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Affimer

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The Affimer protein scaffold – showing the two loops and the amino terminus where designer or random peptides can be inserted to create a target-specific binding surface

Affimer molecules are small, highly stable proteins that bind with high specificity and affinity to a range of target molecules.These engineered non-antibody binding proteins mimic the molecular recognition characteristics of monoclonal antibodies, but with improved properties, being highly stable,[1] experimentally robust,[2] monomeric, soluble, small,[3] and easy to express at high yields in E.coli and mammalian cells.

Development

Affimer proteins were developed initially at the MRC Cancer Cell Unit in Cambridge then across two laboratories at the University of Leeds.[4][5][6][7] Derived from the cysteine protease inhibitor family of cystatins,[8] which function in nature as cysteine protease inhibitors,[9][10] these 12-14 kDa proteins share the common tertiary structure of an alpha-helix lying on top of an anti-parallel beta-sheet.[11]

Affimer proteins display two peptide loops and an N-terminal sequence that can all be randomised to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilisation of the two peptides by the protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides.

Production

Based on the cystatin protein fold, the Affimer protein is easy to modify with different tags and fusion proteins and can be expressed in various host cells.

Phage display libraries of 1010 randomised potential target interaction sequences are generated and screened to identify an Affimer protein with high-specificity binding to the target protein and binding affinities in the nM range. The use of in vitro screening techniques allows affinity maturation to be performed to achieve even greater binding affinities and means that the target space is not limited by an animal host’s immune system. Due to an Affimer protein being generated using recombinant systems, their generation is significantly more rapid and reproducible[12] compared to the production of antibodies.

Multimeric forms of Affimer proteins have been generated and shown to be easily produced with good production characteristics in bacterial host systems. Multimeric forms of Affimer proteins with the same target specificity provided avidity effects in target binding, while fusion of Affimer proteins of different target specificities enables multi-specific affinity proteins.[13]

Affimer binders have been produced to a large number of targets including ubiquitin chains,[14] immunoglobulins,[15] C-reactive protein,[16] interleukin-8,[1] complement C3[17][18] and magnetite nanoparticles[19] for use in a number of molecular recognition applications.

Properties

Affimer binders are recombinant proteins. They display the robust characteristics of high thermostability, with a melting temperature over 80 °C,[20] resistance to extremes of pH, (pH 2-13.7)[20] freeze-thaw cycles and lyophilisation. The low molecular weight[21] of Affimer binders means that problems of steric hindrance, typically observed with antibodies, are avoided across a number of binding applications.

These synthetic antibodies were engineered to be stable, non-toxic, biologically neutral and contain no post-translational modifications or disulfide bridges. Affimer technology makes use of two separate loop sequences, incorporating a total of 12 to 36 amino acids, to create a large potential target interaction surface of 650 to 1000 Å2, allowing for highly-specific, high affinity binding to target proteins.[4][7] Consequently, Affimer molecules can distinguish between proteins that differ by only a single amino acid,[22] can detect subtle changes in protein expression levels even in a multiplexed format and can distinguish between multiple closely related protein domains.[23]

Applications

Affimer binders have been shown to function as research tools across a number of platforms, including ELISA,[24] surface plasmon resonance, affinity purification, immunohistochemistry[25] and flow cytometry. Affimer reagents that inhibit protein-protein interactions are readily isolated and can be expressed in mammalian cells to investigate and modify signalling pathways.[26][27] They can also be co-crystallised in complex with target proteins,[28] enabling drug discovery through in silico screening and displacement assays, making Affimer proteins useful tools in drug target validation pipelines.

Affimer technology has been commercialised and developed by Avacta Life Sciences, who are developing these affinity reagents as tools for research and diagnostics and as biotherapeutics.

