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Properties, production, and applications of camelid single-domain antibody fragments
Open accessAbstract
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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“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
Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hammers, C., Songa, E. B., Bendahman, N., & Hammers, R. (1993) · Nature · doi:10.1038/363446a0 · Cited by 3,241 · cited 4×
“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.”
BackgroundInfluentialRapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries
Jostock, T., Vanhove, M., Brepoels, E., van Gool, R., Daukandt, M., Wehnert, A., van Hegelsom, R., Dransfield, D., Sexton, D., Devlin, M., et al. (2004) · Journal of Immunological Methods · doi:10.1016/j.jim.2004.03.014 · Cited by 2,572
Engineered antibody fragments and the rise of single domains
Holliger, P., & Hudson, P. J. (2005) · Nature Biotechnology · doi:10.1038/nbt1142 · Cited by 1,928 · cited 1×
“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).”
BackgroundWard, E. S., Güssow, D., Griffiths, A. D., Jones, P., & Winter, G. P. (1989) · Nature · doi:10.1038/341544a0 · Cited by 1,010 · cited 2×
“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).”
BackgroundGreenberg, A. S., Avila, D. W., Hughes, M. K., Hughes, A. L., McKinney, E. C., & Flajnik, M. F. (1995) · Nature · doi:10.1038/374168a0 · Cited by 783 · cited 2×
“Although single-domain antibodies later were also identified in particular cartilaginous fish (Greenberg et al. 1995), most research on the biotechnological application of singledomain antibodies was done using camelids because of Appl Microbiol Biotechnol (2007) 77:13–22”
BackgroundSelection and identification of single domain antibody fragments from camel heavy‐chain antibodies
Ghahroudi, M. A., Desmyter, A., Wyns, L., HAMERS, R., & Muyldermans, S. (1997) · FEBS Letters · doi:10.1016/s0014-5793(97)01062-4 · Cited by 775 · cited 1×
“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.”
BackgroundTargeting and tracing antigens in live cells with fluorescent nanobodies
Rothbauer, U., Zolghadr, K., Tillib, S. V., Nowak, D., Schermelleh, L., Gahl, A., Backmann, N., Conrath, K., Muyldermans, S., Cardoso, M. C., et al. (2006) · Nature Methods · doi:10.1038/nmeth953 · Cited by 715 · cited 1×
“Because of their stability, VHHs are especially suited for intrabody production because this requires expression in the reducing environment of the cytoplasm (Gueorguieva et al. 2006; Rothbauer et al. 2006).”
BackgroundMolecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies
Genst, E. D., Silence, K., Decanniere, K., Conrath, K., Loris, R., Kinne, J., Muyldermans, S., & Wyns, L. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0505379103 · Cited by 681 · cited 4×
“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).”
BackgroundInfluentialSingle‐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×
“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).”
BackgroundInfluentialStructure-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×
“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).”
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- Estell D (2006) Adapting industry practices for the rapid, large-scale manufacture of pharmaceutical proteins. Bridge 36:39–44
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Wang, Y., Wu, J., Huang, X., & Xiao, Y. (2025) · Immunotherapy Advances · doi:10.1093/immadv/ltag012
“These findings not only support the use of CD7 CAR T cells as a bridge to allo-HSCT but also identify antigen loss as a major resistance mechanism [68, 69].”
Wang, B., Lee, W.-H., Hu, Y., Yeap, Y. Y. C., Ngoh, E. Z. X., Soh, M. K., Salleh, S. N. M., Hussein, N. I. N. B., Tan, D., Pavesi, A., et al. (2026) · Molecular Therapy · doi:10.1016/j.ymthe.2026.07.041
Gonzalez-Prada, J. E., Liu, S., Shang, C., Chernoff, C. S., Bright, D. P., Mortensen, M., Jones, C. F., Nestorow, S. A., Kasaragod, V. B., Chen, W.-N., et al. (2026) · Science Advances · doi:10.1126/sciadv.aeg3548
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“Compared with full-length immunoglobulins, single-domain antibody fragments are structurally simpler and do not require assembly of separate heavy and light chains, making microbial production particularly attractive [3, 8, 9].”
Camelid VHHs as a Versatile Platform for Combating Viral Infections
Mufrrih, M., & Alandijany, T. A. (2026) · Pharmaceuticals · doi:10.3390/ph19091408
Kim, K., Chun, W., Yang, H., Lee, Y., & Chung, J. (2026) · Biomolecules · doi:10.3390/biom16101523
BindingDB Entry 50004792: Small molecules that target protein misfolding.
BindingDB (2020) · UC San Diego · doi:10.7270/q2qj7m50
Dovgan, T., Dharadhar, S., Maggio, A. D., Fjelldal, P. G., Wargelius, A., Baum, E., Nijenhuis, W., den Broeder, M. J., Schulz, R. W., Henegouwen, P. V. B. E., et al. (2026) · General and Comparative Endocrinology · doi:10.1016/j.ygcen.2026.115043
Metadata
- Authors
- Harmsen, M. M., & De Haard, H. J.
- Affiliations
- Wageningen University & Research (NL)
- Ablynx (Belgium) (BE)
- Corresponding author
- Harmsen, M. M.
- Venue
- Applied Microbiology and Biotechnology
- Published
- 2007-08-18
- Volume
- 77
- Issue
- 1
- Pages
- 13-22
- ISSN
- 0175-7598 (ISSN-L)
- 1432-0614
- 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
- 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
- Life Sciences › Immunology and Microbiology › Immunology › 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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