On the following day, VAX-IP minicells or the appropriate VAX-I controls (and then allowed to incubate in the presence of minicells for 2C24 hours as described in the Results section and accompanying figures. cells with a unique tumorlytic mechanism. The pharmacological evaluation of VAX-IP minicells as a single agent administered intravesically in two clinically relevant variations of a syngeneic orthotopic model of superficial bladder malignancy results in a significant survival advantage with 28.6% (0.001) and 16.7% (0.003) of animals surviving after early or late treatment initiation, respectively. The results of these preclinical studies warrant further nonclinical and eventual clinical investigation in underserved nonmuscle invasive bladder malignancy individual populations where total cures are achievable. Introduction Bladder malignancy is the second most common urothelial carcinoma worldwide, the GYPA sixth leading cause of cancer death, and the fourth most common malignancy of men in developed countries.1 An estimated 70% of bladder malignancy patients present with nonmuscle invasive disease (NMIBC), with tumors confined to the mucosal surface of the uroepithelium (Ta), tumors invading the but not yet the underlying muscle (T1) and carcinoma (CIS), which can occur concomitant with TaT1 disease.2 Currently, NMIBC patients are stratified into low-, intermediate- and high-risk disease based on tumor stage and grade in addition to other prognostic factors.3 Treatment begins with transurethral resection of bladder tumor (TURBT) followed by risk level-appropriate post-TURBT adjuvant therapy. In intermediate and high-risk NMIBC, including those patients suffering from localized CIS, intravesical immunotherapy with the live bacterial tuberculosis vaccine Bacillus Calmette-Guerin (BCG) is the most effective adjuvant therapy treatment option. While initial responses to BCG have led to its establishment as the standard-of-care, an estimated 50% will recur and face cystectomy.4,5 Adverse side effects with BCG range from local toxicity (occurs in 90% of patients) to more rare ( 5%) but more serious systemic exposure, which can lead to sepsis, organ failure and death.6C9 Taken together, there remains great need for less toxic alternatives to BCG as well as for bladder-sparing second line salvage therapies for use in high-risk NMIBC patients. Bacterial Lapaquistat acetate minicells may provide an intriguing therapeutic option for the intravesical treatment of NMIBC as they symbolize an emerging class of targeted molecular delivery vehicles for therapeutic use in oncology with encouraging applications for tumor-specific targeted delivery of antineoplastic brokers including small molecule drugs, nucleic acids and protein-based payloads.10C12 Minicells are spherical, nano-sized particles best described as miniature versions of the bacterial cells from which they are produced, complete with all parental bacterial components except the bacterial chromosome.13 Missing a chromosome, minicells are inherently incapable of division, replication and persistence, and by definition, are noninfectious. Nonetheless, minicells are as amenable to recombinant engineering as proto-typical bacteria and easily designed to encapsulate specific macromolecular and small molecule therapeutic agents. This work explains the characterization and the and evaluation of VAX-IP minicells as a recombinant bacterial minicell-based therapeutic for the intravesical treatment of NMIBC. VAX-IP minicells are designed to selectively target and deliver the cholesterol-dependent membrane pore-forming protein toxin, perfringolysin O (PFO) to malignancy cells expressing unligated 5 1 (51) or 3 1 (31) integrin heterodimers and results presented here demonstrate quick, selective tumoricidal effects across a representative panel of human and murine urothelial cell carcinoma (UCC) cell lines work characterizes novel target cell plasma membrane permeabilization effects elicited by the PFO component of VAX-IP minicells, occurring in parallel with the initiation of apoptosis. The ability of VAX-IP minicells to prevent tumor growth and prolong survival after intravesical administration was evaluated using two clinically relevant variations of the syngeneic Lapaquistat acetate orthotopic murine MB49 bladder malignancy model.16 In both variations of the MB49 model, VAX-IP minicells were demonstrated to have significant dose-dependent effects around the respective growth of newly-established or well-established tumors while conferring a survival advantage with complete tumor regressions observed at the optimal therapeutic regimen. These results, along with a favorable toxicity profile, suggest the potential clinical application of VAX-IP minicells Lapaquistat acetate in a variety of NMIBC patient populations in addition to potential growth into other oncology indications. Results Strain construction, minicell generation, security feature assessments, and final characterization All minicell-producing strains and plasmids used in this study are outlined in Table 1. The same genetic strategy previously explained to generate the IPTG-inducible minicell-producing strain VAX8I3 was used to create a second-generation strain, coined VAX12B4, which contains and gene deletions as additional safety features launched using a combination of targeted chromosomal gene deletion techniques employing the Red recombinase system.17,18 After confirming that both genes had been properly deleted and the minicell-producing and suicide genes had been properly integrated into the bacterial chromosome, the ability of the resulting VAX12B4 strain to produce minicells was demonstrated as shown in Determine 1a. The minicell-producing strains, VAX13E8 (gene renders minicell-producing parental cells auxotrophic for the essential bacterial cell.