Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors.

BACKGROUND: Immunologically cold tumors with an 'immune desert' phenotype lack tumor-infiltrating lymphocytes (TILs) and are typically impervious to systemic immune checkpoint blockade (ICB). Intratumoral treatment of tumors with immunomodulatory agents can promote local tumor inflammation leading to improved T cell responses in injected tumors. Addition of systemic ICB increases response frequency and immune-mediated clearance of injected and distal non-injected lesions, and this promising approach is being widely investigated clinically. In this work, we evaluate and characterize the local and systemic antitumor immunotherapeutic activity of VAX014, a novel non-viral targeted oncolytic agent based on recombinant bacterial minicells, following intratumoral administration and in combination with systemic ICB. METHODS: The immunotherapeutic activity of VAX014 following weekly intratumoral administration was investigated in multiple preclinical tumor models with B16F10 murine melanoma serving as the primary model for evaluation of immune desert tumors. Mice bearing a single intradermal tumor were used to evaluate tumor response and overall survival (OS), assess changes in immune cell populations, and explore global changes to immunotranscriptomes of injected tumors. Mice bearing bilateral intradermal tumors were then used to evaluate non-injected tumors for changes in TIL populations and phenotypes, compare immunotranscriptomes across treatment groups, and assess distal non-injected tumor response in the context of monotherapy or in combination with ICB. RESULTS: VAX014 demonstrated strong immune-mediated tumor clearance of injected tumors coinciding with significantly elevated CD8+ TILs and upregulation of multiple immune pathways essential for antitumor immune responses. Modest activity against distal non-injected immune desert tumors was observed despite elevated levels of systemic antitumor lymphocytes. Combination with systemic CTLA-4 blockade improved survival and elevated TILs but did not improve clearance rates of non-injected tumors. Immunotranscriptomes of non-injected tumors from this treatment combination group exhibited upregulation of multiple immune pathways but also identified upregulation of PD-1. Further addition of systemic PD-1 blockade led to rapid clearance of non-injected tumors, enhanced OS, and provided durable protective immunological memory. CONCLUSIONS: Intratumoral administration of VAX014 stimulates local immune activation and robust systemic antitumor lymphocytic responses. Combination with systemic ICB deepens systemic antitumor responses to mediate clearance of injected and distal non-injected tumors.

VAX014, an Oncolytic Therapy, Reduces Adenomas and Modifies Colon Microenvironment in Mouse Model of CRC.

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-CreXApcfl468 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 TH1 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.

Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer.

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 α3ß1 and α5ß1 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.

White paper on microbial anti-cancer therapy and prevention.

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.

A Single Intravesical Instillation of VAX014 Inhibits Orthotopic Superficial Bladder Tumor Implantation to Increase Survival.

BACKGROUND/AIM: VAX014 minicells (VAX014) have been previously characterized as an integrin-specific oncolytic biotherapeutic agent. The present study was designed to evaluate the potential of VAX014 as an immediate post-operative intravesical adjuvant therapy in the treatment of non-muscle invasive bladder cancer (NMIBC). MATERIALS AND METHODS: The ability of VAX014 to kill a panel of dissociated urothelial carcinoma cell lines was tested in vitro. In vivo experiments were conducted using a single intravesical dose of VAX014 in the anti-implantation variation of the MB49 syngeneic orthotopic bladder cancer model with tumor implantation and overall survival rates serving as study endpoints. RESULTS: VAX014 rapidly killed dissociated urothelial carcinoma cells, while single dose in vivo pharmacology studies demonstrated the dose-dependent ability of VAX014 to prevent tumor implantation and development, ultimately resulting in a significant survival advantage compared to controls. CONCLUSION: These results suggest that VAX014 holds potential as an immediate post-operative adjuvant therapy in NMIBC.

Preclinical evaluation of VAX-IP, a novel bacterial minicell-based biopharmaceutical for nonmuscle invasive bladder cancer.

