Affimer

Affimer proteins were developed initially at the MRC Cancer Cell Unit in Cambridge then across two laboratories at the University of Leeds.{{cite journal | vauthors = Woodman R, Yeh JT, Laurenson S, Ko Ferrigno P | title = Design and validation of a neutral protein scaffold for the presentation of peptide aptamers | journal = Journal of Molecular Biology | volume = 352 | issue = 5 | pages = 1118–33 | date = October 2005 | pmid = 16139842 | doi = 10.1016/j.jmb.2005.08.001 }}{{cite journal | vauthors = Hoffmann T, Stadler LK, Busby M, Song Q, Buxton AT, Wagner SD, Davis JJ, Ko Ferrigno P | title = Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant | journal = Protein Engineering, Design & Selection | volume = 23 | issue = 5 | pages = 403–13 | date = May 2010 | pmid = 20179045 | pmc = 2851446 | doi = 10.1093/protein/gzq012 }}{{cite journal | vauthors = Stadler LK, Hoffmann T, Tomlinson DC, Song Q, Lee T, Busby M, Nyathi Y, Gendra E, Tiede C, Flanagan K, Cockell SJ, Wipat A, Harwood C, Wagner SD, Knowles MA, Davis JJ, Keegan N, Ferrigno PK | title = Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant | journal = Protein Engineering, Design & Selection | volume = 24 | issue = 9 | pages = 751–63 | date = September 2011 | pmid = 21616931 | doi = 10.1093/protein/gzr019 | doi-access = }}{{cite journal | vauthors = Tiede C, Tang AA, Deacon SE, Mandal U, Nettleship JE, Owen RL, George SE, Harrison DJ, Owens RJ, Tomlinson DC, McPherson MJ | title = Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications | journal = Protein Engineering, Design & Selection | volume = 27 | issue = 5 | pages = 145–55 | date = May 2014 | pmid = 24668773 | pmc = 4000234 | doi = 10.1093/protein/gzu007 }} Derived from the cysteine protease inhibitor family of cystatins, which function in nature as cysteine protease inhibitors,{{cite journal | vauthors = Turk V, Stoka V, Turk D | title = Cystatins: biochemical and structural properties, and medical relevance | journal = Frontiers in Bioscience | volume = 13 | issue = 13 | pages = 5406–20 | date = May 2008 | pmid = 18508595 | doi = 10.2741/3089 | doi-access = free }}{{cite journal | vauthors = Kondo H, Abe K, Emori Y, Arai S | title = Gene organization of oryzacystatin-II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins | journal = FEBS Letters | volume = 278 | issue = 1 | pages = 87–90 | date = January 1991 | pmid = 1993479 | doi = 10.1016/0014-5793(91)80090-p | s2cid = 40605067 | doi-access = free }} these 12–14 kDa proteins share the common tertiary structure of an alpha-helix lying on top of an anti-parallel beta-sheet.{{cite journal | vauthors = Turk V, Bode W | title = The cystatins: protein inhibitors of cysteine proteinases | journal = FEBS Letters | volume = 285 | issue = 2 | pages = 213–9 | date = July 1991 | pmid = 1855589 | doi = 10.1016/0014-5793(91)80804-C | s2cid = 40444629 }}

Affimer proteins display two peptide loops that can all be randomized to bind to desired target proteins, in a similar manner to monoclonal antibodies. Stabilization of the two peptides by the protein scaffold constrains the possible conformations that the peptides can take. This increases the binding affinity and specificity compared to libraries of free peptides, though can limit the target repertoire of Affimers.

