren, X. Z., Yan, J., Wu, D., Wei, Q. L., & Wan, Y. (2017) · ACS Sensors · doi:10.1021/acssensors.7b00495 · Cited by 143 · see citing works
Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection
Tiwari, P. M., Vanover, D. A., Lindsay, K. E., Bawage, S. S., Kirschman, J. L., Bhosle, S. M., Lifland, A. W., Zurla, C., & Santangelo, P. J. (2018) · Nature Communications · doi:10.1038/s41467-018-06508-3 · Cited by 139 · see citing works
Affinity separation and enrichment methods in proteomic analysis
Fang, X., & Zhang, W. (2008) · Journal of Proteomics · doi:10.1016/j.jprot.2008.06.011 · Cited by 140 · see citing works
VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI′
Hmila, I., Abderrazek, R. B., Saerens, D., Benlasfar, Z., Conrath, K., Ayeb, M. E., Muyldermans, S., & Bouhaouala‐Zahar, B. (2008) · Molecular Immunology · doi:10.1016/j.molimm.2008.04.011 · Cited by 140 · see citing works
Nanobodies®: New ammunition to battle viruses
Vanlandschoot, P., Stortelers, C., Beirnaert, E., Ibañez, L. I., Schepens, B., Depla, E., & Saelens, X. (2011) · Antiviral Research · doi:10.1016/j.antiviral.2011.09.002 · Cited by 135 · see citing works
Nanobodies – from llamas to therapeutic proteins
Kolkman, J. A., & Law, D. A. (2010) · Drug Discovery Today Technologies · doi:10.1016/j.ddtec.2010.03.002 · Cited by 136 · see citing works
Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments
Sandomenico, A., Sivaccumar, J. P., & Ruvo, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176324 · Cited by 134 · see citing works
“Moreover, the high-affinity binding of VHH and V-NAR domains is likely supported by an increased length of the majority of the CDR3 loops (3–28 amino acids) compared to that of the VH domains [13].”
BackgroundInfluential
Therapeutic Antibodies: Current State and Future Trends – Is a Paradigm Change Coming Soon?
Dimitrov, D. S., & Marks, J. D. (2009) · Methods in Molecular Biology™ · doi:10.1007/978-1-59745-554-1_1 · Cited by 132 · see citing works
Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy
Sun, S., Ding, Z., Yang, X., Zhao, X., Zhao, M., Gao, L., Chen, Q., Xie, S., Liu, A., Yin, S., et al. (2021) · International Journal of Nanomedicine · doi:10.2147/ijn.s297631 · Cited by 131 · see citing works
Immuno-imaging using nanobodies
Vaneycken, I., D’Huyvetter, M., Hernot, S., Vos, J. D., Xavier, C., Devoogdt, N., Caveliers, V., & Lahoutte, T. (2011) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2011.06.009 · Cited by 130 · see citing works
“These VHHs or nanobodies have an excellent expression yield in both Escherichia coli and yeast and a good solubility compared to most mouse or human two-chains-derived antibody-fragments [7,8].”
Background
mRNA as novel technology for passive immunotherapy
Schlake, T., Theß, A., Thran, M., & Jordan, I. (2018) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-018-2935-4 · Cited by 132 · see citing works
“It has been shown that small single-domain antibodies could even cross the blood-brain barrier [83] .”
Background
Molecular Targeting of Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor (VEGFR)
Kaufman, N. E. M., Dhingra, S., Jois, S. D. S., & Vicente, M. G. H. (2021) · Molecules · doi:10.3390/molecules26041076 · Cited by 134 · see citing works
“This single Ig domain is stable and can be generated rapidly and cheaply with simple expression systems [74] and are called VHH domains.”
