Purpose: Stimulation of toll-like receptor-9 (TLR9) by CpG oligodeoxynucleotides (CpG) has been shown to counteract the immunosuppressive microenvironment and to inhibit tumor growth in glioma models. Because TLR9 is located intracellularly, we hypothesized that methods that enhance its internalization may also potentiate its immunostimulatory response. The goal of this study was to evaluate carbon nanotubes (CNT) as a CpG delivery vehicle in brain tumor models. Experimental Design: Functionalized single-walled CNTs were conjugated with CpG (CNT–CpG) and evaluated in vitro and in mice bearing intracranial GL261 gliomas. Flow cytometry was used to assess CNT–CpG uptake and antiglioma immune response. Tumor growth was measured by bioluminescent imaging, histology, and animal survival. Results: CNT–CpG was nontoxic and enhanced CpG uptake both in vitro and intracranial gliomas. CNT-mediated CpG delivery also potentiated proinflammatory cytokine production by primary monocytes. Interestingly, a single intracranial injection of low-dose CNT–CpG (but not free CpG or blank CNT) eradicated intracranial GL261 gliomas in half of tumor-bearing mice. Moreover, surviving animals exhibited durable tumor-free remission (>3 months), and were protected from intracranial tumor rechallenge, demonstrating induction of long-term antitumor immunity. Conclusions: These findings suggest that CNTs can potentiate CpG immunopotency by enhancing its delivery into tumor-associated inflammatory cells. Clin Cancer Res; 17(4); 1–12. ©2010 AACR.

Generating antitumor responses through the inhibition of tumor-derived immune suppression represents a promising strategy in the development of cancer immunotherapeutics. Here, we present a strategy incorporating delivery of the bacterium Salmonella typhimurium (ST), naturally tropic for the hypoxic tumor environment, transformed with a small hairpin RNA (shRNA) plasmid against the immunosuppressive molecule indoleamine 2,3-dioxygenase 1 (shIDO). When systemically delivered into mice, shIDO silences host IDO expression and leads to massive intratumoral cell death that is associated with significant tumor infiltration by polymorphonuclear neutrophils (PMN). shIDO-ST treatment causes tumor cell death independently of host IDO and adaptive immunity, which may have important implications for use in immunosuppressed patients with cancer. Furthermore, shIDO-ST treatment increases reactive oxygen species (ROS) produced by infiltrating PMNs and, conversely, PMN immunodepletion abrogates tumor control. Silencing of host IDO significantly enhances S. typhimurium colonization, suggesting that IDO expression within the tumor controls the immune response to S. typhimurium. In summary, we present a novel approach to cancer treatment that involves the specific silencing of tumor-derived IDO that allows for the recruitment of ROS-producing PMNs, which may act primarily to clear S. typhimurium infection, but in the process also induces apoptosis of surrounding tumor tissue resulting in a vigorous antitumor effect. Cancer Res; 72(24); 6447–56. ©2012 AACR.

Cancer vaccine therapies have only achieved limited success when focusing on effector immunity with the goal of eliciting robust tumor-specific T-cell responses. More recently, there is an emerging understanding that effective immunity can only be achieved by coordinate disruption of tumor-derived immunosuppression. Toward that goal, we have developed a potent Salmonella-based vaccine expressing codon-optimized survivin (CO-SVN), referred to as 3342Max. When used alone as a therapeutic vaccine, 3342Max can attenuate growth of aggressive murine melanomas overexpressing SVN. However, under more immunosuppressive conditions, such as those associated with larger tumor volumes, we found that the vaccine was ineffective. Vaccine efficacy could be rescued if tumor-bearing mice were treated initially with Salmonella encoding a short hairpin RNA (shRNA) targeting the tolerogenic molecule STAT3 (YS1646-shSTAT3). In vaccinated mice, silencing STAT3 increased the proliferation and granzyme B levels of intratumoral CD4⁺ and CD8⁺ T cells. The combined strategy also increased apoptosis in tumors of treated mice, enhancing tumor-specific killing of tumor targets. Interestingly, mice treated with YS1646-shSTAT3 or 3342Max alone were similarly unsuccessful in rejecting established tumors, whereas the combined regimen was highly potent. Our findings establish that a combined strategy of silencing immunosuppressive molecules followed by vaccination can act synergistically to attenuate tumor growth, and they offer a novel translational direction to improve tumor immunotherapy. Cancer Res; 71(12); 4183–91. ©2011 AACR.

Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for more than 25 years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This observation was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not seen using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex vivo through mechanisms involving FasL and serine proteases. In addition, CD8⁺ T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC. Cancer Immunol Res; 3(9); 1096–107. ©2015 AACR.

Vaccines explored for cancer therapy have been based generally on injectable vector systems used to control foreign infectious pathogens, to which the immune system evolved to respond naturally. However, these vectors may not be effective at presenting tumor-associated antigens (TAA) to the immune system in a manner that is sufficient to engender antitumor responses. We addressed this issue with a novel orally administered Salmonella-based vector that exploits a type III secretion system to deliver selected TAA in the cytosol of professional antigen-presenting cells in situ. A systematic comparison of candidate genes from the Salmonella Pathogenicity Island 2 (SPI2) locus was conducted in the vaccine design, using model antigens and a codon-optimized form of the human TAA survivin (coSVN), an oncoprotein that is overexpressed in most human cancers. In a screen of 20 SPI2 promoter:effector combinations, a P_sifB::sseJ combination exhibited maximal potency for antigen translocation into the APC cytosol, presentation to CD8 T cells, and murine immunogenicity. In the CT26 mouse model of colon carcinoma, therapeutic vaccination with a lead P_sifB::sseJ-coSVN construct (p8032) produced CXCR3-dependent infiltration of tumors by CD8 T cells, reversed the CD8:Treg ratio at the tumor site, and triggered potent antitumor activity. Vaccine immunogenicity and antitumor potency were enhanced by coadministration of the natural killer T-cell ligand 7DW8-5, which heightened the production of IL12 and IFNγ. Furthermore, combined treatment with p8032 and 7DW8-5 resulted in complete tumor regression in A20 lymphoma-bearing mice, where protective memory was demonstrated. Taken together, our results demonstrate how antigen delivery using an oral Salmonella vector can provide an effective platform for the development of cancer vaccines. Cancer Res; 74(21); 6260–70. ©2014 AACR.