Affibody molecule

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Affibody molecules are small proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies, and are therefore a member of the family of antibody mimetics. Affibody molecules are used in biochemical research and are being developed as potential new biopharmaceutical drugs.


As with other antibody mimetics, the idea behind developing the Affibody molecule was to apply a combinatorial protein engineering approach on a small and robust protein scaffold. The aim was to generate new binders capable of specific binding to different target proteins, while retaining the favorable folding and stability properties, and ease of bacterial expression of the parent molecule.[1][2]

The original Affibody protein scaffold was designed based on the Z domain (the immunoglobulin G binding domain) of protein A. In contrast to antibodies, Affibody molecules are composed of alpha helices and lack disulfide bridges. The parent three-helix bundle structure is currently the fastest folding protein structure known.[3]

Affibody molecules with unique binding properties are acquired by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. Lately, amino acids outside of the binding surface have been substituted in the scaffold to create a surface entirely different from the ancestral protein A domain.

Specific Affibody molecules binding a desired target protein can be “fished out” from pools (libraries) containing billions of different variants, using phage display.


Affibody molecules are based on a three-helix bundle domain, which can be expressed in soluble and proteolytically stable forms in various host cells on its own or via fusion with other protein partners.[4]

They tolerate modification and are independently folding when incorporated into fusion proteins. Head-to-tail fusions of Affibody molecules of the same specificity have proven to give avidity effects in target binding, and head-to-tail fusion of Affibody molecules of different specificities makes it possible to get bi- or multi-specific affinity proteins. Fusions with other proteins can also be created genetically[5][6] or by spontaneous isopeptide bond formation.[7] A site for site-specific conjugation is facilitated by introduction of a single cysteine at a desired position.

A number of different Affibody molecules have been produced by chemical synthesis. Since they do not contain cysteines or disulfide bridges, they fold spontaneously and reversibly into the correct three-dimensional structures when the protection groups are removed after synthesis.[8][9] In some studies, temperatures above the melting temperature have been used, with retained binding properties following return to ambient conditions.[10] Cross-linked variants have been produced as well.


An Affibody molecule consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa. A monoclonal antibody, for comparison, is 150 kDa, and a single-domain antibody, the smallest type of antigen-binding antibody fragment, 12–15 kDa.

Affibody molecules have been shown to withstand high temperatures (90 °C) or acidic and alkaline conditions (pH 2.5 or pH 11, respectively).[11][12][13]

Binders with an affinity of down to sub-nanomolar have been obtained from naïve library selections, and binders with picomolar affinity have been obtained following affinity maturation.[14] Affibodies conjugated to weak electrophiles bind their targets covalently.[15]


Affibody molecules can be used for protein purification,[8] enzyme inhibition,[10] research reagents for protein capture and detection,[16][17] diagnostic imaging[14] and targeted therapy.[18] The HER2/neu specific Affibody ABY-025 is in clinical development for tumor diagnosis.[19]


