Improved Synthesis andIn VitroEvaluation of an Aptamer Ribosomal Toxin Conjugate

Abstract

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5¢ terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamertoxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC 50 of *60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t 1/2 of *4 h, paving the way for future in vivo experiments.

Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers

“…Previously, we streamlined conjugation of an aptamer to a protein toxin by chemically synthesizing a minimized aptamer bearing a 5′ thiol and generating an activated pyridyl disulfide for subsequent toxin conjugation. 21 Here, we exploited the maleimide functional groups on commercially available MMAE and MMAF derivatives to further simplify the process of conjugating aptamers to these small molecule toxins. Unlike our previous work, these aptamer-small molecule toxin conjugates are chemicals, not biologics.…”

“…Previously, our lab demonstrated the ability to generate aptamer-toxin conjugates using a recombinant variant of the ribosomal toxin gelonin and the minimized anti-prostate-specific membrane antigen (PSMA) aptamer, A9.min. 21 Here, we assessed the ability to generate small molecule aptamer-toxin conjugates. Using thiol-reactive variants of the auristatin toxins MMAE and monomethyl auristatin F (MMAF), we generated aptamer-toxin conjugates using both the anti-EGFR aptamer, E07, and the anti-TfR aptamer, Waz, and tested their cytotoxicity on three distinct PDAC cell lines: Panc-1, MIA PaCa-2, and BxPC3.…”

Effect of Chemical Modifications on Aptamer Stability in Serum

“…All other synthesis reagents were purchased from Glen Research (Sterling, VA). A9.min, an anti-PSMA aptamer [16,17], was synthesized along with eight variants of C36, a nontargeting control (Table 1 and Fig. 1A).…”

“…Stability assay was performed as previously described [17]. Ten microliters of 25 mM DyLight 650 labeled oligonucleotide was added to 90 mL of serum and incubated at 37°C for up to 7 days.…”

Targeting prostate cancer: Prostate‐specific membrane antigen based diagnosis and therapy

“…A recent study further pursued this strategy and modified the aptamer-toxin conjugate by reducing its length and optimizing the chemical synthesis. 80 The miniaturization of the aptamer slightly reduced its binding capability-IC 50 of 60 nM for the smaller one versus 27 nM for the longer one-but it was still able to selectively kill PSMA-positive cells.…”