Laurence M. Wood

Listeria and Salmonella bacterial vectors of tumor-associated antigens for cancer immunotherapy.

This review covers the use of the facultative intracellular bacteria, Listeriamonocytogenes and Salmonella enterica serovar typhimurium as delivery systems for tumor-associated antigens in tumor immunotherapy. Because of their ability to infect and survive in antigen presenting cells, these bacteria have been harnessed to deliver tumor antigens to the immune system both as bacterially expressed proteins and encoded on eukaryotic plasmids. They do this in the context of strong innate immunity, which provides the required stimulus to the immune response to break tolerance against those tumor-associated antigens that bear homology to self. Here we describe differences in the properties of these bacteria as vaccine vectors, a summary of the major therapies they have been applied to and their advancement towards the clinic.

Attenuated Listeria monocytogenes: a powerful and versatile vector for the future of tumor immunotherapy

For over a century, inactivated or attenuated bacteria have been employed in the clinic as immunotherapies to treat cancer, starting with the Coleys vaccines in the 19th century and leading to the currently approved bacillus Calmette-Guérin vaccine for bladder cancer. While effective, the inflammation induced by these therapies is transient and not designed to induce long-lasting tumor-specific cytolytic T lymphocyte (CTL) responses that have proven so adept at eradicating tumors. Therefore, in order to maintain the benefits of bacteria-induced acute inflammation but gain long-lasting anti-tumor immunity, many groups have constructed recombinant bacteria expressing tumor-associated antigens (TAAs) for the purpose of activating tumor-specific CTLs. One bacterium has proven particularly adept at inducing powerful anti-tumor immunity, Listeria monocytogenes (Lm). Lm is a gram-positive bacterium that selectively infects antigen-presenting cells wherein it is able to efficiently deliver tumor antigens to both the MHC Class I and II antigen presentation pathways for activation of tumor-targeting CTL-mediated immunity. Lm is a versatile bacterial vector as evidenced by its ability to induce therapeutic immunity against a wide-array of TAAs and specifically infect and kill tumor cells directly. It is for these reasons, among others, that Lm-based immunotherapies have delivered impressive therapeutic efficacy in preclinical models of cancer for two decades and are now showing promise clinically. In this review, we will provide an overview of the history leading up to the development of current Lm-based immunotherapies, the advantages and mechanisms of Lm as a therapeutic vaccine vector, the preclinical experience with Lm-based immunotherapies targeting a number of malignancies, and the recent findings from clinical trials along with concluding remarks on the future of Lm-based tumor immunotherapies.

Targeting tumor vasculature with novel Listeria-based vaccines directed against CD105

The FDA approval of bevacizumab (Avastin®, Genentech/Roche), a monoclonal antibody raised against human VEGF-A, as second-line therapy for colon and lung carcinoma validated the approach of targeting human tumors with angiogenesis inhibitors. While the VEGF/VEGFR pathway is a viable target for anti-angiogenesis tumor therapy, additional targets involved in tumor neovascularization have been identified. One promising target present specifically on tumor vasculature is endoglin (CD105), a member of the TGF-β receptor complex expressed on vascular endothelium and believed to play a role in angiogenesis. Monoclonal antibody therapy and preventive vaccination against CD105 has met with some success in controlling tumor growth. This report describes the in vivo proof-of-concept studies for two novel therapeutic vaccines, Lm-LLO-CD105A and Lm-LLO-CD105B, directed against CD105 as a strategy to target neovascularization of established tumors. Listeria-based vaccines directed against CD105 lead to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous Listeria-based vaccines targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Support is provided for epitope spreading to endogenous tumor antigens and reduction in tumor vascularity after vaccination with Listeria-based CD105 vaccines. Reported here, these CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

A novel role for ATM in regulating proteasome- mediated protein degradation through suppression of the ISG15 conjugation pathway

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

Liposome promotion of tumor growth is associated with angiogenesis and inhibition of antitumor immune responses

UNLABELLED Liposomes have tremendous potential as drug carriers in the treatment of cancer. However, despite enhanced tumor drug delivery and decreased toxicity, patient survival rates have not improved significantly compared to corresponding free drug treatments. Importantly, we found that a liposomal nanoparticle currently used as a drug carrier in cancer patients enhanced tumor growth in an immune competent murine model of cancer. This was associated with increased tumor angiogenesis and suppression of antitumor immune responses as indicated by decreased cytokine production by tumor macrophages and cytotoxic T cells, diminished tumor infiltration of tumor-specific T cells, and decreased number of dendritic cells in tumor draining lymph nodes. These results suggest that carrier-induced immunosuppression and angiogenesis have the potential to reduce the antitumor effects of drugs loaded within. These findings may have significant implications for the current use and future development of anticancer nanoparticles and further investigations are urgently needed. FROM THE CLINICAL EDITOR This study discusses important implications of nanoliposome-based drug delivery systems in cancer therapy, and demonstrates that nanoliposomes may have immunosuppressive and angiogenetic properties, directly counterbalancing their anti-cancer activity, which may also have important clinical implications related to more widespread applications of such systems.

