Increasing chemical diversity to overcome the pharmacokinetic limitations of Aptamers
In 2004, the first and so far only therapeutic aptamer, pegaptanib for the treatment of neovascular (wet) age-related macular degeneration, was approved by the FDA. Since then, only a small number of therapeutic aptamers have entered clinical trials, largely because potential applications have been limited by the inherent physicochemical properties and ensuing pharmacokinetic profile of aptamers.
As Dr. Sarah Shigdar, Senior Lecturer at Deakin University, Australia and President of the International Society on Aptamers says:
“Two issues that have prevented some aptamers from progressing further into clinical trials are their short half-life and rapid renal clearance. While some applications, such as drug delivery, do not require long plasma stability, ways to prevent rapid renal clearance are necessary to ensure that an effective therapeutic dose is delivered. Enhancing chemical diversity to slow renal clearance, is certain to enhance the applicability of aptamers to systemic delivery rather than limit it to local delivery.”
Like other oligonucleotide therapeutics, unmodified aptamers are susceptible to nuclease-mediated degradation and rapid renal clearance due to their small size. Commonly used chemical modifications and addition of ligands such as pegylation can alleviate these issues but may present their own challenges.1,2
One issue specific to aptamers is the limited chemical repertoire of the natural nucleobases in DNA or RNA compared to that of proteins.3 Even when starting with libraries of 1015 randomized nucleic acid sequences for SELEX (Systematic evolution of ligands by exponential enrichment), a sizeable fraction of proteins are resistant to successful aptamer selection. Increasing chemical diversity within the random sequence selection pool can have benefits beyond increasing the number of addressable targets, by increasing affinity, nuclease resistance and plasma residence time.
Scientists at SomaLogic, one of the companies at the forefront of developing aptamers, have developed SOMAmers (slow off-rate modified DNA aptamers) containing modified deoxyuracils with amino acid-like side chains at the C5 position. According to Dr. Nebojsa Janjic, the Chief Science Officer of SomaLogic, they made “enormous progress with improving the efficiency of SELEX against protein targets by introducing modifications at a single base in the starting DNA libraries. But we found out empirically that it mattered very much which modifications we chose: hydrophobic aromatic side chains clearly produced the best results. In contrast, more hydrophilic side chains were generally not that different from unmodified DNA. It turns out that amino acids with the same kind of hydrophobic aromatic side chains (phenylalanine, tyrosine and tryptophan) are over-represented in the parts of antibodies that bind to protein antigens, so our observations, in the context of an entirely different class of ligands, kind of made sense.”
In a recently published study, Gupta and colleagues from SomaLogic and Otsuka Pharmaceutical report on the pharmacokinetic profile of these SOMAmers.4 To investigate this, they designed a series of aptamers with varying side chain hydrophobicity, number of side chains, and overall oligonucleotide length based on the previously described SL1026 and SL1033 interleukin-6 SOMAmers.5 All test sequences contained a 3′-idT cap, and a 5′-PEG (40 kDa) and were heavily modified with 2′-OMe to inactivate binding and thus eliminate potential on-target effects on the PK properties.
Dr. Janjic, the senior author of this study, remarked that when they measured plasma half-life and clearance in rats following an intravenous bolus dose “we found to our surprise that the very same types of side chains [hydrophobic] that gave us the best aptamers in terms of binding affinity, also reduced residence time of aptamers in plasma the most.” Although this effect is analogous to what has been seen with excessive loading of antibodies with hydrophobic small molecule drugs, Dr. Janjic says that he “found this kind of shocking, because antibodies are so big (150,000 Da) and small molecules are comparatively so small (around 350-600 Da), but there it is, that is what has been observed in the antibody-drug conjugate field.”
The study also showed that the reduction in plasma residence time was correlated with the number of hydrophobic side chains and length of the aptamer but that the correlation was significantly weaker in shorter sequences. This means that plasma clearance of these modified aptamers can be slowed by using shorter (<24 bases) sequences with fewer side chains, “a key observation for the field which kind of made for a happy ending” (Dr. Janjic). With certain combinations of the amino acid-like side chains in the starting library, two such modifications are enough to get increased nuclease resistance as well as high affinity in shorter sequences as Gawande et al. from SomaLogic reported last year.6
Dr. Janjic believes that “the biggest impact [of the study] will be with being able to get aptamers with high affinity and specificity to many additional protein targets. Most drug targets are proteins, so this is highly relevant for drug discovery. The modified side chains also represent convenient sites of SAR-type optimization of aptamers. We typically start with uniformly-modified libraries at each of the positions that have modified side chains (for example, benzyl side chain), but once we know the sequence of the individual aptamer, we can systematically modify these side chains, the way a medicinal chemist would with a small molecule, to get to the optimal properties of the aptamer-based drug candidate. The field will mature, and we will have many additional aptamer-based drugs in the future. Anything that resembles a true discovery platform, including some of the components I mentioned above related to PK and metabolic stability, will certainly help. Aptamer therapeutics also need to be aimed at the right indications, that is, indications for which specific properties of aptamers are advantageous over other therapeutic classes.”
Vittorio de Franciscis Research Director at CNR-IEOS, Italy says the study “is a comprehensive work that offers the blueprint to enhance the pharmacokinetic properties of aptamers paving the way to substitute therapeutic antibodies with this new class of safer and precise RNA-based drugs.”
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- Chemically modified DNA aptamers bind interleukin-6 with high affinity and inhibit signaling by blocking its interaction with interleukin-6 receptor.
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