Although antibodies continue to be the most frequently used tool for biomedical discovery, antibody technology is not problem-free. As such, newer alternatives have been developed as part of efforts to overcome some of the limitations of conventional antibodies. Included among these, nanobodies, Aptamers, and Affimer® proteins have all shown considerable promise for research, diagnostic, and therapeutic applications, not least within the realm of coronavirus research. This article discusses the benefits of antibody alternatives and comments on how they are being used.
Early discovery must confirm target relevance
According to Carl Ascoli, chief science officer at Rockland Immunochemicals, while it is widely recognized that developing new technologies is essential to push the boundaries of science, tried and trusted polyclonal antibodies still represent the best choice for early discovery. “The primary aim of early discovery is to confirm whether a target or a modification on a target is relevant,” he says. “This screening process is most effectively conducted using polyclonal antibodies that are relatively straightforward and inexpensive to produce and are not necessarily needed in vast supply. Only once the significance of a target has been confirmed is there a need for reagents involving more complex production methods such as conventional hybridoma-based monoclonal or recombinant monoclonal antibodies. Generating these is justified once it is known that the target they bind has value, but it remains of paramount importance to consider the expense and complexity of a technology when choosing an approach that best meets the needs of the job in hand.”
Nanobodies offer application-dependent advantages
Unlike conventional antibodies, nanobodies are heavy-chain-only antibodies that do not require a paired light chain for binding. As a result, the smallest antigen-binding construct is just a single immunoglobulin domain. This small size improves tissue permeability, which can be advantageous for in vivo imaging and super-resolution imaging, or when targeting specific cells within a tumor. In addition, nanobodies benefit from several other application-dependent advantages. “Because antigen-binding loops from only one Ig domain interact with the target, nanobodies bind to antigens differently than conventional antibodies,” explains Dr. Michael Fiebig, vice president product portfolio and innovation at Absolute Antibody. “This appears to offer particular advantages when targeting structural motifs, giving researchers the ability to detect and affect more diverse epitopes. Nanobodies also form excellent building blocks for creating multi-specific antibodies as there is no potential for light chain mispairing, as well as for producing engineered antibodies with Fc domains of different species and isotypes.”
Image: Nanobodies provide ample opportunities for engineering. Image provided by Absolute Antibody.
Aptamers support a wide range of applications
Aptamers are nucleic acid-based affinity reagents with a flexible backbone capable of folding into highly organized, complex structures. These structures wrap around all or part of their target (in the case of small molecules) or fit snugly into clefts and gaps within the surface of much larger target molecules (in the case of proteins, cells, or tissues), allowing for selection based on specificity.
“Aptamers are used to support applications ranging from imaging (fluorescence microscopy and IHC), through quantification (ELISA), to binding studies (label-free detection, surface plasmon resonance, biolayer interferometry),” notes Derek Smith, director of global sales at Aptamer Group. “They are also used for purification, especially in cases requiring tight control over binding and release, as well as being employed for rapid diagnostics applications such as lateral flow and biosensing, and for the development of therapeutics such as aptamer-drug conjugates. This versatility is achieved by tailoring the selection process to the end application, which is in stark contrast to conventional antibody production where reagents are only assessed for application suitability after they have been produced.”
Affimer® technology is well-suited to detect more challenging targets
Affimers are non-antibody affinity reagents produced using a proprietary technology. At approximately one-tenth the size of a conventional antibody, they comprise a protein scaffold based on either the human protease inhibitor Stefin A or on a consensus sequence of Cystatin A from several plant species, giving them a large binding surface that equates to exceptional affinity and selectivity for antigenic targets.
“A major advantage of Affimer technology is that it offers highly specific target recognition without limitation by the immune system,” reports David Wilson, commercial director, reagents and diagnostics, at Avacta. “This makes Affimer proteins ideal to detect targets for which antibody generation is challenging: targets that may be toxic to the host; targets sharing a high degree of homology with host proteins; conformational or post-translational epitopes that may be lost during antigen processing; or targets where it is difficult to source the antigen in sufficient quantities to generate an immune response. Moreover, compared to antibodies, Affimer proteins are far less sensitive to the assay environment, making them better able to tolerate extremes of pH or temperature and allowing for long-term storage.” Affimer technology also benefits from extremely rapid production times, with high yields achieved using just sub-milligram quantities of antigen.
Pulling together in the fight against COVID-19
With the current pandemic situation continuing to unfold, the need for reagents to study SARS-CoV-2 is critical. Irrespective of whether those reagents are antibodies or antibody alternatives, companies worldwide are united in their drive to help researchers understand and tackle the virus. Rockland has already produced a polyclonal antibody proven to detect SARS-CoV-2 in patient samples, while Absolute Antibody has manufactured a range of recombinant SARS-CoV-2 antibodies, including engineered synthetic nanobodies against the receptor binding domain of the virus that can be used as serological controls and have shown promise for the development of inhalable drugs.
Avacta was able to identify and characterize highly specific, reproducible SARS-CoV-2 Affimer binders within just four weeks and is currently working with multiple partners to develop laboratory, point-of-care, and self-tests to increase global testing capacity. Aptamer Group is likewise instigating various partnership opportunities as well as seeing the utility of its products to detect SARS-CoV-2 in wastewater. As awareness grows around how antibody alternatives can diversify and strengthen the portfolio of reagents available to researchers, it is widely agreed that their use will increase significantly in the near future.