In 1957 Francis Crick proposed the central dogma, which has since acted as the fundamental law of molecular biology. All genetic information follows the same path: deoxyribonucleic acid (DNA) is transcribed into ribonucleic acid (RNA) which is then translated into an amino-acid sequence, ultimately leading to a functional peptide or protein.

The central dogma is largely unidirectional, however, on rare occasions, certain entities will reverse the flow of information; such is the case with the human immunodeficiency virus (HIV). Once inside a cell, the HIV particle will reverse transcribe its RNA genome into DNA so as for it to be integrated within the host cell’s genome, ultimately being copied with each ensuing cellular division.

Looking at the central dogma from a molecular evolution perspective leads us to ponder upon the creation of cells in the first place: for transcription to take place, as well as for DNA replication, various proteins and peptides are required. On the other hand, in order to make proteins, DNA is needed to provide the correct recipe of amino acids. A real chicken and the egg problem, so which came first?

From a quick look at the central dogma, it appears as if the sole purpose of RNA is to act as a messenger between DNA and proteins; but looks can be deceiving. Back in the 1950s, it had been established that the catalysis of peptide bonds formed between amino acids during protein production took place in the ribosome’s large subunit. More information was elucidated from various in vitro experiments performed during the late 1960s. By stripping a ribosome of its proteins, scientists noticed a catalytic RNA core (23S for prokaryotes and 28S for eukaryotes), and ever since we have assumed the origin of an “RNA world”. RNAs would have come long before the advent of DNA or proteins and have functions well beyond that of a simple messenger molecule.

Over the years, functional RNAs have become of interest to the biopharmaceutical field and are now the basis for numerous up and coming therapeutics. Aptamers, which can be of DNA or RNA origin, have been suggested to be nucleotide analogues of antibodies or “chemical antibodies” and can specifically bind to their intended target. Spanning from 20 to 100 nucleotides in length, these single nucleotide strands will fold onto themselves (forming characteristic shapes such as loops and hairpin structures) to allow binding to various epitopes and have shown promise on both the diagnostic and therapeutic sides of medicine.

Presently, Macugen® (pegaptanib) is the only FDA approved aptamer. As a 27 nucleotide-long RNA ophthalmic drug, pegaptanib targets the vascular endothelial growth factor (VEGF), more specifically isoform 165, a key player in the development of wet age-related macular degeneration (WAMD). The macula is the functional center of the retina (a thin layer of tissue on the inside of the back of the eyes that converts incoming light into neural signals which ultimately allow for visual recognition). WAMD is characterized by leaky blood vessels that grow under the retina, in time causing loss in the center of the field of vision. Although this condition cannot be cured, treatments such as Macugen can help prevent any further vision loss by preventing VEGF from binding to its intended receptor on endothelial cells and preventing further growth of blood vessels.

Concurrently, various antibody-related products targeting VEGF have also been approved. Such is the case with Genentech’s hybridoma derived Avastin® (bevacizumab) approved for various oncologic indications and off-label prescriptions which include WAMD, as well as Lucentis® (ranibizumab), a Fab derived and optimized from Avastin, which is approved to treat WAMD.

Head-to-head comparisons between Macugen and Lucentis have provided quite a bit of insight (pardon the pun). Lucentis, which binds to all isoforms of VEGF has proven more efficacious (determined by the best-corrected visual acuity score, or BCVA) but also slightly more dangerous, leading to suspected major cardiovascular events (such as hypertension) experienced by a small number of patients. On the other hand, Macugen, which is more selective as it binds to a single isoform of VEGF, has been shown to be less effective but deemed safer. Individuals suffering from WAMD oftentimes experience co-morbid vascular events and so, proper patient screening prior to drug administration will be required for treatment optimization.

Other aptamers currently in development face similar issues by targeting proteins for which monoclonal antibody therapies already exist; aptamers will have to fight to demonstrate a clear clinical benefit. Nevertheless, several unique challenges have also surfaced, potentially further limiting aptamers’ regular use in a clinical setting.

One such issue remains the short half-life of aptamers within the bloodstream. Oligonucleotides are often targeted by exonucleases that cleave the phosphodiester bonds present between nucleotides, breaking them down to their monomeric state, and rendering them non-functional. Depending on the concentration, length, and structure, oligonucleotides often degrade within several minutes, making their clinical applicability quite limited. One reason for antibodies having been such a success is their extended serum half-life (some reaching up to three weeks).

Beyond exonucleases, the aptamer’s small size dramatically impacts their serum half-life. When it comes to therapeutics, the smaller the better (such as improved penetration into the retina), but too small of a size can be an impeding factor as therapeutics are often filtered out of the body via the renal system. Reaching well below the glomerular filtration size (30-50 kDa), aptamers (6-30 kDa) are easily excreted from the human body. At the current time, beyond Lucentis, only two other antibody fragments have reached the clinic; one reason being their relatively small size (50 kDa) when compared to a full-length antibody (150-180 kDa). As previously mentioned, antibodies often have a half-life of several days even weeks. Antibody fragments suffer equally from their smaller size by being lower than the glomerular filtration threshold and only reaching several hours in circulation following injection.

Regardless, both aptamers and antibody fragments have found a similar solution: the addition of polyethylene glycol (PEG). The addition of PEG to these small molecular entities enables an increase in serum half-life by preventing glomerular filtration, drastically altering their pharmacokinetic profile. Such is the case with Macugen, which now benefits from an average half-life of 10 days, and Cimzia® (certolizumab pegol), one of the two other approved antibody fragments which has been reported to have a half-life of close to two weeks with its PEG addition.

Beyond these potential setbacks, the relatively small size of aptamers can be a double-edged sword. With antibody fragments now being preferred when it comes to receptor blocking in human tumors, smaller sized aptamers should accumulate more efficiently and in greater number within a tumor’s limited and highly compact space. Several aptamers targeting the EGFR family, such as HER2, HER3 and HER4, as well as various well-known immunotherapeutic targets such as PD-1, remain in development.

Following recent healthcare reform initiatives, decreasing the cost of drug manufacturing has been a major goal for the biopharmaceutical industry. Antibody fragments have become of preference over full length antibodies (especially when effector functions are not needed) for their ease of production in prokaryotic rather than eukaryotic cells. Taking this one step further, aptamers are developed in vitro by a process called systematic evolution of ligands by exponential enrichment, or SELEX for short, and can be further synthesized in vitro. Making full use of a cell-free assembly results in even more cost-friendly manufacturing.

Only time will tell whether aptamers will make their mark on the biotech world. However, with close to 35 years of antibodies and antibody fragments being a staple in the diagnostic and therapeutic markets, they are set to sustain their lead for now.

Guillaume Trusz

Author Guillaume Trusz

Guillaume Trusz received his B.S. in Molecular, Cell, and Developmental Biology from the University of California, Los Angeles (UCLA) in 2015 and his M.S. in Biomedical Imaging from the University of California, San Francisco (UCSF) in 2018. Prior to working as an Associate Scientist in the Discovery Immunology Group at LakePharma, Guillaume contributed to various academic and industry related research projects pertaining to small molecules, nanoparticles, as well as biosimilars.

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