Single-domain antibody


Single-domain antibody
Ribbon diagram of a llama VHH domain.
The extended CDR3 loop is coloured orange.

A single-domain antibody (sdAb, called Nanobody by Ablynx, the developer[1]) is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies (150–160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain).[2]

The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids; these are called VHH fragments. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to bind specifically to target epitopes.[3]

Single-domain antibodies are being researched for multiple pharmaceutical applications and have potential for use in the treatment of acute coronary syndrome, cancer and Alzheimer's disease.[4][5]

Contents

Properties

A single-domain antibody is a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG. These peptides have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high concentrations of urea. Those derived from camelid and fish antibodies are less lipophilic and better soluble in water, owing to their complementarity determining region 3 (CDR3), which forms an extended loop (coloured orange in the ribbon diagram above) covering the lipophilic site that normally binds to a light chain.[6][7] In contrast to common antibodies, two out of six single-domain antibodies survived a temperature of 90 °C (194 °F) without losing their ability to bind antigens in a 1999 study.[8] Stability towards gastric acid and proteases depends on the amino acid sequence. Some species have been shown to be active in the intestine after oral application,[9][10] but their low absorption from the gut impedes the development of systemically active single-domain antibodies.

The comparatively low molecular mass leads to a better permeability in tissues, and to a short plasma half-life since they are eliminated renally.[2] Unlike whole antibodies, they do not show complement system triggered cytotoxicity because they lack an Fc region. Camelid and fish derived sdAbs are able to bind to hidden antigens that are not accessible to whole antibodies, for example to the active sites of enzymes. This property has been shown to result from their extended CDR3 loop, which is able to penetrate such sites.[7][11]

Production

A shark (left) and a camelid (middle) heavy-chain antibody in comparison to a common antibody (right). Heavy chains are shown in a darker shade, light chains in a lighter shade. VH and VL are the variable domains.

From heavy-chain antibodies

A single-domain antibody can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. By reverse transcription and polymerase chain reaction, a gene library of single-domain antibodies containing several million clones is produced. Screening techniques like phage display and ribosome display help to identify the clones binding the antigen.[12]

A different method uses gene libraries from animals that have not been immunized beforehand. Such naïve libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step.[13]

When the most potent clones have been identified, their DNA sequence is optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization is unproblematic because of the homology between camelid VHH and human VH fragments.[13] The final step is the translation of the optimised single-domain antibody in E. coli, Saccharomyces cerevisiae or other suitable organisms.

From conventional antibodies

Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains.[14] The process is similar, comprising gene libraries from immunized or naïve donors and display techniques for identification of the most specific antigens. A problem with this approach is that the binding region of common IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization is usually accomplished by replacing lipophilic by hydrophilic amino acids, but often results in a loss of affinity to the antigen.[15] If affinity can be retained, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other organisms.

Potential applications

Orally available single-domain antibodies against E. coli-induced diarrhoea in piglets have been developed and successfully tested.[10] Other diseases of the gastrointestinal tract, such as inflammatory bowel disease and colon cancer, are also possible targets for orally available sdAbs.[16]

Detergent stable species targeting a surface protein of Malassezia furfur have been engineered for the use in anti-dandruff shampoos.[6]

ALX-0081, a single-domain antibody targeting von Willebrand factor is in clinical trials for the prevention of thrombosis in patients with acute coronary syndrome.[17] A Phase II study examining ALX-0081 in high risk percutaneous coronary intervention has started in September 2009.[18]

Ablynx expects that their Nanobodies might cross the blood-brain barrier and permeate into large solid tumours more easily than whole antibodies, which would allow for the development of drugs against brain cancers.[16]