  1. ^ Nord, K; Nilsson, J; Nilsson, B; Uhlén, M; Nygren, P-A (1995). "A combinatorial library of an α-helical bacterial receptor domain". Prot. Eng. 8 (6): 601–608. doi:10.1093/protein/8.6.601. PMID 8532685. 
  2. ^ Nord, K; Gunneriusson, E; Ringdahl, J; Ståhl, S; Uhlén, M; Nygren, P-A (1997). "Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain". Nature Biotechnology. 15 (8): 772–777. doi:10.1038/nbt0897-772. PMID 9255793. 
  3. ^ Arora, P; Oas, T; Myers, J (2004). "Fast and faster: A designed variant of the B-domain of protein A folds in 3 μsec". Protein Sci. 13 (4): 847–853. doi:10.1110/ps.03541304. PMC 2280057Freely accessible. PMID 15044721. 
  4. ^ Ståhl, S; Nygren, P-A (1997). "The use of gene fusions to protein A and protein G in immunology and biotechnology". Pathol. Biol. Paris. 45 (1): 66–76. PMID 9097850. 
  5. ^ Rönnmark, J; Hansson, M; Nguyen, T; Uhlén, M; Robert, A; Ståhl, S; Nygren, P-A (2002). "Construction and characterization of affibody-Fc chimeras produced in Escherichia coli". J. Immunol. Methods. 261 (1–2): 199–211. doi:10.1016/S0022-1759(01)00563-4. PMID 11861078. 
  6. ^ Rönnmark, J; Kampf, C; Asplund, A; Höiden-Guthénberg, I; Wester, K; Pontén, F; Uhlén, M; Nygren, P-A (2003). "Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol". J. Immunol. Methods. 281 (1–2): 149–160. doi:10.1016/j.jim.2003.06.001. PMID 14580889. 
  7. ^ Veggiani, G; Nakamura, T; Brenner, M; Gayet, R; Yan, J; Robinson, C; Howarth, M (2016). "Programmable polyproteams built using twin peptide superglues". PNAS. 113 (5): 1202–1207. doi:10.1073/pnas.1519214113. PMID 26787909. 
  8. ^ a b Nord, K; Nord, O; Uhlén, M; Kelley, B; Ljungqvist, C; Nygren, P-A (2001). "Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A". Eur. J. Biochem. 268 (15): 1–10. doi:10.1046/j.1432-1327.2001.02344.x. PMID 11488921. 
  9. ^ Engfeldt, T; Renberg, B; Brumer, H; Nygren, P-A; Karlström, EA (2005). "Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein". Chem. BioChem. 6 (6): 1043–1050. doi:10.1002/cbic.200400388. PMID 15880677. 
  10. ^ a b "Phusion Hot Start High-Fidelity DNA Polymerase". Finnzymes. 
  11. ^ Ahlgren, S; Wållberg, H; Tran, TA; Widström, C; Hjertman, M; Abrahmsén, L; Berndorff, D; Dinkelborg, LM; et al. (2009). "Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine". J. Nucl. Med. 50 (5): 781–789. doi:10.2967/jnumed.108.056929. PMID 19372467. 
  12. ^ Orlova, A; Rosik, D; Sandström, M; Lundqvist, H.; Einarsson, L; Tolmachev, V (2007). "Evaluation of [(111/114m)In]CHX-A"-DTPA-ZHER2:342, an affibody ligand conjugate for targeting of HER2-expressing malignant tumors". Q. J. Nucl. Med. Mol. Imaging. 51 (4): 314–23. PMID 17464277. 
  13. ^ Tran, T; Engfeldt, T; Orlova, A; Sandström, M; Feldwisch, J; Abrahmsén, L; Wennborg, A; Tolmachev, V; et al. (2007). "(99m)Tc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors". Bioconjug. Chem. 18 (6): 1956–64. doi:10.1021/bc7002617. PMID 17944527. 
  14. ^ a b Orlova, A; Magnusson, M; Eriksson, TL; Nilsson, M; Larsson, B; Höidén-Guthenberg, I; Widström, C; Carlsson, J; et al. (2006). "Tumor imaging using a picomolar affinity HER2 binding affibody molecule". Cancer Res. 66 (8): 4339–48. doi:10.1158/0008-5472.CAN-05-3521. PMID 16618759. 
  15. ^ Holm, L; Moody, P; Howarth, M (2009). "Electrophilic Affibodies Forming Covalent Bonds to Protein Targets". The Journal of Biological Chemistry. 284 (47): 32906–13. doi:10.1074/jbc.M109.034322. PMC 2781706Freely accessible. PMID 19759009. 
  16. ^ Renberg, B; Nordin, J; Merca, A; Uhlén, M; Feldwisch, J; Nygren, P-A; Karlström, AE (2007). "Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats". J. Proteome Res. 6 (1): 171–179. doi:10.1021/pr060316r. PMID 17203961. 
  17. ^ Lundberg, E; Höidén-Guthenberg, I; Larsson, B; Uhlén, M; Gräslund, T (2007). "Site-specifically conjugated anti-HER2 Affibody molecules as one-step reagents for target expression analyses on cells and xenograft samples". J. Immunol. Methods. 319 (1–2): 53–63. doi:10.1016/j.jim.2006.10.013. PMID 17196217. 
  18. ^ Tolmachev, V; Orlova, A; Pehrson, R; Galli, J; Baastrup, B; Andersson, K; Sandström, M; Rosik, D; et al. (2007). "Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule". Cancer Res. 67 (6): 2773–82. doi:10.1158/0008-5472.CAN-06-1630. PMID 17363599. 
  19. ^ Gebauer, M; Skerra, A (2009). "Engineered protein scaffolds as next-generation antibody therapeutics". Current Opinion in Chemical Biology. 13 (3): 245–55. doi:10.1016/j.cbpa.2009.04.627. PMID 19501012. 

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