Affibody molecule

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Affibody molecules are small, robust 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. [1] These molecules can be used for molecular recognition in diagnostic and therapeutic applications. [2] [3]

Contents

Development

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 with almost good affinity, while retaining the favorable folding and stability properties, and ease of bacterial expression of the parent molecule. [4] [5]

The original Affibody protein scaffold was designed based on the Z domain (the immunoglobulin G binding domain) of protein A. These molecules are the newly developed class of scaffold proteins derived from the 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.

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

Production

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. [7]

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 [8] [9] or by spontaneous isopeptide bond formation. [10] A site for site-specific conjugation is facilitated by introduction of a single cysteine at a desired position, therefore this engineered protein can be used to conjugate to radionuclides such as technetium-99m and indium-111 to visualize receptor-overexpressing tumors. [11] [12]

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. [13] [14] In some studies, temperatures above the melting temperature have been used, with retained binding properties following return to ambient conditions. [15] Cross-linked variants have been produced as well.

Properties

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 (194 °F)) or acidic and alkaline conditions (pH  2.5 or pH 11, respectively). [16] [17] [18] Affibody molecules have ashort plasma half-life because of their small size.While this is advantageous for imaging,as the unbound tracer is rapidly cleared from the bloodstream, a longer plasma half-life is generally preferred for therapeutic applications. One approach to extend the plasma half-life of small molecules, such as affibody molecules,is to link them to an albumin-binding domain (ABD). [19] A highly effective ABD is the 46-amino acid G148-GA3 domain, which is derived from streptococcal protein G, along with its engineered variant ABD035. This version has a strong femtomolar affinity for human serum albumin (HSA) [28]. The ABD can help prolong the serum half-life by forming a complex with serum albumin (SA) in the bloodstream, increasing the size of the complex and preventing it from being filtered by the kidneys. [20]

Binders with an affinity of down to sub-nanomolar have been obtained from native library selections, and binders with picomolar affinity have been obtained following affinity maturation. [21] Affibodies conjugated to weak electrophiles bind their targets covalently. [22] Combination of small size, ease of engineering, high affinity and specificity makes Affibody molecules suitable alternative as monoclonal antibodies for both molecular imaging and therapeutical applications, especially for the receptor-overexpressing tumors. [23] [24] These proteins are characterized by a high rate of extravasation and rapid clearance of non-bound tracer from the circulation, as well as other nonspecific compartments, when compared to antibodies and their fragments

Applications

Affibody molecules can be used for protein purification, [13] enzyme inhibition, [15] research reagents for protein capture and detection, [25] [26] diagnostic imaging [21] and targeted therapy. [27] The second generation of Affibody molecule, ABY-025, binds selectively to HER2 receptors with picomolar affinity. These Affibody molecules are in clinical development for tumor diagnosis. [28] [29] [30] [31] Anti-HER2 Affibody molecule, fused with albumin binding domain (ABD), denoted as ABY-027, labeled with Lutetium-177 provided reduction of renal and hepatic uptake of radioactivity in mice xenografts. [32] Recently, anti-ZEGFR Affibody ZEGFR:2377 labeled with technetium-99m was successfully used to visualize ZEGR expressing tumor in mice xenograft also. [33]

