Patrick Lizotte

In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer

Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The “in situ vaccination” immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic anti-tumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from Cowpea Mosaic Virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic anti-tumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity, and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the anti-tumour immune response. CPMV also exhibited clear treatment efficacy and systemic anti-tumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer.

Immune-Mediated Regression of Established B16F10 Melanoma by Intratumoral Injection of Attenuated Toxoplasma gondii Protects against Rechallenge

Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8+ T cell–mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8+ T cells and NK cells, but did not require CD4+ T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.

Avirulent Toxoplasma gondii Generates Therapeutic Antitumor Immunity by Reversing Immunosuppression in the Ovarian Cancer Microenvironment

Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c(+) antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c(+) cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c(+) cells regained the ability to efficiently cross-present antigen and prime CD8(+) T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8(+) T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8(+) T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors.

Attenuated Listeria monocytogenes reprograms M2-polarized tumor-associated macrophages in ovarian cancer leading to iNOS-mediated tumor cell lysis

A principal mechanism by which tumors evade immune-mediated elimination is through immunosuppression. Previous approaches to tumor immunotherapy have focused on modifying the immunosuppressive environment with immune checkpoint inhibitors, cytokine therapy, and other modalities with the intent to generate T-cell based anti-tumor immunity. We hypothesized that transformation of the suppressive ovarian cancer microenvironment could be achieved by introduction of the attenuated ΔactA/ΔinlB strain of Listeria monocytogenes. ΔactA/ΔinlB introduced into the microenvironment of the aggressive ID8-Defb29/Vegf-A murine ovarian carcinoma is preferentially phagocytosed by tumor-associated macrophages (TAMs) and reprograms that population from one of suppression to immunostimulation. TAMs in the peritoneum upregulated their co-stimulatory molecules CD80 and CD86, increased transcription of inflammatory cytokines, and downregulated transcription of suppressive effector molecules. Surprisingly, therapeutic benefit was not mediated by T- or NK-cell activity. ΔactA/ΔinlB-induced repolarization of TAMs activated direct tumor cell lysis via Nos2 production of nitric oxide. Modulation of the immunosuppressive nature of the ID8-Defb29/Vegf-A microenvironment, specifically by reprogramming of the TAM suppressive population from M2 to M1 polarization, is critical for our observed immune-mediated survival benefit.

Stimulating antitumor immunity with nanoparticles

A variety of strategies, have been applied to cancer treatment and the most recent one to become prominent is immunotherapy. This interest has been fostered by the demonstration that the immune system does recognize and often eliminate small tumors but tumors that become clinical problems block antitumor immune responses with immunosuppression orchestrated by the tumor cells. Methods to reverse this tumor-mediated immunosuppression will improve cancer immunotherapy outcomes. The immunostimulatory potential of nanoparticles (NPs), holds promise for cancer treatment. Phagocytes of various types are an important component of both immunosuppression and immunostimulation and phagocytes actively take up NPs of various sorts, so NPs are a natural system to manipulate these key immune regulatory cells. NPs can be engineered with multiple useful therapeutic features, such as various payloads such as antigens and/or immunomodulatory agents including cytokines, ligands for immunostimulatory receptors or antagonists for immunosuppressive receptors. As more is learned about how tumors suppress antitumor immune responses the payload options expand further. Here we review multiple approaches of NP-based cancer therapies to modify the tumor microenvironment and stimulate innate and adaptive immune systems to obtain effective antitumor immune responses.

Abstract A140: Viral-like nanoparticles for tumor immunotherapy by in situ vaccination mediate potent antitumor immunity

Tumors are recognized by the immune system, but tumors that reach clinically relevant size have established mechanisms to suppress the immune response and protect themselves from immune attack. One approach to enable effective immune response is to change the immunosuppressive tumor microenvironment into an immunostimulatory environment by introducing immunostimulatory reagents directly into tumors. This is essentially an antitumor therapeutic “in situ vaccination”, because the tumors provide the antigens and the adjuvants are the immunostimulatory reagents. The overall goal of the approach is to not only stimulate an anti-tumor immune response against the directly treated tumor, but more importantly to stimulate a systemic anti-tumor response to treat unseen metastases. There are many immunostimulatory reagents that can be used and each has different capabilities. Here we report on plant-derived viral-like nanoparticles from Cowpea Mosaic Virus used in mouse cancer models. These particles are only composed of viral capsid proteins, have no nucleic acids and have no recognized immunostimulatory reagents. However, they are strongly immunostimulatory through unknown pathways and cause dramatic changes in the tumor microenvironment that lead to primary tumor reduction and resistance to metastatic tumors. The treatment is immune-mediated since it requires IFN-γ, IL-12, and adaptive immunity and also involves neutrophils. Tumor reduction or elimination occurs in many anatomic locations and with multiple tumor types. Most importantly, treatment of a primary tumor by direct intratumoral injection mediates robust rejection of a rechallenge with the same tumor. The mechanisms and pathways of immunostimulation are under investigation. In addition to the inherent immunostimulatory adjuvant properties of these nanoparticles, they are a versatile platform to which other reagents for immune modulation can be attached. This demonstration of the value of viral-like nanoparticles for treatment of cancer opens a new avenue of cancer immunotherapy. Citation Format: Patrick Lizotte, Amy Wen, NIcole Steinmetz, Steven Fiering. Viral-like nanoparticles for tumor immunotherapy by in situ vaccination mediate potent antitumor immunity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A140.