Background
IgA Nephropathy: Molecular Mechanisms of the Disease
Mestecky, J. F., Raška, M., Julian, B. A., Gharavi, A. G., Renfrow, M. B., Moldoveanu, Z., Novak, L., Matoušovic, K., & Novák, J. (2012) · Annual Review of Pathology Mechanisms of Disease · doi:10.1146/annurev-pathol-011110-130216 · Cited by 129 · see citing works
Recent advances in the selection and identification of antigen-specific nanobodies
Liu, W., Song, H., Chen, Q., Yu, J., Xian, M., Nian, R., & Feng, D. (2018) · Molecular Immunology · doi:10.1016/j.molimm.2018.02.012 · Cited by 127 · see citing works
“With a molecular weight of approximately 12–15 kDa, nanobodies are reported to be the smallest intact antigen-binding single polypeptide chains found in any natural antibody (Arbabi Ghahroudi et al., 1997; Harmsen and De Haard, 2007; Muyldermans, 2013).”
In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT
Vaneycken, I., Govaert, J., Vincke, C., Caveliers, V., Lahoutte, T., BAETSELIER, P. D., Raes, G., Bossuyt, A. M., Muyldermans, S., & Devoogdt, N. (2010) · Journal of Nuclear Medicine · doi:10.2967/jnumed.109.069823 · Cited by 126 · see citing works
“Because of their small size and the high degree of identity of their framework sequence to human VH proteins, it is expected that Nanobodies will exhibit a low immunogenicity (23).”
Background
Engineered affinity proteins—Generation and applications
Grönwall, C., & Ståhl, S. (2009) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2009.01.014 · Cited by 122 · see citing works
Single-Domain Antibodies or Nanobodies: A Class of Next-Generation Antibodies
Khodabakhsh, F., Behdani, M., Rami, A., & Kazemi‐Lomedasht, F. (2018) · International Reviews of Immunology · doi:10.1080/08830185.2018.1526932 · Cited by 119 · see citing works
“Additional structural features of HCAbs such as high percentage of hydrophilic amino acids in variable domains, high number of mutational hot spots and the disulfide bonds in antigen-binding site discriminate them from conventional antibodies [4].”
Background
Immunoglobulin domains inEscherichia coliand other enterobacteria: from pathogenesis to applications in antibody technologies
Bodelón, G., Palomino, C. M., & Fernández, L. Á. (2012) · FEMS Microbiology Reviews · doi:10.1111/j.1574-6976.2012.00347.x · Cited by 115 · see citing works
Pre-Clinical Intravenous Serum Pharmacokinetics of Albumin Binding and Non-Half-Life Extended Nanobodies®
Hoefman, S., Ottevaere, I., Baumeister, J., & Sargentini‐Maier, M. L. (2015) · Antibodies · doi:10.3390/antib4030141 · Cited by 117 · see citing works
G Protein–Coupled Receptors in Asthma Therapy: Pharmacology and Drug Action
Wendell, S. G., Fan, H., & Zhang, C. (2019) · Pharmacological Reviews · doi:10.1124/pr.118.016899 · Cited by 117 · see citing works
“…are associated with several advantages, such as better solubility and tissue penetration, low immunogenicity, and high physical stability, and they can be easily produced in prokaryotic or eukaryotic host organisms (Harmsen and De Haard, 2007; Joost and Kolkman, 2010; Klarenbeek et al., 2012).”
Background
High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models
Li, W., Schäfer, A., Kulkarni, S. S., Liu, X., Martinez, D. R., Chen, C., Sun, Z., Leist, S. R., Drelich, A. K., Zhang, L., et al. (2020) · Cell · doi:10.1016/j.cell.2020.09.007 · Cited by 117 · see citing works
Therapeutic Antibodies against Intracellular Tumor Antigens
Trenevska, I., Li, D., & Banham, A. H. (2017) · Frontiers in Immunology · doi:10.3389/fimmu.2017.01001 · Cited by 113 · see citing works
“These can be engineered from conventional human Igs, or also from those from camelids (camel or llama) and cartilaginous fish (carpet or nurse sharks), whose immune systems were found to have evolved high-affinity V-like domains fused to a conserved framework that is reflective of the constant Fc region found in human…”
Background
Nanobody; an Old Concept and New Vehicle for Immunotargeting
Rahbarizadeh, F., Ahmadvand, D., & Sharifzadeh, Z. (2011) · Immunological Investigations · doi:10.3109/08820139.2010.542228 · Cited by 116 · see citing works
Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis
Yu, X., Yang, Y.-P., Dikici, E., Deo, S. K., & Daunert, S. (2017) · Annual Review of Analytical Chemistry · doi:10.1146/annurev-anchem-061516-045205 · Cited by 113 · see citing works
“This is also possible because nanobodies are capable of recognizing concave-shaped antigens (15).”
