A New Alternative to Antibodies

Avacta Life Sciences recently invested £10 million in launching Affimers, an engineered protein alternative to antibodies, which have potential in a wide range of applications.

To learn about some of the features of Affimers, and how they can be of benefit to researchers, we spoke to Alastair Smith, Chief Executive Officer, Avacta Group plc.

AS: Affimers were developed initially at the MRC Cancer Cell Unit in Cambridge then across two laboratories at the University of Leeds. The technology was then acquired, developed and commercialised by Avacta Group plc, through Avacta Life Sciences Ltd. 

Affimers are derived from cystatin proteins and make use of two separate loop sequences, incorporating a total of 12 to 36 random amino acids, to create a large potential target interaction surface. We make large libraries of these that we screen for highly-specific, high affinity binding to target proteins or other molecules. 

AS: Although antibodies are well established affinity reagents, they have a number of issues for researchers, such as long developmental lead times, being difficult to express, being difficult to engineer, batch reproducibility, rarely being mono-specific, not being able to be generated to some targets (e.g. those that are toxic to the host animal), being limited by the animal’s immune system and a complicated IP landscape. 

Affimers have been engineered to address these issues. Their key benefits are:

• Quick to develop – custom generated in just 7 weeks, compared to 6 - 12 months for a good antibody of comparable quality

• Specificity can be controlled during the library screen

• Small, robust and stable

• Consistent batch reproducibility

• Easily functionalised

• Not limited by the immune system, generated in vitro

• No use of animals

• Can be lyophilised for shipping and storage

• Straightforward licensing

• Quality guaranteed by rigorous QC

• A direct replacement for antibodies – no process or workflow change required

AS: Aptamers are based on nucleic acids such as DNA.  They are physically limited to a small range of conformations, internal bonds often need to be broken in order to form bonds with the target, and they are chemically limited in the type of bonds that can be formed with the target. Their highly charged backbone will repel targets with like charge, and they bind non-specifically to the numerous nucleic-acid binding proteins that are naturally present in cells and tissues, making specificity and high background a problem. Also, nucleic acids can be very sensitive to chemicals and some conditions make them vulnerable to degradation and impractical for some samples.

Affimers, in contrast, are based on proteins that have a favourable conformation and a wide range of chemical interactions with the target through their amino acids in the same way an antibody does. The Affimer scaffold is engineered to be biologically inert (i.e. it has no affinity for most human proteins), biophysically stable within a wide pH and temperature range, making Affimers suitable for a wide range of assay conditions. 

From a business perspective Affimers are not easily reverse-engineered and Avacta Life Sciences owns the scaffold intellectual property rights (IPR) and has licensed all necessary screening IPR.

AS: The process of ubiquitination (also called ubiquitylation) is still not fully understood, and, because of its relevance in many diseases is therefore an area of intense academic and commercial interest. But there has been a huge shortage of antibodies to aid researchers in this field. In 10 years since the Nobel prize for chemistry was awarded “for the discovery of ubiquitin-mediated protein degradation”, just two ubiquitin binding antibodies have been successfully developed. Yet Avacta’s scientists have produced four Affimers in a matter of months, and more are in the pipeline!  Affimers will open up the ubiquitination research field because they will provide scientists with the tools to ask vital questions about ubiquitination and its role in many diseases. 

AS: In ELISAs, one of the main issues is specificity. Antibodies are rarely mono-specific which means that  cross-reactivity with an irrelevant protein can cause a signal in the assay or a false positive may also arise if the labelled secondary antibody binds non-specifically to the surface on which the assay is being carried out.

Affimers are selected in vitro on the basis of their specificity so they are unlikely to bind to irrelevant antigens in the sample. The Affimer scaffold has also been engineered to have no natural binding partners in human samples which means that the incidence of false positive results is greatly reduced- this is what we called ‘biological neutrality’ earlier. Finally, Affimers remain functional when immobilised on any one of a range of solid supports and can be functionalised with enzymes such as HRP, allowing a standard ELISA format to be performed.

In Western blots, the well-known non-specific reactivity of an antibody can lead to false positives by cross-reacting with a protein band at the same location as the true target, especially when probing for common post-translational modifications. False negatives may be caused by physical loss of a conformational epitope or by post-translational modifications that mask the epitope.

Custom Affimers are validated in applications that are relevant to a customer’s ultimate application. Although there can be no guarantees in biology, we are able to maximise the probability of finding a specific binder to any target by using counter-selection against confounding targets to generate Affimers for western blots with no cross-reactivity.

For more information on Affimers visit www.avactalifesciences.com.