Developing RNA aptamers for binding cell surface targets on prostate cancer cells

Agents able to bind tightly and specifically to the surface of malignant cells would greatly benefit cancer diagnosis and treatment. While antibodies have the ability to specifically recognize some tumor cell markers, their large size and own immunogenicity markedly limit their pharmacological value. The development of nuclease stable RNA molecules, termed “aptamers”, has provided a new alternative to antibodies. These molecules are in vitro selected from a large synthetic library of random sequences of RNA molecules, a small number of which bind tightly and specifically to almost any ligand. Since these aptamers can be stabilized against nuclease degradation, they can be administered in vivo with extended half-lives. Here we describe the first identification of nuclease resistant RNA aptamers that bind to and inhibit the enzymatic activity of a specific cancer cell membrane antigen, the Prostate Specific Membrane Antigen (PSMA). A synthetic library of 6 × 1014 random sequence RNA molecules, containing 2′-fluoro-modified pyrimidines for nuclease stability, was evolved in vitro to bind a baculovirus purified PSMA fusion protein, termed xPSM. The xPSM target was designed without native cytoplasmic and transmembrane domains in order to avoid raising aptamers to epitopes unavailable on whole cells. The purified xPSM protein is folded into its native conformation by evidence of N-acetylated-α-linked-acidic-dipeptidase (NAALADase) enzymatic activity. Six rounds of in vitro selecting aptamers that bind xPSM, bound to magnetic beads, evolved the original library down to two individual aptamer sequences, xPSM-A9 and xPSM-A10. These aptamers are unique and bind PSMA with affinities of 1.1 nM and 11.9 nM, respectively, by evidence of PSMA NAALADase inhibition. Further, both aptamers inhibit native PSMA NAALADase activity from LNCaP cell membranes. Kinetic analysis suggests that xPSM-A10 binds an epitope in the active site of PSMA, where aptamer xPSM-A9 binds a separate epitope, outside of the active site. The full-length aptamers were 71 bases in length, or approximately 23.4 kilodaltons (kD). One aptamer, xPSM-A10-3, was truncated to only 56 bases or 18.5 kD. Later, these aptamers may be applied clinically as PSMA inhibitors, or may be modified to carry imaging or therapeutic agents.