Antibody alternatives in specific aptamer 3-D scaffold binding
Curator: Larry H. Bernstein, MD, FCAP
New Proteomics Tools to Open Up Drug Discovery
Dr. Paul Ko Ferrigno, Chief Scientific Officer, and Dr. Jane McLeod, of Avacta Life Sciences
Size matters: Smaller molecules allow a tighter packing density on solid surfaces for improved signal-to-noise in assays, such as SPR or ELISA.
In the current genomic era of very accurate DNA analyses by in situ hybridization, DNA chip analyses, and deep sequencing, it is often assumed that antibodies have an analogous ability to identify molecular targets accurately. Nothing could be further from the truth. Proteomics as a field is still lagging behind its genomic counterpart in the level of detail we can achieve, the level of data we can collect and the overall levels of accuracy and reliability that the collected data represent.
From the estimated 20,000 human genes 100,000 different possible proteins have been predicted. What is more, the variation achieved via the post-translational modification of these proteins brings another layer of complexity to cellular signalling. All this means that while studying the genetic blueprint can offer insights into the cell, it is only through examining the functional protein units that we can comprehensively map the dynamic interactions that occur within the cell to drive an organism or disease process.
Why not use antibodies?
Antibodies form the basis of molecular recognition in proteomics, whether this is to identify a protein within a complex mixture or label a specific protein within a cell. Both their target specificity and high binding capacity have made these molecules fantastically useful tools within diagnostics. However, the large majority of commercially available antibodies are for use as reagents in research and development, where they are simply not as well validated and issues with their manufacture have created problems that have hindered drug development.
It has proven next to impossible to develop antibodies to certain targets. This may be because of their high homology to the host protein, so the immune system fails to recognise them as different, or due to antigen processing resulting in the loss of post-translational modifications or discontinuous epitopes. However, without the necessary antibodies to investigate these targets the corresponding research avenues have remained closed and key drug targets may have been missed.
Almost worse than lacking the necessary research tools is the problem of antibody reproducibility. Matthias Uhlen revealed that of the 5,436 antibodies tested as part of the Protein Atlas project over 50 percent failed to recognize their target in at least one of two commonly used assays. Antibodies that are not specific to their target or do not recognize their target at all are responsible for increasing the cost of biological research, with an estimated $800 million spent globally every year on bad antibodies. Many published studies have had to be retracted due to antibody-derived error and those that remain unidentified in the literature will continue to lead researchers down blind alleys. This level of misinformation in the published literature is not just hindering the progression of the field, but possibly even sending it backwards and costing more than is needed — an issue not to be taken lightly when so many research budgets are coming under the knife.
More recently there has been a call from a number of leading scientists for the use of polyclonal antibodies, considered to be the worst offenders in terms of batch-to-batch irreproducibility, to be abandoned. They suggest a move towards recombinant systems of production, which would remove the restrictions of the immune system on antibody production. Yet, they state that $1 billion dollars investment would be required to re-route antibody production down this path and suggest that a period of five to ten years may be required to bring about these changes.
Simply producing recombinant antibodies rather than animal-derived affinity reagents will still leave us with a number of problems regarding their use. Antibodies are simply too large to target many smaller, hidden epitopes and the presence of key disulphide bonds within their structure makes them all too susceptible to reduction within the cell, rendering them useless for applications such as live cell imaging of molecules within the cytoplasm. Moreover, can we afford to wait another decade for a solution to this problem before we pursue protein targets for basic understanding and drug development?
Antibody alternatives for new targets and techniques
Antibody alternatives are already available to researchers in the life sciences field. They are produced either from nucleic acid or protein molecules. Aptamers are short, single-stranded RNA or DNA molecules that fold to form 3D scaffolds, which can present a specific interaction surface to allow specific binding to its target molecule. Protein scaffolds are formed from parts of or whole proteins modified to present a peptide sequence. This peptide sequence works in a similar manner to present a specific interaction surface for specific binding to a desired target.
These antibody alternatives are produced in recombinant systems, minimizing batch-to-batch variation and allowing them to be produced to theoretically any target. Additionally, as they do not use animals in their production they are generally less expensive to produce than traditional antibodies.
While companies such as Affibody and Avacta Life Sciences are aiming to open up the drug pipeline, by offering these alternative affinity reagents to previously inaccessible targets for use in research and development, many have moved into exploiting their therapeutic potential. Noxxon produce an RNA-based scaffold, Spiegelmers, which are currently in phase 2 clinical trials for diabetic nephropathy, and Molecular Partners have reached phase 3 clinical trials with their protein scaffold DARPins, for wet age-related macular degeneration.
These new antibody alternatives are smaller than traditional antibodies. This opens up the use of new technologies, such as super resolution microscopy. While the diffraction limit remained at about 250 nmm the length of the antibody at 15 nm was of little importance, tagging your molecule as accurately as necessary. Removing this limit in super resolution microscopy has meant that antibodies are now too large to provide the required level of accuracy. Instead, using an antibody alternative of only 2nm to tag your protein of interest opens up this new technique offering greater precision to intracellular localization.
As these scaffolds have been engineered to be fit-for-purpose many contain no intramolecular disulphide bonds and so are not sensitive to the reducing environment of the cell. This function enables their use as intracellular reporters of molecular conformation, as well as in standard assays like IHC or western blotting, so allowing scientists to use the same reagent across both intracellular and biochemical assays, thus bridging the gap between cell biology and biochemical studies.
Offering increased reproducibility, access to an increased range of applications, and the opportunity to hit previously inaccessible targets, antibody alternatives are opening up potential new avenues of drug discovery.
About the Authors
Dr. Paul Ko Ferrigno is Chief Scientific Officer at Avacta Life Sciences and a Visiting Professor at the University of Leeds. He has been working on peptide aptamers since 2001 in Leeds and at the MRC Cancer Cell Unit in Cambridge, UK where his team developed the Affimer scaffold technology.
Dr. Jane McLeod is a Scientific Writer at Avacta Life Sciences.