Affimer

Affimer molecules are small proteins that bind to target molecules with similar specificity and affinity to that of antibodies. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. In addition, these affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E.coli and mammalian cells. Affimer molecules are small proteins that bind to target molecules with similar specificity and affinity to that of antibodies. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. In addition, these affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E.coli and mammalian cells. Affimer proteins were developed initially at the MRC Cancer Cell Unit in Cambridge then across two laboratories at the University of Leeds. Derived from the cysteine protease inhibitor family of cystatins, which function in nature as cysteine protease inhibitors, these 12–14 kDa proteins share the common tertiary structure of an alpha-helix lying on top of an anti-parallel beta-sheet. 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. Phage display libraries of 1010 randomised potential target interaction sequences are used to screen for Affimer proteins that exhibit high-specificity binding to the target protein with binding affinities in the nM range. The ability to direct in vitro screening techniques allows the identification of highly specific, high affinity Affimer binders, negating requirements for affinity maturation of these reagents. In vitro screening and development also mean that the target space for Affimer binders is not limited by an animal host’s immune system. Due to Affimer proteins being generated using recombinant systems, their generation is significantly more rapid and reproducible compared to the production of traditional antibodies. Multimeric forms of Affimer proteins have been generated and shown to yield titrimetric volumes in the range of 200–400 mg/L under small scale culture using bacterial host systems. Multimeric forms of Affimer proteins with the same target specificity provided avidity effects in target binding, while fusion of different Affimer proteins with different target specificities would enable multi-specific affinity proteins. Many different tags and fusion proteins, such as fluorophores, single-stranded DNA, His, and c-Myc tags can be easily conjugated to Affimer proteins. Specific cysteine residues can be introduced to the protein to allow thiol chemistry to uniformly orient Affimer proteins on a solid support for the purposes of increasing target capture in ligand binding assays and biosensors. This flexible functionalisation of the Affimer molecule maximises the performance of Affimer reagents across multiple applications and assay formats. Affimer binders are recombinant proteins. They display the robust characteristics of high thermostability, with melting temperatures over 80 °C, resistance to extremes of pH (2–13.7), freeze-thaw cycles and lyophilisation. The low molecular weight of Affimer binders means that problems of steric hindrance, that are typically observed with antibodies, may be avoided. As they are manufactured using recombinant bacterial production processes, the batch-to-batch consistency is maintained for Affimer reagents, overcoming some of the issues of reproducibility and security of supply. 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 is purported to result in highly-specific, high affinity binding to target proteins. As a result, Affimer molecules can distinguish between proteins that differ by only a single amino acid, can detect subtle changes in protein expression levels even in a multiplexed format and can distinguish between multiple closely related protein domains.