Phage display libraries of 10⁹ randomized sequences{{cite journal | vauthors = Arrata I, Barnard A, Tomlinson DC, Wilson AJ | title = Interfacing native and non-native peptides: using Affimers to recognise α-helix mimicking foldamers | journal = Chemical Communications | volume = 53 | issue = 19 | pages = 2834–2837 | date = March 2017 | pmid = 28217789 | doi = 10.1039/c6cc09395g | url = http://eprints.whiterose.ac.uk/112780/3/Chem%20Commun%20Foldamer-Adhirons%2009-01-17.pdf }} are used to screen for Affimer proteins that exhibit high-specificity binding to the target protein with binding affinities in the nM range.{{cite journal | vauthors = Zhurauski P, Arya SK, Jolly P, Tiede C, Tomlinson DC, Ko Ferrigno P, Estrela P | title = Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection | journal = Biosensors & Bioelectronics | volume = 108 | pages = 1–8 | date = June 2018 | pmid = 29482002 | doi = 10.1016/j.bios.2018.02.041 | s2cid = 3549374 | url = http://eprints.whiterose.ac.uk/127708/1/Zhurauski%20_Tomlinson%20-%20manuscript%20revised.pdf }}{{Cite journal|last1=Basran|first1=Amrik|last2=Stanley|first2=Emma | name-list-style = vanc |date=2018-07-01|title=Abstract 3776: Generation and formatting of an Affimer® biotherapeutic for the inhibition of the PD-L1/PD-1 pathway: Proof of concept in mouse|url=http://cancerres.aacrjournals.org/content/78/13_Supplement/3776|journal=Cancer Research | volume=78|issue=13 Supplement|pages=3776|doi=10.1158/1538-7445.AM2018-3776|s2cid=81376936 |issn=0008-5472|url-access=subscription}} The ability to direct in vitro screening techniques allows the identification of specific, high affinity Affimers. In vitro screening and development also mean that the target space for Affimers is not limited by the animal immune system. Affimers are generated using recombinant systems, so their generation is more rapid and reproducible compared to the production of polyclonal antibodies.{{cite journal | vauthors = Xie C, Tiede C, Zhang X, Wang C, Li Z, Xu X, McPherson MJ, Tomlinson DC, Xu W | title = Development of an Affimer-antibody combined immunological diagnosis kit for glypican-3 | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 9608 | date = August 2017 | pmid = 28852111 | pmc = 5575301 | doi = 10.1038/s41598-017-10083-w | bibcode = 2017NatSR...7.9608X }}

Multimeric forms Affimers have been generated and shown to yield titres in the range of 200–400 mg/L under small-scale culture using bacterial host systems. Multimeric forms of Affimers with the same target specificity provide avidity effects in target binding.

Many different tags and fusion proteins, such as fluorophores, single-stranded DNA, His, and c-Myc tags can be conjugated to Affimers.{{cite journal|vauthors=Schlichthaerle T, Eklund AS, Schueder F, Strauss MT, Tiede C, Curd A, Ries J, Peckham M, Tomlinson DC, Jungmann R|date=August 2018|title=Site-Specific Labeling of Affimers for DNA-PAINT Microscopy|journal=Angewandte Chemie|volume=57|issue=34|pages=11060–11063|doi=10.1002/anie.201804020|pmid=29873161|s2cid=46950252 |url=http://eprints.whiterose.ac.uk/131878/1/Schlichthaerle_et_al-2018-Angewandte_Chemie_International_Edition.pdf}}{{cite journal | vauthors = Bedford R, Tiede C, Hughes R, Curd A, McPherson MJ, Peckham M, Tomlinson DC | title = Alternative reagents to antibodies in imaging applications | journal = Biophysical Reviews | volume = 9 | issue = 4 | pages = 299–308 | date = August 2017 | pmid = 28752365 | pmc = 5578921 | doi = 10.1007/s12551-017-0278-2 }}{{cite journal | vauthors = Lopata A, Hughes R, Tiede C, Heissler SM, Sellers JR, Knight PJ, Tomlinson D, Peckham M | title = Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells | journal = Scientific Reports | volume = 8 | issue = 1 | pages = 6572 | date = April 2018 | pmid = 29700342 | pmc = 5920084 | doi = 10.1038/s41598-018-24953-4 | bibcode = 2018NatSR...8.6572L }}{{cite journal | vauthors = Wang W, Guo Y, Tiede C, Chen S, Kopytynski M, Kong Y, Kulak A, Tomlinson D, Chen R, McPherson M, Zhou D | title = Ultraefficient Cap-Exchange Protocol To Compact Biofunctional Quantum Dots for Sensitive Ratiometric Biosensing and Cell Imaging | journal = ACS Applied Materials & Interfaces | volume = 9 | issue = 18 | pages = 15232–15244 | date = May 2017 | pmid = 28421739 | pmc = 5432960 | doi = 10.1021/acsami.6b13807 }}{{cite journal | vauthors = Fisher MJ, Williamson DJ, Burslem GM, Plante JP, Manfield IW, Tiede C, Ault JR, Stockley PG, Plein S, Maqbool A, Tomlinson DC, Foster R, Warriner SL, Bon RS | title = Trivalent Gd-DOTA reagents for modification of proteins | journal = RSC Advances | volume = 5 | issue = 116 | pages = 96194–96200 | date = December 2015 | pmid = 27019702 | pmc = 4786947 | doi = 10.1039/c5ra20359g | bibcode = 2015RSCAd...596194F }} Specific cysteine residues can be introduced to the protein to allow thiol chemistry to uniformly orient Affimers on a solid support eg ELISA plates.{{cite journal | vauthors = Weckman NE, McRae C, Ko Ferrigno P, Seshia AA | title = Comparison of the specificity and affinity of surface immobilised Affimer binders using the quartz crystal microbalance | journal = The Analyst | volume = 141 | issue = 22 | pages = 6278–6286 | date = October 2016 | pmid = 27704086 | doi = 10.1039/c6an01602b | bibcode = 2016Ana...141.6278W }}{{cite journal | vauthors = Koutsoumpeli E, Tiede C, Murray J, Tang A, Bon RS, Tomlinson DC, Johnson S | title = Antibody Mimetics for the Detection of Small Organic Compounds Using a Quartz Crystal Microbalance | journal = Analytical Chemistry | volume = 89 | issue = 5 | pages = 3051–3058 | date = March 2017 | pmid = 28192970 | doi = 10.1021/acs.analchem.6b04790 | url = http://eprints.whiterose.ac.uk/112585/ }} This flexible functionalisation of the Affimer molecule allows functionality across multiple applications and assay formats.