Background
Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview
Goldman, E. R., Liu, J. L., Zabetakis, D., & Anderson, G. P. (2017) · Frontiers in Immunology · doi:10.3389/fimmu.2017.00865 · Cited by 111 · see citing works
“For example, ~90% of VHHs contain a Trp at position 118 (20, 55).”
Background
Fundamentals and application of ordered molecular assemblies to affinity biosensing
Matharu, Z., Bandodkar, A. J., Gupta, V., & Malhotra, B. D. (2011) · Chemical Society Reviews · doi:10.1039/c1cs15145b · Cited by 109 · see citing works
Clostridium difficile infection: molecular pathogenesis and novel therapeutics
Rineh, A., Kelso, M. J., Vatansever, F., Tegos, G. P., & Hamblin, M. R. (2014) · Expert Review of Anti-infective Therapy · doi:10.1586/14787210.2014.866515 · Cited by 111 · see citing works
From Desert to Medicine: A Review of Camel Genomics and Therapeutic Products
Ali, A., Baby, B., & Vijayan, R. (2019) · Frontiers in Genetics · doi:10.3389/fgene.2019.00017 · Cited by 108 · see citing works
Advances in the design of nanomaterial-based electrochemical affinity and enzymatic biosensors for metabolic biomarkers: A review
Farzin, L., Shamsipur, M., Samandari, L., & Sheibani, S. (2018) · Microchimica Acta · doi:10.1007/s00604-018-2820-8 · Cited by 108 · see citing works
Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody
Pruszyński, M., Koumarianou, E., Vaidyanathan, G., Revets, H., Devoogdt, N., Lahoutte, T., & Zalutsky, M. R. (2012) · Nuclear Medicine and Biology · doi:10.1016/j.nucmedbio.2012.08.008 · Cited by 105 · see citing works
“With a molecular weight of 12–15 kDa, Nbs are an order of magnitude smaller than intact mAbs, and smaller than Fab (~50 kDa) and scFv (~25 kDa)fragments [8].”
Antibody Fragments and Their Purification by Protein L Affinity Chromatography
Rodrigo, G., Gruvegård, M., & Alstine, J. M. V. (2015) · Antibodies · doi:10.3390/antib4030259 · Cited by 103 · see citing works
Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy
Sha, H., Zou, Z., Xin, K., Bian, X., Cai, X., Lu, W., Chen, J., Chen, G., Huang, L., Blair, A. M., et al. (2014) · Journal of Controlled Release · doi:10.1016/j.jconrel.2014.12.039 · Cited by 103 · see citing works
Ozoralizumab, a Humanized Anti-TNFα NANOBODY® Compound, Exhibits Efficacy Not Only at the Onset of Arthritis in a Human TNF Transgenic Mouse but Also During Secondary Failure of Administration of an Anti-TNFα IgG
Ishiwatari-Ogata, C., Kyuuma, M., Ogata, H., Yamakawa, M., Iwata, K., Ochi, M., Hori, M., Miyata, N., & Fujii, Y. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.853008 · Cited by 102 · see citing works
“NANOBODY®, the variable heavy chain domains of heavy-chain antibody (VHH), retains all of the antigen-binding activities of heavy-chain antibodies and possesses unique characteristics, including low molecular weight (12–15 kDa), rapid tissue penetration, and low immunogenicity (19, 20).”