References

  1. ^ a b Sharma R., Deacon S.E., Nowak D., George S.E., Szymonik M.P., Tang A.A.S., Tomlinson D.C., Davis A.G., McPherson M.J., Wälti C. (2016). "Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity". Biosensors and Bioelectronics. 80: 607–13. doi:10.1016/j.bios.2016.02.028.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  2. ^ Rawlings A.E., Bramble J.P., Tang A.A.S., Somner L.A., Monnington A.E., Cooke D.J., McPherson M.J., Tomlinson D.C., Staniland S.S. (2015). "Phage display selected magnetite interacting adhirons for shape controlled nanoparticle synthesis". Chem. Sci. 6: 5586–94. doi:10.1039/C5SC01472G.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  3. ^ Roberts, Josh P. (2013). "Biomarkers Take Center Stage". GEN. 33.
  4. ^ a b Woodman R., Yeh J. T-H., Laurenson S., Ko Ferrigno P. (2005). "Design and validation of a neutral protein scaffold for the presentation of peptide aptamers". J. Mol. Biol. 352 (5): 1118–33. doi:10.1016/j.jmb.2005.08.001.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ^ Hoffman T., Stadler L.K.J., Busby M., Song Q., Buxton A.T., Wagner S.D., Davis J.J., Ko Ferrigno P. (2010). "Structure-function studies of an engineered scaffold protein derived from stefin A. I:Development of the SQT variant". Protein Eng. Des. Sel. 23 (5): 403–13. doi:10.1093/protein/gzq012.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  6. ^ Stadler L.K., Hoffman T., Tomlinson D.C., Song Q., Lee T., Busby M., Nyathi Y., Gendra E., Tiede C., Flanagan K., Cockell S.J., Wipat A., Harwood C., Wagner S.D., Knowles M.A., Davis J.J., Keegan N. Ko Ferrigno P. (2011). "Structure-function studies of an engineered scaffold protein derived from stefin A. II: Development and applications of the SQT variant". Protein Eng. Des. Sel. 24 (9): 751–63. doi:10.1093/protein/gzr019.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  7. ^ a b Tiede C., Tang A.A., Deacon S.E., Mandal U., Nettleship J.E., Owen R.L., George S.E., Harrison D.J., Owens R.J., Tomlinson D.C., McPherson M.J. (2014). "Adhiron: A stable and versatile peptide display scaffold for molecular recognition applications". Protein Eng. Des. Sel. 27 (5): 145–55. doi:10.1093/protein/gzu007.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ^ "Affimers – Next Generation Affinity Reagents". Avacta Life Sciences. Retrieved 22 May 2014.
  9. ^ Turk V., Stoka V., Turk D. (2008). "Cystatins: Biochemical and structural properties, and medical releavnce". Front Biosci. 1 (13): 5406–5420.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  10. ^ Kondo H., Abe K., Emori Y., Arai S. (1991). "Gene organization of oryzastatin II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins". FEBS Lett. 278: 87–90. doi:10.1016/0014-5793(91)80090-p.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  11. ^ Turk V. and W. Bode (1991). "The cystatins: protein inhibitors of cysteine proteinases". FEBS Lett. 285 (2): 213–219. doi:10.1016/0014-5793(91)80804-C. PMID 1855589.
  12. ^ Avacta Life Sciences. "Key Benefits of Affimers - Rapid Generation".
  13. ^ Avacta Life Sciences. "Multimeric Affimer binders".
  14. ^ Avacta Life Sciences. "Anti-diUbiquitin K48-linkage Affimer (36-28)".
  15. ^ Avacta Life Sciences. "Anti-Immunoglobulin Research Area of Affimers".
  16. ^ Johnson A, Song Q, Ko Ferrigno P, Bueno PR, Davis JJ. (Aug 7, 2012). "Sensitive Affimer and antibody based impedimetric label-free assays for C-reactive protein". Anal. Chem. 84 (15): 6553–60. doi:10.1021/ac300835b. PMID 22789061.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ^ Avacta Life Sciences. "ALS application data- Affinity purification".
  18. ^ Avacta Life Sciences. "ALS application data protein-protein interactions".
  19. ^ Rawlings A.E., Bramble J.P., Tang A.A.S., Somner L.A., Monnington A.E., Cooke D.J., McPherson M.J., Tomlinson D.C., Staniland S.S. (2015). "Phage display selected magnetite interacting adhirons for shape controlled nanoparticle synthesis". Chem. Sci. 6: 5586–94. doi:10.1039/C5SC01472G.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  20. ^ a b Avacta Life Sciences. "Key benefits of Affimers - Robustness".
  21. ^ Avacta Life Sciences. "Key Benefits of Affimers - Small Size".
  22. ^ Avacta Life Sciences. "ALS applications data- Difficult targets".
  23. ^ Avacta Life Sciences. "ALS applications data- SH2 domains".
  24. ^ Avacta Life Sciences. "ALS applications data - ELISA".
  25. ^ Avacta Life Sciences. "ALS applications data - IHC".
  26. ^ Avacta Life Sciences. "ALS applications data - Protein-protein interactions".
  27. ^ Kyle H.F., Wickson K.F., Stott J. Burslem G.M., Breeze A.L., Tiede C., Tomlinson D.C., Warriner S.L., Nelson A., Wilson A.J., Edwards T.A. (2015). "Exploration of the HIF-1α/p300 interface using peptide and adhiron phage display technologies". Mol. Biosyst. 11 (10): 2738–49. doi:10.1039/c5mb00284b.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  28. ^ Avacta Life Sciences. "ALS application data- Allosteric inhibition of FcγRIIIa-IgG interactions".
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