Affimers are recombinant proteins. As they are manufactured using recombinant bacterial production processes, the batch-to-batch consistency for Affimers is improved compared to polyclonal antibodies, overcoming some of the issues of reproducibility and security of supply.{{Cite web|url=https://www.avacta.com/resources/anti-idiotypic-binders-trastuzumab-validated-regulatory-bioanalysis-assay-collaboration|title=Anti-idiotypic binders for Trastuzumab validated in regulatory bioanalysis assay in collaboration with Covance|work=Avacta|access-date=2018-10-19|archive-date=2018-10-19|archive-url=https://web.archive.org/web/20181019205624/https://www.avacta.com/resources/anti-idiotypic-binders-trastuzumab-validated-regulatory-bioanalysis-assay-collaboration|url-status=dead}}{{Cite web|url=https://www.avacta.com/resources/development-and-validation-anti-idiotypic-affmers-trastuzumab-pharmacokinetic-assay|title=Development and validation of anti-idiotypic Affmers to trastuzumab in a pharmacokinetic assay {{!}} Avacta|website=www.avacta.com|access-date=2018-10-19|archive-date=2018-10-19|archive-url=https://web.archive.org/web/20181019164028/https://www.avacta.com/resources/development-and-validation-anti-idiotypic-affmers-trastuzumab-pharmacokinetic-assay|url-status=dead}}

These synthetic antibodies were engineered to be stable, non-toxic, biologically neutral and contain no post-translational modifications or disulfide bridges. Two separate loop sequences, incorporating a total of 12 to 36 amino acids, form the target interaction surface so interaction surfaces can range form 650–1000 Å. The large interaction surface results allows binding to target proteins.{{cite journal | vauthors = Davis JJ, Tkac J, Laurenson S, Ko Ferrigno P | title = Peptide aptamers in label-free protein detection: 1. Characterization of the immobilized scaffold | journal = Analytical Chemistry | volume = 79 | issue = 3 | pages = 1089–96 | date = February 2007 | pmid = 17263340 | doi = 10.1021/ac061863z }}{{cite journal | vauthors = Davis JJ, Tkac J, Humphreys R, Buxton AT, Lee TA, Ko Ferrigno P | title = Peptide aptamers in label-free protein detection: 2. Chemical optimization and detection of distinct protein isoforms | journal = Analytical Chemistry | volume = 81 | issue = 9 | pages = 3314–20 | date = May 2009 | pmid = 19320493 | doi = 10.1021/ac802513n }}{{Cite web|url=https://www.avacta.com/resources/affimer-binders-improve-diagnostic-assay-performance-cdifficile-detection|title=Affimer binders improve diagnostic assay performance for C.difficile detection|work=Avacta|access-date=2018-10-19|archive-date=2018-10-19|archive-url=https://web.archive.org/web/20181019164036/https://www.avacta.com/resources/affimer-binders-improve-diagnostic-assay-performance-cdifficile-detection|url-status=dead}}{{Cite web|url=https://www.avacta.com/resources/development-and-characterisation-affimer-binders-4-leading-therapeutic-antibodies|title=Development and characterisation of Affimer binders to 4 leading therapeutic antibodies {{!}} Avacta|website=www.avacta.com|access-date=2018-10-19|archive-date=2018-10-19|archive-url=https://web.archive.org/web/20181019163956/https://www.avacta.com/resources/development-and-characterisation-affimer-binders-4-leading-therapeutic-antibodies|url-status=dead}}

