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.Aptamers to several molecular targets of cancer treatment, including HER2, VEGF and EGFR have been reported, as well as aptamers to extracellular domains of the insulin receptor, the IL-4 receptor, and the transferrin receptor. Aptamers may also play a role in DNA-encoded chemistry and antisense therapeutics. As a consequence, aptamers may find application in a number of therapeutic areas.
The application of aptamers in vivo has been limited by their relatively short half-life and lack of efficient systemic delivery. Conjugation of aptamers with polyethylene glycol (PEG) facilitates uptake of aptamers by macrophages via receptor mediated endocytosis, but results in a decrease in cell binding efficacy, as measured by dissociation constants, and a reduction in in vivo stability. Similarly, conjugation of aptamers to proteins, lipids and other molecules may also compromise aptamer activity in vivo.
Bifunctional PEG, which is typically based on PEG polymers with a terminal primary amino group (N-PEG) and a terminal carboxyl group (C-PEG), as well as a PEG with terminal amine and carboxyl end groups (N-C-PEG) have been used to prepare aptamer-PEG conjugates with improved in vivo stability, however, the use of these compounds to prepare conjugates is inefficient.
Another approach for preparing aptamer-PEG conjugates has been reported. Specifically, Herdewinkel et al. reported preparing an aptamer-PEG conjugate by reacting the 5′ end of an aptamer with a cysteine residue present in the protein to which the aptamer was originally bound. Although aptamers modified in this manner exhibited improved resistance to nuclease digestion and prolonged half-lives in plasma in vivo, the preparation of the conjugate was inefficient.
There is a need in the art for a method of preparing improved aptamer-PEG conjugates, as well as pharmaceutical compositions incorporating such improved conjugates. The present disclosure meets this need and provides further related advantages.A
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