References

  1. ^ Gibbs, W. Wayt (August 2005). "Nanobodies". Scientific American Magazine. http://www.sciam.com/article.cfm?chanID=sa006&articleID=0004C949-F886-12DB-B88683414B7F0000. 
  2. ^ a b Harmsen MM, De Haard HJ (November 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. PMC 2039825. PMID 17704915. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2039825. 
  3. ^ Möller, A.; Pion, E; Narayan, V; Ball, KL (September 2010). "Intracellular activation of interferon regulatory factor-1 by nanobodies to the multi-functional (Mf1) domain". The Journal of biological chemistry (J Biol Chem) 285 (49): 38348–38361. doi:10.1074/jbc.M110.149476. PMC 2992268. PMID 20817723. http://www.jbc.org/content/early/2010/09/03/jbc.M110.149476.abstract. 
  4. ^ "Nanobodies herald a new era in cancer therapy". The Medical News. 12 May 2004. http://www.news-medical.net/?id=1477. 
  5. ^ "Pipeline". Ablynx. http://www.ablynx.com/research/pipeline.htm. Retrieved 20 January 2010. 
  6. ^ a b Dolk, E.; Van Der Vaart, M.; Lutje Hulsik, D.; Vriend, G.; De Haard, H.; Spinelli, S.; Cambillau, C.; Frenken, L. et al. (2005). "Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo". Applied and Environmental Microbiology 71 (1): 442–450. doi:10.1128/AEM.71.1.442-450.2005. PMC 544197. PMID 15640220. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=544197.  edit
  7. ^ a b Stanfield, R.; Dooley, H.; Flajnik, M.; Wilson, I. (2004). "Crystal structure of a shark single-domain antibody V region in complex with lysozyme". Science 305 (5691): 1770–1773. Bibcode 2004Sci...305.1770S. doi:10.1126/science.1101148. PMID 15319492.  edit
  8. ^ Van Der Linden, R.; Frenken, L.; De Geus, B.; Harmsen, M.; Ruuls, R.; Stok, W.; De Ron, L.; Wilson, S. et al. (1999). "Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies". Biochimica et Biophysica Acta 1431 (1): 37–46. doi:10.1016/S0167-4838(99)00030-8. PMID 10209277.  edit
  9. ^ Harmsen, M. .; Vansolt, C. .; Hoogendoorn, A. .; Vanzijderveld, F. .; Niewold, T. .; Vandermeulen, J. . (2005). "Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea". Veterinary Microbiology 111 (1–2): 89. doi:10.1016/j.vetmic.2005.09.005. PMID 16221532.  edit
  10. ^ a b Harmsen, M. M.; Solt, C. B.; Zijderveld-Van Bemmel, A. M.; Niewold, T. A.; Zijderveld, F. G. (2006). "Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy". Applied Microbiology and Biotechnology 72 (3): 544–551. doi:10.1007/s00253-005-0300-7. PMID 16450109.  edit
  11. ^ Desmyter, A.; Transue, T. R.; Ghahroudi, M. A.; Thi, M. H.; Poortmans, F.; Hamers, R.; Muyldermans, S.; Wyns, L. (1996). "Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme". Nature structural biology 3 (9): 803–811. doi:10.1038/nsb0996-803. PMID 8784355.  edit
  12. ^ Arbabi Ghahroudi, M.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. (1997). "Selection and identification of single domain antibody fragments from camel heavy-chain antibodies". FEBS letters 414 (3): 521–526. doi:10.1016/S0014-5793(97)01062-4. PMID 9323027.  edit
  13. ^ a b Saerens, D.; Ghassabeh, G.; Muyldermans, S. (2008). "Single-domain antibodies as building blocks for novel therapeutics". Current Opinion in Pharmacology 8 (5): 600–608. doi:10.1016/j.coph.2008.07.006. PMID 18691671.  edit
  14. ^ Holt, L. J.; Herring, C.; Jespers, L. S.; Woolven, B. P.; Tomlinson, I. M. (2003). "Domain antibodies: proteins for therapy". Trends in biotechnology 21 (11): 484–490. doi:10.1016/j.tibtech.2003.08.007. PMID 14573361.  edit
  15. ^ Borrebaeck, C. A. K.; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189. doi:10.1038/nbt1202-1189. PMID 12454662.  edit
  16. ^ a b "Nanobodies". Nanobody.org. 2006. http://www.nanobody.org. 
  17. ^ "Ablynx Announces Interim Results of First Nanobody Phase I Study of,ALX-0081 (ANTI-VWF)". Bio-Medicine.org. 2 July 2007. http://www.bio-medicine.org/medicine-technology/Ablynx-Announces-Interim-Results-of-First-Nanobody-Phase-I-Study-of-0AALX-0081--28ANTI-VWF-29-135-1. 
  18. ^ ClinicalTrials.gov NCT01020383 Comparative Study of ALX-0081 Versus GPIIb/IIIa Inhibitor in High Risk Percutaneous Coronary Intervention (PCI) Patients

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