Affimer technology has been commercialised and developed by Avacta, which is developing these affinity reagents as tools for diagnostics and as biotherapeutics.

Affimer binders have been used across a number of platforms, including ELISA,{{Cite web|url=https://www.avacta.com/resources/avacta-scientist-dr-geoff-platt-presents-affimer-data-webinar|title=Avacta Scientist Dr Geoff Platt presents Affimer data in webinar {{!}} Avacta|website=www.avacta.com|language=en|access-date=2018-10-22|archive-date=2018-10-22|archive-url=https://web.archive.org/web/20181022153525/https://www.avacta.com/resources/avacta-scientist-dr-geoff-platt-presents-affimer-data-webinar|url-status=dead}} surface plasmon resonance,{{cite journal|vauthors=Robinson JI, Baxter EW, Owen RL, Thomsen M, Tomlinson DC, Waterhouse MP, Win SJ, Nettleship JE, Tiede C, Foster RJ, Owens RJ, Fishwick CW, Harris SA, Goldman A, McPherson MJ, Morgan AW|date=January 2018|title=Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=115|issue=1|pages=E72–E81|doi=10.1073/pnas.1707856115|pmc=5776790|pmid=29247053|doi-access=free}}{{cite journal | vauthors = Michel MA, Swatek KN, Hospenthal MK, Komander D | title = Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling | journal = Molecular Cell | volume = 68 | issue = 1 | pages = 233–246.e5 | date = October 2017 | pmid = 28943312 | pmc = 5640506 | doi = 10.1016/j.molcel.2017.08.020 }}{{cite journal | vauthors = Johnson A, Song Q, Ko Ferrigno P, Bueno PR, Davis JJ | title = Sensitive affimer and antibody based impedimetric label-free assays for C-reactive protein | language = EN | journal = Analytical Chemistry | volume = 84 | issue = 15 | pages = 6553–60 | date = August 2012 | pmid = 22789061 | doi = 10.1021/ac300835b }} affinity purification.{{cite web |url= https://www.drugtargetreview.com/whitepaper/11094/affimer-reagents-facilitate-affinity-chromatography-purification-of-proteins/ |title=Affimer® reagents facilitate affinity chromatography purification |website=www.drugtargetreview.com |access-date=2018-10-22}}{{Cite journal |last1=Pechlivani |first1=Nikoletta |last2=Kearney |first2=Katherine J. |last3=Tiede |first3=Christian |last4=Cheah |first4=Ramsah |last5=Phoenix |first5=Fladia |last6=Ponnambalam |first6=Sreenivasan |last7=Ault |first7=James R. |last8=McPherson |first8=Michael J. |last9=Tomlinson |first9=Darren C. |last10=Ajjan |first10=Ramzi A. |date=2022-05-01 |title=Affinity purification of fibrinogen using an Affimer column |url=https://www.sciencedirect.com/science/article/pii/S0304416522000332 |journal=Biochimica et Biophysica Acta (BBA) - General Subjects |language=en |volume=1866 |issue=5 |pages=130115 |doi=10.1016/j.bbagen.2022.130115 |pmid=35240235 |s2cid=247199635 |issn=0304-4165|doi-access=free }} Affimers that inhibit protein-protein interactions can be produced with the potential to express these inhibitors in mammalian cells modify signalling pathways as cell therapies.{{cite journal | vauthors = Kyle HF, Wickson KF, Stott J, Burslem GM, Breeze AL, Tiede C, Tomlinson DC, Warriner SL, Nelson A, Wilson AJ, Edwards TA | title = Exploration of the HIF-1α/p300 interface using peptide and Adhiron phage display technologies | journal = Molecular BioSystems | volume = 11 | issue = 10 | pages = 2738–49 | date = October 2015 | pmid = 26135796 | doi = 10.1039/c5mb00284b | doi-access = free }}{{cite journal | vauthors = Heidelberger JB, Voigt A, Borisova ME, Petrosino G, Ruf S, Wagner SA, Beli P | title = Proteomic profiling of VCP substrates links VCP to K6-linked ubiquitylation and c-Myc function | journal = EMBO Reports | volume = 19 | issue = 4 | pages = e44754 | date = April 2018 | pmid = 29467282 | pmc = 5891417 | doi = 10.15252/embr.201744754 }}

