Abstract A117: Rapid uptake of a streptavidin conjugate into prostate cancer cells: A model of macromolecule targeting delivery.

Introduction: Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, has been confirmed to be an important tumor-marker indicating prostate cancer progression, and is becoming an active target for imaging or therapy for prostate cancer. Furthermore, the streptavidin-biotin coupling pair has been successfully employed in pretargeting therapy for multiple cancers. In the present study, we describe the synthesis and in vitro performance evaluation of a biotinylated PSMA inhibitor (biotin-PEG12-CTT54) possessing two functional motifs separated by a PEG linker: 1) a PSMA targeting core (CTT-54) that specifically binds enzyme-biomarker PSMA and 2) biotin for binding streptavidin. Results and Discussion: Biotin-PEG12-CTT54 was confirmed by MALDI-TOF analysis: calculated 1267.52, found 1267.5221. This bifunctional compound was further evaluated for its inhibitory potency against purified PSMA and in vitro cell imaging efficiency. PSMA inhibition study confirmed that conjugation of CTT-54 to biotin through PEG linker (biotinylated inhibitor, IC50 = 10 nM) had no adverse effect on the inhibitory potency of the parent inhibitor core CTT-54 (IC50 = 14 nM). In a two-step pre-targeting imaging experiment, 3-day grown PSMA-positive LNCaP cells was treated with this biotinylated compound, then followed by the incubation with Cy5-conjugated streptavidin, our data demonstrated specifically labeling PSMA-positive prostate cancer cells, but not for cells treated with Cy5-conjugated streptavidin only. Additionally, the complex between the biotinylated compound and Cy5-conjugated streptavidin was prepared and purified prior to cell labeling. The purified pre-formed complex was found to contain three biotinylated inhibitors for each streptavidin using a newly-developed digitally-operated linear quadrupole ion trap orthogonal acceleration time-of-flight mass spectrometer, which is designed to analyze non-covalently bound complex by confidentially maintaining its integrity through the analytical process. The preformed complex was tested for its inhibitory potency against purified PSMA and the cell-labeling of PSMA-positive cells, the experimental results revealed an improved inhibitory potency (IC50 = 1.9 nM) against PSMA and rapid uptake of streptavidin into PSMA-positive prostate cancer cells within 10 min. To better understand the intracellular fate of Cy5-streptavidin once internalized in LNCaP cells, co-localization studies were conducted with transferrin-fluorescein, which can be tracked as an endosomal marker for the ligand-receptor internalization via clathrin-coated pits. Co-localization of internalized streptavidin-Cy5 with transferrin-fluorescein appeared strongly in perinuclear region after 2h-incubation. The pattern of PSMA internalization induced by preformed complex is consistent with antibody or small-molecule inhibitors alone. Conclusions: We have successfully demonstrated that macromolecules such as streptavidin can be specifically delivered into prostate cancer cells. These results support a proof-of-concept that a combination of streptavidin (serving as a drug or imaging agent carrier) and a biotinylated PSMA inhibitor may lead to the development of a novel strategy of tumor-targeting imaging or drug delivery for prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A117.