SignificanceThe development of therapeutic cancer vaccines using recombinant microorganisms has been pursued for many decades. However, the underlying mechanisms of therapeutic cancer vaccines remain unclear. Here we compare recombinant Listeria-based cancer vaccines to synthetic long peptide and adenovirus delivery systems for tumor antigens, and describe immunologic correlates of antitumor efficacy of Listeria-based cancer vaccines. Our results show that the profound antitumor efficacy requires tumor microenvironment (TME) remodeling that depends on tumor-specific CD8+ T cells induced by live-attenuated double-deleted Listeria monocytogenes expressing cognate tumor antigens. Together, this work highlights the importance of cognate tumor antigen expression by cancer vaccines and pinpoints the relationship between induced tumor antigen-specific immunity and the TME.

SummaryAblation of cells by the controlled expression of a lethal gene can be used to engineer plant traits such as male sterility and disease resistance. However, it may not be possible to achieve suf®cient speci®city of expression to prevent secondary effects in non-targeted tissues. In this paper we demonstrate that the extracellular ribonuclease, barnase, can be engineered into two complementary fragments, allowing overlapping promoter speci®city to be used to enhance targeting speci®city. Using a transient system, we ®rst show that barnase can be split into two inactive peptide fragments, that when co-expressed can complement each other to reconstitute barnase activity. When a luciferase reporter gene was introduced into plant cells along with genes encoding both partial barnase peptides, a substantial reduction in luciferase activity was seen. Cytotoxicity of the reconstituted barnase was demonstrated by crossing together parents constitutively expressing each of the barnase fragments, then assaying their progeny for the presence of both partial barnase genes. None of over 300 tomato seeds planted resulted in a viable progeny that inherited both transgenes. When expression of the partial barnase genes was instead targeted to the tapetum, male sterility resulted. All 13 tomato progeny that inherited both transgenes were male sterile, whereas the three progeny inheriting only the N-terminal barnase gene were male fertile. Finally, we describe how male sterility generated by this type of two-component system can be used in hybrid seed production.

Live-attenuated Listeria monocytogenes has shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenes recombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens. Lm-RIID grows normally in broth but commits suicide inside host cells by inducing Cre recombinase and deleting essential genes flanked by loxP sites, resulting in a self-limiting infection even in immunocompromised mice. Lm-RIID vaccination of mice induces potent CD8+ T cells and protects against virulent challenges, similar to live L. monocytogenes vaccines. When combined with α-PD-1, Lm-RIID is as effective as live-attenuated L. monocytogenes in a therapeutic tumor model. This impressive efficacy, together with the increased clearance rate, makes Lm-RIID ideal for prophylactic immunization against diseases that require T cells for protection.

Modification of the tumor microenvironment (TME) to promote immune-mediated tumor cell destruction is considered to be an essential step for effective immunotherapy. We are evaluating recombinant live-attenuated, double deleted Listeria monocytogenes (LADD) as an immunotherapy platform for the treatment of cancer in several clinical trials in diverse indications. One LADD strain, known as CRS-207, has been engineered to express the tumor-associated antigen mesothelin and is being tested in pancreatic, ovarian and mesothelioma malignancies. Using multi-dimensional immunohistochemistry of paired biopsies from three patients with mesothelioma, we demonstrate the recruitment and expansion of effector tumor-infiltrating lymphocytes, including CD8+ T cells, mature DCs, CD163− macrophages and NK cells, following two prime infusions of CRS-207. In several different syngeneic mouse tumor models, we demonstrate that treatment with LADD engineered to express endogenous tumor antigens also induced significant changes in the TME that were consistent with changes observed in cancer patients, including enhanced CD8+ T cell effector function, recruitment of critical antigen presenting cells and reduction of regulatory T cells, and these changes correlated with significant therapeutic benefit in the mouse. LADD-induced changes to the TME were required for synergistic therapeutic antitumor efficacy combined with immune checkpoint blockade, including targeting MC38 tumor-specific neoantigens. Together, these findings demonstrate that intravenous administration of recombinant LADD therapy induces favorable changes in the tumor microenvironment in mice and humans with promise for effective outcomes in human clinical trials. Citation Format: Weiwen Deng, Takahiro Tsujikawa, Nitya Nair, Thomas Hudson, Weiqun Liu, Chris S. Rae, Edward E. Lemmens, Anthony W. Desbien, William Hanson, Peter Lauer, Lisa M. Coussens, Dirk G. Brockstedt, Thomas W. Dubensky, Jr., Meredith L. Leong. Favorable changes in tumor microenvironment following intravenous dosing with live attenuated Listeria monocytogenes-based immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A013.