References

  1. ↑ Frejd FY, Kim KT (2017). "Affibody molecules as engineered protein drugs". Exp Mol Med . 49 (3): e306. doi:10.1038/emm.2017.35. PMC   5382565 . PMID   28336959.
  2. ↑ Garousi J, Andersson K, Mitran B, Pichl ML, Ståhl S, Orlova A, Löfblom J, Tolmachev V (2016). "PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 Affibody molecules". Int J Oncol . 48 (4): 1325–1332. doi:10.3892/ijo.2016.3369. PMC   4777594 . PMID   26847636 – via SPANDIDOS PUBLICATIONS.
  3. ↑ Sörensen J, Sandberg D, Sandström M, Wennborg A, Feldwisch J, Tolmachev V, Åström G, Lubberink M, Garske-Román U, Carlsson J, Lindman H (2014). "First-in-human molecular imaging of HER2 expression in breast cancer metastases using the 111In-ABY-025 affibody molecule". J Nucl Med . 55 (5): 730–735. doi: 10.2967/jnumed.113.131243 . PMID   24665085.
  4. ↑ Nord, K; Nilsson, J; Nilsson, B; Uhlén, M; Nygren, P-A (1995). "A combinatorial library of an α-helical bacterial receptor domain". Protein Engineering, Design and Selection . 8 (6): 601–608. doi:10.1093/protein/8.6.601. PMID   8532685.
  5. ↑ 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. S2CID   25252394.
  6. ↑ 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   2280057 . PMID   15044721.
  7. ↑ Ståhl, S; Nygren, P-A (1997). "The use of gene fusions to protein A and protein G in immunology and biotechnology". Pathol. Biol. 45 (1): 66–76. PMID   9097850.
  8. ↑ 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.
  9. ↑ 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.
  10. ↑ 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. Bibcode:2016PNAS..113.1202V. doi: 10.1073/pnas.1519214113 . PMC   4747704 . PMID   26787909.
  11. ↑ Altai M, Wållberg H, Orlova A, Rosestedt M, Hosseinimehr SJ, Tolmachev V, Ståhl S (2012). "Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules". Amino Acids. 42 (5): 1975–1985. doi:10.1007/s00726-011-0927-x. PMID   21573874. S2CID   7995180.
  12. ↑ Tolmachev V, Friedman M, Sandström M, Eriksson TL, Rosik D, Hodik M, Ståhl S, Frejd FY, Orlova A (2009). "Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry". J Nucl Med. 50 (2): 274–283. doi: 10.2967/jnumed.108.055525 . PMID   19164241.
  13. 1 2 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.
  14. ↑ 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". ChemBioChem . 6 (6): 1043–1050. doi:10.1002/cbic.200400388. PMID   15880677. S2CID   5895074.
  15. 1 2 "Phusion Hot Start High-Fidelity DNA Polymerase". Finnzymes. Archived from the original on 2009-03-28.
  16. ↑ 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.
  17. ↑ 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.
  18. ↑ 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.
  19. ↑ Yin, Wen; Orlova, Anna; Altai, Mohamed; Oroujeni, Maryam (21 November 2021). "The Influence of Domain Permutations of an Albumin-Binding Domain-Fused HER2-Targeting Affibody-Based Drug Conjugate on Tumor Cell Proliferation and Therapy Efficacy". Pharmaceutics. 13 (1974): 1974. doi: 10.3390/pharmaceutics13111974 . PMC   8617914 . PMID   34834389.
  20. ↑ Yin, Wen (21 November 2021). "The Influence of Domain Permutations of an Albumin-Binding Domain-Fused HER2-Targeting Affibody-Based Drug Conjugate on Tumor Cell Proliferation and Therapy Efficacy". Pharmaceutics. 13 (1974): 1974. doi: 10.3390/pharmaceutics13111974 . PMC   8617914 . PMID   34834389.
  21. 1 2 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.
  22. ↑ 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   2781706 . PMID   19759009.
  23. ↑ Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY (2010). "Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications". FEBS Lett . 584 (12): 2670–2680. Bibcode:2010FEBSL.584.2670L. doi: 10.1016/j.febslet.2010.04.014 . PMID   20388508 – via Elsevier, Science Direct.
  24. ↑ Tolmachev V, Orlova A, Nilsson FY, Feldwisch J, Wennborg A, Abrahmsen L (2007). "Affibody molecules: potential for in vivo imaging of molecular targets for cancer therapy". Expert Opin Biol Ther . 7 (4): 555–568. doi:10.1517/14712598.7.4.555. PMID   17373906. S2CID   29621968.
  25. ↑ 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.
  26. ↑ 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.
  27. ↑ 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.
  28. ↑ Feldwisch, Joachim; Tolmachev, Vladimir; Lendel, Christofer; Herne, Nina; Sjöberg, Anna; Larsson, Barbro; Rosik, Daniel; Lindqvist, Eva; Fant, Gunilla; Höidén-Guthenberg, Ingmarie; Galli, Joakim (2010-04-30). "Design of an optimized scaffold for affibody molecules". Journal of Molecular Biology. 398 (2): 232–247. doi:10.1016/j.jmb.2010.03.002. ISSN   1089-8638. PMID   20226194.
  29. ↑ 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.
  30. ↑ Sörensen J, Velikyan I, Sandberg D, Wennborg A, Feldwisch J, Tolmachev V, Orlova A, Sandström M, Lubberink M, Olofsson H, Carlsson J, Lindman H (2016). "Measuring HER2-Receptor Expression In Metastatic Breast Cancer Using [68Ga]ABY-025 Affibody PET/CT". Theranostics. 6 (2): 262–271. doi:10.7150/thno.13502. PMC   4729774 . PMID   26877784.
  31. ↑ Sörensen J, Sandberg D, Sandström M, Wenn-borg A, Feldwisch J, Tolmachev V, Åström G, Lubberink M, Garske-Román U, Carlsson J, Lindman H (2014). "First-in-human molecular imaging of HER2 expression in breast cancer metastases using the 111In-ABY-025 affibody molecule" (PDF). J Nucl Med. 55 (5): 730–735. doi: 10.2967/jnumed.113.131243 . PMID   24665085.
  32. ↑ Orlova, Anna; Jonsson, Andreas; Rosik, Daniel; Lundqvist, Hans; Lindborg, Malin; Abrahmsen, Lars; Ekblad, Caroline; Frejd, Fredrik Y.; Tolmachev, Vladimir (June 2013). "Site-specific radiometal labeling and improved biodistribution using ABY-027, a novel HER2-targeting affibody molecule-albumin-binding domain fusion protein". Journal of Nuclear Medicine. 54 (6): 961–968. doi: 10.2967/jnumed.112.110700 . ISSN   1535-5667. PMID   23528382. S2CID   25959793.
  33. ↑ Andersson KG, Oroujeni M, Garousi J, Mitran B, Ståhl S, Orlova A, Löfblom J, Tolmachev V (2016). "Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator". Int J Oncol. 49 (6): 2285–2293. doi:10.3892/ijo.2016.3721. PMC   5118000 . PMID   27748899.
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