Emerging clinical evidence indicates that the combination of local administration of immunotherapy with systemic immune-checkpoint blockade targeting the PD-1/PD-L1 pathway improves response rates in select solid tumor indications; however, limited clinical experience with this approach exists in advanced bladder cancer patients. VAX014 is a novel bacterial minicell-based, integrin-targeted oncolytic agent undergoing clinical investigation for intravesical (IVE) treatment of nonmuscle-invasive bladder cancer. Here, we demonstrated that the antitumor activity of VAX014 following IVE administration was dependent upon CD4+ and CD8+ T cells in two syngeneic orthotopic bladder tumor models (MB49 and MBT-2). PD-L1 upregulation was found to be an acquired immune-resistance mechanism in the MB49 model, and the combination of VAX014 with systemic PD-L1 blockade resulted in a significant improvement in bladder tumor clearance rates and development of protective antitumor immunologic memory. Combination treatment also led to enhanced systemic antitumor immune responses capable of clearing distal intradermal tumors and controlling pulmonary metastasis. Distal tumors actively responding to combination therapy demonstrated a phenotypic shift from regulatory T cell to Th1 in intratumoral CD4+ T cells, which was accompanied by a higher percentage of activated CD8+ T cells and higher IFNγ. Finally, VAX014’s target integrins α3β1 and α5β1 were overexpressed in tumor biopsies from advanced-stage bladder cancer patients, as well as in both the MB49 and MBT-2 orthotopic mouse models of bladder cancer. These collective findings provide a rationale for the clinical investigation of VAX014 and systemic PD-1/PD-L1 blockade in advanced-stage bladder cancer.

Colorectal cancer (CRC) remains the third most common form of cancer and, despite its reduced mortality, results in over 50,000 deaths annually, highlighting the need for novel therapeutic approaches. VAX014 is a novel clinical-stage, oncolytic bacterial minicell-based therapy shown to elicit protective antitumor immune responses in cancer, but it has not been fully evaluated in CRC. Here, VAX014 was demonstrated to induce oncolysis in CRC cell lines in vitro and was evaluated in vivo, both as a prophylactic (before spontaneous development of adenomatous polyps) and as a neoadjuvant treatment using the Fabp-CreXApc^fl468 preclinical animal model of colon cancer. As a prophylactic, VAX014 significantly reduced the size and number of adenomas without inducing long term changes in the gene expression of inflammatory, T helper 1 antitumor, and immunosuppression markers. In the presence of adenomas, a neoadjuvant VAX014 treatment reduced the number of tumors, induced the gene expression of antitumor T_H1 immune markers in adenomas, and promoted the expansion of the probiotic bacterium Akkermansia muciniphila. The neoadjuvant VAX014 treatment was associated with decreased Ki67 proliferation in vivo, suggesting that VAX014 inhibits adenoma development through both oncolytic and immunotherapeutic effects. Combined, these data support the potential of VAX014 treatment in CRC and “at risk” polyp-bearing or early adenocarcinoma populations.

In situ immunization (ISI) has emerged as a promising approach to bolster early phases of the cancer immunity cycle through improved T-cell priming. One class of ISI agents, oncolytic viruses (OV), has demonstrated clinical activity, but overall benefit remains limited. Mounting evidence suggests that due to their inherent vulnerability to antiviral effects of type I IFN, OVs have limited activity in solid tumors expressing stimulator of interferon genes (STING) and/or retinoic acid–inducible gene I (RIG-I). Here, using a combination of pharmacologic, genetic, and in vivo approaches, we demonstrate that VAX014, a bacterial minicell–based oncolytic ISI agent, activates both STING and RIG-I and leverages this activity to work best in STING-positive and/or RIG-I–positive tumors. Intratumoral treatment of established syngeneic tumors expressing STING and RIG-I with VAX014 resulted in 100% tumor clearance in two mouse models. Antitumor activity of VAX014 was shown to be dependent on both tumor-intrinsic STING and RIG-I with additive activity stemming from host-intrinsic STING. Analysis of human solid tumor datasets demonstrated STING and RIG-I co-expression is prevalent in solid tumors and associates with clinical benefit in many indications, particularly those most amenable to intratumoral administration. These collective findings differentiate VAX014 from OVs by elucidating the ability of this agent to elicit antitumor activity in STING-positive and/or RIG-I–positive solid tumors and provide evidence that STING/RIG-I agonism is part of VAX014’s mechanism of action. Taken together, this work supports the ongoing clinical investigation of VAX014 treatment as an alternative to OV therapy in patients with solid tumors.

Supplemental Figure S12. Knockout of RIG-I does not dampen VAX014-mediated oncolysis in MB49 cells. MB49WT (WT) and MB49RIG-I KO (RIG-I KO) cells were plated (5,000 cells/well) and treated with a titration (MOI 10,000:1 to 5:1) of VAX014 rBMCs for 2 hours and assayed for cell killing and release of LDH. A) EC50 values and B) LDHhalf-max values calculated by GraphPad Prism 10. Individual data points are plotted with the mean (n= 3). ns = not significant, compared to MB49 WT. #indicates which RIG-I KO clone is represented in Figure 4.

Supplemental Figure S22. Expression of type I IFN transcripts in tumors treated with VAX014 in vivo. A) Expression of IFN-β and several isoforms of IFN-α transcripts in excised i.d. MB49WT tumors treated with i.t. VAX014 at various treatment timepoints. Dashed line represents baseline normalized expression. B) Heatmap of IFN transcript log2 normalized expression at various treatment timepoints (n= 3). Violin plots show log2 normalized expression values as calculated by Rosalind and IFN transcript heatmap was generated using GraphPad Prism 10.

Supplemental Figure S20. Efficacy of i.t. VAX014 in i.d. MC38 WT tumors. 7-10-week-old female C57BL/6 mice were implanted with a single i.d. MC38 WT tumor on Day 0. Animals received weekly i.t. injections of saline or VAX014 starting on Day 5 until complete response (CR) was achieved or termination criteria were reached. A) Mean tumor growth rates and B) Kaplan-Meier survival curves for saline- and VAX014-treated animals (n= 14/group). Mean tumor growth rates (mean ± SEM) were plotted until the median survival of the saline group (Day 25). C) Kaplan-Meier survival curves for mice treated with saline or VAX014 in the presence or absence of depletion antibodies against CD4, CD8, and NK cells (n= 6/group). Unless otherwise indicated, the saline group was used as the comparator for statistical testing. D) Representative data of depletion confirmation in blood from mice in C) analyzed by flow cytometry (n= 3/group). Student’s t test was used to analyze the significance of tumor growth rates on Day 25.