The development of new therapies that can prevent recurrence and progression of nonmuscle invasive bladder cancer remains an unmet clinical need. The continued cost of monitoring and treatment of recurrent disease, along with its high prevalence and incidence rate, is a strain on healthcare economics worldwide. The current work describes the characterization and pharmacological evaluation of VAX-IP as a novel bacterial minicell-based biopharmaceutical agent undergoing development for the treatment of nonmuscle invasive bladder cancer and other oncology indications. VAX-IP minicells selectively target two oncology-associated integrin heterodimer subtypes to deliver a unique bacterial cytolysin protein toxin, perfringolysin O, specifically to cancer cells, rapidly killing integrin-expressing murine and human urothelial cell carcinoma cells with a unique tumorlytic mechanism. The in vivo 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 cancer results in a significant survival advantage with 28.6% (P = 0.001) and 16.7% (P = 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 cancer patient populations where complete cures are achievable.

Immunization with non-replicating E. coli minicells delivering both protein antigen and DNA protects mice from lethal challenge with lymphocytic choriomeningitis virus.

In the midst of new investigations into the mechanisms of both delivery and protection of new vaccines and vaccine carriers, it has become clear that immunization with delivery mechanisms that do not involve living, replicating organisms are vastly preferred. In this report, non-replicating bacterial minicells simultaneously co-delivering the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) and the corresponding DNA vaccine were tested for the ability to generate protective cellular immune responses in mice. It was found that good protection (89%) was achieved after intramuscular administration, moderate protection (31%) was achieved after intranasal administration, and less protection (7%) was achieved following gastric immunization. These results provide a solid foundation on which to pursue the use of bacterial minicells as a non-replicating vaccine delivery platform.

The use of bacterial minicells to transfer plasmid DNA to eukaryotic cells.

The delivery of DNA to mammalian cells is of critical importance to the development of genetic vaccines, gene replacement therapies and gene silencing. For these applications, targeting, effective DNA transfer and vector safety are the major roadblocks in furthering development. In this report, we present a novel DNA delivery vehicle that makes use of protoplasted, achromosomal bacterial minicells. Transfer of plasmid DNA as measured by green fluorescent protein expression was found to occur in as high as 25% of cultured Cos-7 cells when a novel chimeric protein containing the D2-D5 region of invasin was expressed and displayed on the surface of protoplasted minicells. Based on endoplasmic reticulum stress and other responses, protoplasted minicells were non-toxic to recipient eukaryotic cells as a consequence of the transfection process. Taken together, these results suggest that bacterial minicells may represent a novel and promising gene delivery vehicle.

Immune responses elicited by bacterial minicells capable of simultaneous DNA and protein antigen delivery.

Recent events surrounding emerging infectious diseases, bioterrorism and increasing multidrug antibiotic resistance in bacteria have drastically increased current needs for effective vaccines. Many years of study have shown that live, attenuated pathogens are often more effective at delivering heterologous protein or DNA to induce protective immune responses. However, these vaccine carriers have inherent safety concerns that have limited their development and their use in many patient populations. Studies using nonliving delivery mechanisms have shown that providing both protein antigen and DNA encoding the antigen to an individual induces an improved, more protective immune response but rarely, if ever, are both delivered simultaneously. Here, non-replicating bacterial minicells derived from a commensal E. coli strain are shown to effectively induce antigen-specific immune responses after simultaneous protein and DNA delivery. These data demonstrate the potential use of achromosomal bacterial minicells as a vaccine carrier.

Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system.

The refinement of tightly regulated prokaryotic expression systems that permit functional expression of toxic recombinant proteins is a continually evolving process. Unfortunately, the current best promoter options are either tightly repressed and produce little protein, or produce substantial protein but lack the necessary repression to avoid mutations stimulated by leaky expression in the absence of inducer. In this report, we present three novel prokaryotic expression constructs that are tightly regulated by L-rhamnose and D-glucose. These expression vectors utilize the Escherichia coli rhaT promoter and corresponding regulatory genes to provide titratable, high-level protein yield without compromising clone integrity. Together, these components may enable the stable cloning and functional expression of otherwise toxic proteins.