Development of an immunotherapeutic adenovirus targeting hormone-independent prostate cancer

Abstract

Jae Sik Kim,1 Sang Don Lee,2 Sang Jin Lee,3 Moon Kee Chung21Department of Urology, The Catholic University of Korea Incheon St Mary's Hospital, Incheon, 2Pusan National University Yangsan Hospital and Research Institute for Convergence of Biomedical Science and Technology, Yangsan, 3Genitourinary Cancer Branch, National Cancer Center, Goyang, KoreaBackground: To develop a targeting therapy for hormone-independent prostate cancer, we constructed and characterized conditionally replicating oncolytic adenovirus (Ad) equipped with mRFP(monomeric red fluorescence protein)/ttk (modified herpes simplex virus thymidine kinase) This construct was then further modified to express both mRFP/ttk and a soluble form of cytokine FLT3L (fms-related tyrosine kinase 3 ligand) simultaneously.Methods: To construct the recombinant oncolytic adenovirus, E1a and E4 genes, which are necessary for adenovirus replication, were controlled by the prostate-specific enhancer sequence (PSES) targeting prostate cancer cells expressing prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). Simultaneously, it expressed the mRFP/ttk fusion protein in order to be able to elicit the cytotoxic effect.Results: The Ad5/35PSES.mRFP/ttk chimeric recombinant adenovirus was generated successfully. When replication of Ad5/35PSES.mRFP/ttk was evaluated in prostate cancer cell lines under fluorescence microscopy, red fluorescence intensity increased more in LNCaP cells, suggesting that the mRFP/ttk fusion protein was folded functionally. In addition, the replication assay including wild-type adenovirus as a positive control showed that PSES-positive cells (LNCaP and CWR22rv) permitted virus replication but not PSES-negative cells (DU145 and PC3). Next, we evaluated the killing activity of this recombinant adenovirus. The Ad5/35PSES.mRFP/ttk killed LNCaP and CWR22rv more effectively. Unlike PSES-positive cells, DU145 and PC3 were resistant to killing by this recombinant adenovirus. Finally, in order to potentiate therapeutic efficacy, we developed a recombinant adenovirus expressing multiexogenous genes, mRFP/ttk and sFLT3L.Conclusion: In the present study, a replication-competent adenovirus was successfully designed to replicate conditionally in PSA-positive and PSMA-positive prostate cancer cells. This recombinant adenovirus is equipped with the fusion protein of suicidal and red-fluorescence fusion protein together with sFLT3L. This construct would be expected to have potent antitumor effects and deserves more extensive investigation.Keywords: adenovirus, prostate cancer, hormone-independent, suicide gene