Towards Effective, Stable Aptamer Drug Conjugates for the Treatment of Solid Tumors
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Traditional chemotherapeutic agents are limited in their utility due to their lack of specificity; their administration often results in highly damaging off-target effects. This prevents the administration of curative doses and may result in the development of drug resistance. In response, there has been much interest in the development of ligand drug conjugates which deliver toxins to cancer cells while bypassing healthy tissue. By far, the most developed ligand drug conjugates are the antibody drug conjugates. However, antibody production result in a heterogeneous mixture of final products, and antibodies are considered too large for optimal solid tumor penetration. By contrast, aptamers are single stranded nucleic acid ligands with similar affinities and specificities to antibodies. However, they are roughly one tenth the size of antibodies. Additionally, they are chemically synthesized which affords precise control over their production and conjugation to toxins. Thus, we explored the use of aptamers for targeted delivery of toxins to solid tumors.;Initially, we streamlined a method for conjugating recombinant gelonin (rGel), a ribosomal toxin, to A9, an anti-prostate specific membrane antigen (PSMA) aptamer for the treatment of prostate cancer. Thought A9-rGel conjugates specifically killed PSMA expressing prostate cancer cells in vitro , the conjugates were ineffective in vivo. Further investigation revealed that A9 was unable to target to in vivo and that A9-rGel conjugates had a half life in mouse serum of only 2.9 hours. Furthermore, gelonin is expressed in E. coli rendering it a biologic, which is a hurdle when seeking regulatory approval. In response, we turned our efforts to prostate cancer, a much more difficult to treat cancer. We conjugated Waz, an anti-transferrin receptor aptamer and E07, an anti-epidermal growth factor receptor aptamer to the auristatin modified toxins monomethyl auristatin E and F. We achieved an excellent therapeutic window when comparing the IC50 values on pancreatic cancer cells to off target cells.;Though we show good targeting and delivery in vitro, aptamer experiments in vivo are affected by the relatively short half-lives of their aptamers. The aptamer field has widely adopted the use of fYrR modified RNA (2'-fluorocytidine and 2'-fluorouridine in combination with 2'-hydroxyadenosine and 2'-hydroxyguanosine) for the development of nuclease stabilized aptamers for in vivo applications. However, reported half lives are often only on the order of hours. We explored the stability of this chemistry along with DNA, 2'-O-methyl, and fGmH (2'fluoro guanosine in combination with 2'-0-methyl adenosine, cytodine, and uracil). Our results show that fYrR and DNA oligonucleotides generally have half lives of 10 h or less while fGmH and 2'-0-methyl oligonucleotides are significantly more stable. Hence, we conclude that they are likely more appropriate for the development of aptamer drug conjugates.
- Theses and Dissertations