Abstract A68: Local tumor treatments to simulate systemic antitumor immune responses

The generation of effective systemic antitumor immune response is the central goal of tumor immunotherapy. The majority of efforts are focused on systemic treatments that inject therapeutic reagents IV. This is an excellent approach and will be a central facet of tumor immunotherapy, however there is also an opportunity to develop therapies that involve direct intratumoral treatments of recognized tumors with the goal of stimulating systemic antitumor immune responses. Such approach can be used in conjunction with systemically applied therapies and have advantages because of the greater ability to control reagent concentrations that are intratumorally injected. We will present data from recent studies that utilize attenuated microorganisms, known TLR agonists and hyperthermia alone or in combinations to treat murine melanoma and ovarian cancer. Citation Format: Patrick Lizotte, Mee Rie Sheen, Amy M. Wen, Nicole F. Steinmetz, Steven Fiering. Local tumor treatments to simulate systemic antitumor immune responses. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A68.

Abstract A36: Treatment of established dermal murine B16F10 melanoma with an attenuated Toxoplasma gondii eliminates the treated tumor and stimulates systemic antitumor immunity.

While the surgical removal of dermal melanoma cures that lesion, it does nothing to stimulate antitumor immunity that could potentially identify and eliminate occult metastatic disease. An immune based treatment that eliminates the primary dermal melanoma could also potentially generate systemic immunity that will protect against metastatic disease. We have utilized an attenuated strain of Toxoplasma gondii (cps) to break tumor-mediated immunosuppression, stimulate an antitumor immune response that eliminates the dermal tumor, and generate systemic antitumor immunity that leads to rejection of subsequent dermal or intravenous challenges with B16F10. T. gondii is an obligate intracellular eukaryotic parasite that infects virtually any mammalian species. The cps strain is a uracil auxotroph that can be grown in vitro but is unable to replicate in vivo. Despite its lack of infectivity, it enters cells and stimulates a strong T-cell mediated immune response characterized by long lasted CD8 effector cells. The presence of cps in the tumor microenvironment modifies the phenotype of tumor infiltrating leukocytes, and along with the expected anti-Toxoplasma immune response there is antigen-spreading so that tumor antigens are responded to and the immune system recognizes subsequent tumor challenges when there is no associated cps. The treatment requires CD8 and NK cells for efficacy but does not require CD4 cells. The treatment also requires that the host express IL-12 and Ifn-g. The treatment requires live cps for efficacy and is effective in mice that are latently infected with another strain of T. gondii, so it could function in the high percentage of humans with latent T. gondii infection and an established immune response against T. gondii. Citation Format: Steven Fiering, Jason R. Baird, Katelyn Byrne, Patrick Lizotte, David Mullins, Mary Jo turk. Treatment of established dermal murine B16F10 melanoma with an attenuated Toxoplasma gondii eliminates the treated tumor and stimulates systemic antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A36.

Abstract B21: Immune-based treatment of ovarian cancer in a mouse model with attenuated Toxoplasma gondii.

Ovarian cancer is generally diagnosed at stage 3 or 4 metastatic disease. The disease course of ovarian cancer is characterized by a remission period following surgery and chemotherapy, but the remission generally only lasts 1-2 years and the relapse is very difficult to treat, so the disease has very poor 5 year survival rates. Effective stimulation of an antitumor immune response during the remission period could eliminate occult metastatic disease and therefore developing immunotherapy approaches for ovarian cancer that can be applied during the remission period is of considerable interest. We have utilized the ID8 based ovarian cancer model in the form of the highly aggressive ID8/Vegf/Defb29 subline to test the ability of an attenuated strain of T. gondii (cps) to stimulate an anti-tumor immune response. We show that following establishment of the tumor by IP injection of tumor cells, introduction of cps into the peritoneal cavity significantly slows the development of the disease. cps predominantly invades immunosuppressive phagocytes in the tumor microenvironment and modifies these and newly recruited phagocytes to be immunostimulatory. The phagocytes are now much more efficient antigen processers and presenters, the immune system is freed from immunosuppression, and the antitumor immune response is activated. The treatment efficacy is associated with a large increase of tumor antigen-specific CD8 T cells and these T cells demonstrate antitumor activity in a transfer experiment and tumor challenge. cps is nontoxic, even in animals with no adaptive immune system, and is a fully attenuated but highly effective platform for developing cancer treatment vaccines. Further developed and applied, cps could be the basis for an autologous tumor vaccine approach that has the potential to improve ovarian cancer outcomes. Citation Format: Steven Fiering, Jason R. Baird, Barbara Fox, Patrick Lizotte, Kiah Sanders, Jose Conjeo-Garcia, David Bzik. Immune-based treatment of ovarian cancer in a mouse model with attenuated Toxoplasma gondii. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B21.