The ubiquitin-like protein, ISG15, is a novel tumor-associated antigen for cancer immunotherapy

The recent announcement of the first FDA-approved therapeutic vaccine for prostate cancer, Sipuleucel-T, is a watershed moment for the field of tumor immunotherapy. However, while Sipuleucel-T provides a powerful tool to clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results with HER2 vaccines appear promising in terms of efficacy; however, the lack of HER2 overexpression by a majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated antigen for cancer immunotherapy.

Listeria monocytogenes-derived listeriolysin O has pathogen-associated molecular pattern-like properties independent of its hemolytic ability.

ABSTRACT There is a constant need for improved adjuvants to augment the induction of immune responses against tumor-associated antigens (TAA) during immunotherapy. Previous studies have established that listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, exhibits multifaceted effects to boost the stimulation of immune responses to a variety of antigens. However, the direct ability of LLO as an adjuvant and whether it acts as a pathogen-associated molecular pattern (PAMP) have not been demonstrated. In this paper, we show that a detoxified, nonhemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy and may activate innate and cellular immune responses by acting as a PAMP. Our investigation of the adjuvant activity demonstrates that dtLLO, either fused to or administered as a mixture with a human papillomavirus type 16 (HPV-16) E7 recombinant protein, can augment antitumor immune responses and facilitate tumor eradication. Further mechanistic studies using bone marrow-derived dendritic cells suggest that dtLLO acts as a PAMP by stimulating production of proinflammatory cytokines and inducing maturation of antigen-presenting cells (APC). We propose that dtLLO is an effective adjuvant for tumor immunotherapy, and likely for other therapeutic settings.

Listeria monocytogenes and its products as agents for cancer immunotherapy.

This review covers the use of Listeria monocytogenes and its virulence factors as cancer immunotherapeutics. We describe their development as vectors to carry protein tumor antigen and eukaryotic DNA plasmids to antigen-presenting cells and efforts to harness their tumor-homing properties. We also describe their use as vectors of angiogenic molecules to induce an immune response that will destroy tumor vasculature. The background knowledge necessary to understand the biology behind the rationale to develop Listeria as a vaccine vector for tumor immunotherapy is included as well as a brief summary of the major therapies that have used this approach thus far.

Interferon γ-induced intratumoral expression of CXCL9 alters the local distribution of T cells following immunotherapy with Listeria monocytogenes

The ability of Listeria monocytogenes-based anticancer vaccines to induce tumor regression depends on the responsiveness of malignant cells to interferon γ (IFNγ). Inhibition of IFNγ limits the recruitment of T cells to the tumors of vaccinated mice. We hypothesized that vaccination with immunotherapeutic L. monocytogenes induces the IFNγ-dependent production of chemokines that regulate the migration of tumor-infiltrating T cells. To gain further insights into this issue, we examined the chemokine responses of a transplantable, human papillomavirus (HPV)-immortalized murine tumor model (TC-1) following the administration of a L. monocytogenes-based immunotherapeutic agent that expresses E7 from HPV-16. Here, we report that the administration of L. monocytogenes-based anticancer vaccines increases the secretion of chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL10 by tumors, hence favoring the recruitment of T cells bearing the cognate chemokine (C-X-C motif) receptor 3 (CXCR3). Furthermore, the expression of CXCL9, but not CXCL10, in TC-1 tumors was significantly reduced upon anti-IFNγ antibody treatment. CXCL9 was highly expressed by TC-1 cells following the administration of IFNγ and tumor necrosis factor α (TNFα), in vitro. Moreover, the inhibition of CXCL9 in TC-1 cells reduced the proportion of CD8+ T cells infiltrating tumors in vaccinated mice, while increasing that of CD4+ T cells, thus altering T-cell subset distribution. We conclude that the administration of L. monocytogenes-based anticancer vaccines regulates TH1 chemokine responses and that malignant cells are an important source of these chemokines.

Listeria-derived ActA is an effective adjuvant for primary and metastatic tumor immunotherapy

Tumor immunotherapy is currently at the cusp of becoming an important aspect of comprehensive cancer treatment in the clinic. However, the need for improved adjuvants to augment immune responses against tumor antigens is always present. In this paper, we characterize the Listeria monocytogenes-derived actin-nucleating protein, ActA, as a novel adjuvant for use in tumor immunotherapy. ActA is a virulence factor that is expressed on the cell surface of L. monocytogenes and facilitates the production of actin tails that propel Listeria throughout the cytosol of an infected host cell. It is believed that this ActA-dependent cytosolic motility allows Listeria to evade adaptive host cell defenses and facilitates its invasion into a proximal uninfected host cell. However, there is evidence that ActA fused to a tumor antigen and delivered by L. monocytogenes can perform a beneficial function in tumor immunotherapy as an adjuvant. Our investigation of this adjuvant activity demonstrates that ActA, either fused to or administered as a mixture with a tumor antigen, can augment anti-tumor immune responses, break immune tolerance and facilitate tumor eradication, which suggests that ActA is not only an effective adjuvant in tumor immunotherapy but can also be applied in a number of therapeutic settings.