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Novel molecular multilevel targeted antitumor agents

Aim: A multifunctional fusion protein, IL-13.E13K-D2-NLS, effectively recognizes glioblastoma (GBM) cells and delivers its portion to the cell nucleus. IL-13.E13K-D2-NLS is composed of a cancer cell targeting ligand (IL-13.E13K), specialized cytosol translocation bacterial toxin domain 2 of Pseudomonas exotoxin A (D2) and SV40 Tantigen nuclear localization signal (NLS). We have now tested whether we can produce proteins that would serve as a delivery vehicle to lysosomes and mitochondria as well. Moreover, we examined whether IL-13.E13K-D2-NLS can deliver anticancer drugs like doxorubicin to their nuclear site of action in cancer cells. Methods: We have thus constructed two novel proteins: IL-13.E13K-D2-LLS which incorporates lysosomal localization signal (LLS) of a human lysosomal-associated membrane protein 1 (LAMP-1) for targeting to lysosomes and IL-13-D2-KK2, which incorporates a pro-apoptotic peptide (KLAKLAK)2 (KK2) exerting its action in mitochondria. Furthermore, we have produced IL-13.E13K-D2-NLS and IL-13.E13K-D2-LLS versions containing a cysteine for site-specific conjugation with a modified doxorubicin, WP936. Results: We found that single-chain recombinant proteins IL-13.E13K-D2-LLS and IL-13-D2-KK2 are internalized and localized mostly to the lysosomal and mitochondrial compartments, respectively, without major trafficking to cells' nuclei. We also determined that IL-13.E13K-D2-NLS-cys[WP936], IL-13.E13K-D2-LAMP-cys[WP936], and IL-13-D2-KK2 were cytotoxic to GBM cells overexpressing interleukin 13 receptor alpha 2, while much less cytotoxic to GBM cell lines expressing low levels of the receptor. IL-13.E13K-D2-NLS-cys[WP936] was the most potent of the tested antitumor agents including free WP936. Conclusion: We believe that our receptor-directed intracellular organelle-targeted proteins can be employed for numerous specific and safer treatment applications when drugs have specific intracellular sites of their action.