Background
Highly Sensitive Single Domain Antibody–Quantum Dot Conjugates for Detection of HER2 Biomarker in Lung and Breast Cancer Cells
Rakovich, T. Y., Mahfoud, O. K., Mohamed, B. M., Prina‐Mello, A., Crosbie-Staunton, K., Broeck, T. V. D., Kimpe, L. D., Sukhanova, A. V., Baty, D., Rakovich, A., et al. (2014) · ACS Nano · doi:10.1021/nn500212h · Cited by 102 · see citing works
A modular IgG-scFv bispecific antibody topology
Orcutt, K. D., Ackerman, M. E., Cieslewicz, M., Quiroz, E., Slusarczyk, A. L., Frangioni, J. V., & Wittrup, K. D. (2009) · Protein Engineering Design and Selection · doi:10.1093/protein/gzp077 · Cited by 101 · see citing works
GPCR-targeting nanobodies: attractive research tools, diagnostics, and therapeutics
Mujić-Delić, A., de Wit, R. H., Verkaar, F., & Smit, M. J. (2014) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2014.03.003 · Cited by 100 · see citing works
Principles and Current Clinical Landscape of Multispecific Antibodies against Cancer
Elshiaty, M., Schindler, H., & Christopoulos, P. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22115632 · Cited by 98 · see citing works
“Interestingly, the antigen specificity of most antibodies shows a predominant dependence on the CDRs contributed by mainly the VH rather than the VL domain, which takes an extreme form in the heavy chain-only antibodies naturally produced by camelids and sharks [29], and is exploited by “singledomain antibodies” (sdAb…”
Background
Anti-CTLA-4 nanobody as a promising approach in cancer immunotherapy
Babamohamadi, M., Mohammadi, N., Faryadi, E., Haddadi, M., Merati, A., Ghobadinezhad, F., Amirian, R., Izadi, Z., & Hadjati, J. (2024) · Cell Death and Disease · doi:10.1038/s41419-023-06391-x · Cited by 101 · see citing works
Anti-EGFR-iRGD recombinant protein modified biomimetic nanoparticles loaded with gambogic acid to enhance targeting and antitumor ability in colorectal cancer treatment
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MINI-REVIEW Properties, production, and applications of camelid single-domain antibody fragments
Harmsen, M. M., & de Haard, H. (2013)
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Authors: Harmsen, M. M., & De Haard, H. J.
Affiliations: Wageningen University & Research (NL)
Corresponding author: Harmsen, M. M.
ORCID: Harmsen, M. M.
Venue: Applied Microbiology and Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 2007-08-18
Volume: 77
Issue: 1
Pages: 13-22
ISSN: 0175-7598 (ISSN-L)
1432-0614
DOI: 10.1007/s00253-007-1142-2
PMC: PMC2039825
OpenAlex: W2147451568
Cited by: 809
References: 96
Citations by year: 2026: 31 · 2025: 43 · 2024: 48 · 2023: 41 · 2022: 41 · 2021: 65 · 2020: 57 · 2019: 49 · 2018: 53 · 2017: 42 · 2016: 46 · 2015: 48 · 2014: 53 · 2013: 40 · 2012: 52
FWCI: 2.67
Type: journal article
Language: en
Open access: hybrid
doi.org
Licence: cc-by
creativecommons.org/licenses/by/4.0 (tdm)
creativecommons.org/licenses/by/4.0 (vor)
Locations: Applied Microbiology and Biotechnology (publishedVersion, cc-by)
PubMed Central (publishedVersion, cc-by)
PubMed (publishedVersion)
Socio-Environmental Systems Modeling (acceptedVersion)
Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
Topics: Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Life Sciences › Agricultural and Biological Sciences › Food Science › Animal Diversity and Health Studies
Keywords: single-domain antibody fragments, VHH, nanobodies, camelid antibodies, recombinant antibody production, yeast expression system, phage display, ribosome display, yeast surface display, therapeutic applications
MeSH terms: Animals, Camelids, New World, Immunoglobulin Fragments, Immunoglobulin Heavy Chains, Models, Biological

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