The small size and stability profile of Affimers combined with their human origin confer drug-like properties. This may represent advantages over antibodies in terms of tissue penetration, for example in solid tumours where Avacta are developing PD-L1 inhibitors as alternatives to Opdivo and Yervoy,{{Cite journal|title=Interview with Amrik Basran at Avacta Life Sciences|url=https://www.drugtargetreview.com/article/11013/interview-amrik-basran-chief-scientific-officer-avacta-life-sciences/|journal=Drug Target Review }} though requires half life modification to prevent rapid excretion through the kidney.

Affimers can be conjugated to form multimers for the design of therapeutics. Examples include the production of multi-specific Affimer molecules to albumin binders to increase their half-life in vivo and for use as the targeting moiety in chimeric receptors or modified to carry a toxin in Affimer-drug conjugates.{{Cite web|url=https://www.avacta.com/resources/tmac-affimer-drug-conjugates|title=TMAC Affimer Drug Conjugates|work=Avacta|access-date=2018-10-22|archive-date=2018-10-22|archive-url=https://web.archive.org/web/20181022153432/https://www.avacta.com/resources/tmac-affimer-drug-conjugates|url-status=dead}}

Affimers as therapeutics are in discovery and preclinical development to tackle cancer, both via CAR-T cell therapy and as immune checkpoint inhibitors.{{Cite news|url=http://sourcelifescience.com/source-clusters/item/16-avacta-and-memorial-sloan-kettering-cancer-center-in-cell-therapy-research-collaboration|title=Avacta and Memorial Sloan Kettering Cancer Center in cell therapy research collaboration|last=Mulligan|first=Tom|access-date=2018-10-22|language=en-gb|archive-date=2022-07-03|archive-url=https://web.archive.org/web/20220703145751/http://sourcelifescience.com/source-clusters/item/16-avacta-and-memorial-sloan-kettering-cancer-center-in-cell-therapy-research-collaboration|url-status=dead}}{{Cite web|url=https://www.avacta.com/therapeutics/pipeline|title=Pipeline {{!}} Avacta|website=www.avacta.com|language=en|access-date=2018-10-22}}{{cite journal | vauthors = King R, Tiede C, Simmons K, Fishwick C, Tomlinson D, Ajjan R | title = Inhibition of complement C3 and fibrinogen interaction: a potential novel therapeutic target to reduce cardiovascular disease in diabetes | journal = Lancet | volume = 385 | pages = S57 | date = February 2015 | issue = Suppl 1 | pmid = 26312879 | doi = 10.1016/s0140-6736(15)60372-5 | s2cid = 35622552 }} Early studies using ex vivo human samples showed low immunogenicity associated with the Affimer scaffold, at levels comparable to a marketed antibody therapeutic.{{Cite web|url=https://www.avacta.com/resources/affimer-technology-results-pbmc-immunogenicity-testing|title=Affimer Technology: Results of PBMC Immunogenicity Testing|work=Avacta|access-date=2018-10-22|archive-date=2018-10-22|archive-url=https://web.archive.org/web/20181022153553/https://www.avacta.com/resources/affimer-technology-results-pbmc-immunogenicity-testing|url-status=dead}} Furthermore, initial preclinical studies showed good efficacy and tolerability of the anti-PDL1 immuno-oncology Affimers in mice. It is anticipated that IND filing for the first Affimer therapeutic will occur in 2023.

{{Reflist|35em}}

• [http://www.avacta.com Avacta]
• [https://avacta.wistia.com/medias/xcwat7zy16 An Introduction to Affimer Technology - video]

{{Engineered antibodies}}

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Category:Antibody mimetics