Affibody molecule explained

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]

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 (90abbr=NaNabbr=) or acidic and alkaline conditions (pH 2.5 or pH 11, respectively).[16] [17] [18]

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.[19] Affibodies conjugated to weak electrophiles bind their targets covalently.[20] 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.[21] [22] 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,[23] [24] diagnostic imaging[19] and targeted therapy.[25] 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.[26] [27] [28] [29] 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.[30] Recently, anti-ZEGFR Affibody ZEGFR:2377 labeled with technetium-99m was successfully used to visualize ZEGR expressing tumor in mice xenograft also.[31]

External links

Notes and References

  1. Frejd FY, Kim KT . 2017 . Affibody molecules as engineered protein drugs . . 49 . 3 . e306 . 10.1038/emm.2017.35 . 5382565 . 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 . . 48 . 4 . 1325–1332 . 10.3892/ijo.2016.3369 . 4777594 . 26847636 . 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 . . 55 . 5 . 730–735 . 10.2967/jnumed.113.131243 . 24665085 . free.
  4. Nord . K . Nilsson . J . Nilsson . B . Uhlén . M . Nygren . P-A . 1995 . A combinatorial library of an α-helical bacterial receptor domain . . 8 . 6 . 601–608 . 10.1093/protein/8.6.601 . 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 . . 15 . 8 . 772–777 . 10.1038/nbt0897-772 . 9255793 . 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 . . 13 . 4 . 847–853 . 10.1110/ps.03541304 . 2280057 . 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. 9097850.
  8. 10.1016/S0022-1759(01)00563-4. 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. 11861078.
  9. 10.1016/j.jim.2003.06.001. 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. 14580889.
  10. Veggiani . G . Nakamura . T . Brenner . M . Gayet . R . Yan . J . Robinson . C . Howarth . M . 2016 . Programmable polyproteams built using twin peptide superglues . . 113 . 5 . 1202–1207 . 2016PNAS..113.1202V . 10.1073/pnas.1519214113 . 4747704 . 26787909 . free.
  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. 10.1007/s00726-011-0927-x. 21573874. 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. 10.2967/jnumed.108.055525. 19164241. free.
  13. 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 . . 268 . 15 . 1–10 . 10.1046/j.1432-1327.2001.02344.x . 11488921 . free.
  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 . . 6 . 6 . 1043–1050 . 10.1002/cbic.200400388 . 15880677 . 5895074.
  15. Web site: Finnzymes . Phusion Hot Start High-Fidelity DNA Polymerase . dead . https://web.archive.org/web/20090328110438/http://www.finnzymes.fi/pcr/phusion_hot_start_high_fidelity_dna_polymerase.html . 2009-03-28 .
  16. 10.2967/jnumed.108.056929. Ahlgren. S. Wållberg. H. Tran. TA. Widström. C. Hjertman. M. Abrahmsén. L. Berndorff. D. Dinkelborg. LM. Cyr. JE. 8. 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. 19372467. free.
  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. 17464277.
  18. Tran . T . Engfeldt . T . Orlova . A . Sandström . M . Feldwisch . J . Abrahmsén . L . Wennborg . A . Tolmachev . V . Karlström . AE . 8 . 2007 . (99m)Tc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors . . 18 . 6 . 1956–64 . 10.1021/bc7002617 . 17944527.
  19. Orlova . A . Magnusson . M . Eriksson . TL . Nilsson . M . Larsson . B . Höidén-Guthenberg . I . Widström . C . Carlsson . J . Tolmachev . V . 8 . 2006 . Tumor imaging using a picomolar affinity HER2 binding affibody molecule . . 66 . 8 . 4339–48 . 10.1158/0008-5472.CAN-05-3521 . 16618759 . free.
  20. Holm . L . Moody . P . Howarth . M . 2009 . Electrophilic Affibodies Forming Covalent Bonds to Protein Targets . . 284 . 47 . 32906–13 . 10.1074/jbc.M109.034322 . 2781706 . 19759009 . free.
  21. Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY . 2010 . Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications . . 584 . 12 . 2670–2680 . 10.1016/j.febslet.2010.04.014 . 20388508 . . free.
  22. 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 . . 7 . 4 . 555–568 . 10.1517/14712598.7.4.555 . 17373906 . 29621968.
  23. 10.1021/pr060316r. 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. 17203961.
  24. 10.1016/j.jim.2006.10.013. 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. 17196217.
  25. 10.1158/0008-5472.CAN-06-1630. Tolmachev. V. Orlova. A. Pehrson. R. Galli. J. Baastrup. B. Andersson. K. Sandström. M. Rosik. D. Carlsson. J. 8. 2007. Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule. Cancer Res.. 67. 6. 2773–82. 17363599. free.
  26. 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 . 10.1016/j.jmb.2010.03.002 . 1089-8638 . 20226194.
  27. Gebauer. M. Skerra. A. Engineered protein scaffolds as next-generation antibody therapeutics. Current Opinion in Chemical Biology. 13. 3. 245–55. 2009. 19501012. 10.1016/j.cbpa.2009.04.627.
  28. 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. 10.7150/thno.13502. 26877784. 4729774.
  29. 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.. J Nucl Med. 55 . 5 . 730–735. 10.2967/jnumed.113.131243. 24665085. free.
  30. 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 . 10.2967/jnumed.112.110700 . 1535-5667 . 23528382. 25959793 . free .
  31. 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. 10.3892/ijo.2016.3721. 27748899. 5118000.

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