Arthee Jahangir

STING Ligand c-di-GMP Improves Cancer Vaccination against Metastatic Breast Cancer

Chandra, Quispe-Tintaya, and colleagues show that stimulator of IFN genes (STING) ligand c-di-GMP activated caspase-3, stimulated T cells, and nearly completely eliminated all metastases in mouse breast cancer model 4T1, when combined with Listeria monocytogenes–based Mage-b vaccine in a therapeutic setting. Cancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)–based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 μmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3–3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 μmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP–killed 4T1 tumor cells, and through c-di-GMP–activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy. Cancer Immunol Res; 2(9); 901–10. ©2014 AACR.

Nontoxic radioactive Listeriaat is a highly effective therapy against metastatic pancreatic cancer

No significant improvement in therapy of pancreatic cancer has been reported over the last 25 y, underscoring the urgent need for new alternative therapies. Here, we coupled a radioisotope, 188Rhenium, to an attenuated (at) live Listeria monocytogenes (Listeriaat) using Listeria-binding antibodies, thus creating a unique radioactive Listeriaat (RL). We then demonstrated in a highly metastatic pancreatic mouse tumor model (Panc-02) that RL delivered radioactivity to the metastases and less abundantly to primary tumors in vivo, without harming normal cells. This result was possible because Listeriaat was efficiently cleared by the immune system in normal tissues but not in the heavily immune-suppressed microenvironment of metastases and primary tumor. Multiple treatments with low doses of the RL resulted in a dramatic decrease in the number of metastases (∼90%) compared with control groups in the Panc-02 model. This is the first report of using live attenuated bacteria delivering a highly radioactive payload to the metastases, resulting in killing tumor cells in vivo without harming normal cells. The nontoxic RL treatment is attractive for clinical development as a therapy to prevent pancreatic cancer recurrence and metastases.

Curcumin improves the therapeutic efficacy of Listeriaat-Mage-b vaccine in correlation with improved T-cell responses in blood of a triple-negative breast cancer model 4T1

Success of cancer vaccination is strongly hampered by immune suppression in the tumor microenvironment (TME). Interleukin (IL)‐6 is particularly and highly produced by triple‐negative breast cancer (TNBC) cells, and has been considered as an important contributor to immune suppression in the TME. Therefore, we hypothesized that IL‐6 reduction may improve efficacy of vaccination against TNBC cancer through improved T‐cell responses. To prove this hypothesis, we investigated the effect of curcumin, an inhibitor of IL‐6 production, on vaccination of a highly attenuated Listeria monocytogenes (Listeriaat), encoding tumor‐associated antigens (TAA) Mage‐b in a TNBC model 4T1. Two therapeutic vaccination strategies with Listeriaat‐Mage‐b and curcumin were tested. The first immunization strategy involved all Listeriaat‐Mage‐b vaccinations and curcumin after tumor development. As curcumin has been consumed all over the world, the second immunization strategy involved curcumin before and all therapeutic vaccinations with Listeriaat‐Mage‐b after tumor development. Here, we demonstrate that curcumin significantly improves therapeutic efficacy of Listeriaat‐Mage‐b with both immunization strategies particularly against metastases in a TNBC model (4T1). The combination therapy was slightly but significantly more effective against the metastases when curcumin was administered before compared to after tumor development. With curcumin before tumor development in the combination therapy, the production of IL‐6 was significantly decreased and IL‐12 increased by myeloid‐derived suppressor cells (MDSC), in correlation with improved CD4 and CD8 T‐cell responses in blood. Our study suggests that curcumin improves the efficacy of Listeriaat‐Mage‐b vaccine against metastases in TNBC model 4T1 through reversal of tumor‐induced immune suppression.

Direct incorporation of the NKT-cell activator α-galactosylceramide into a recombinant Listeria monocytogenes improves breast cancer vaccine efficacy

Background:Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer.Methods:Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined.Results:Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b.Conclusions:Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

Is cancer vaccination feasible at older age

Age-related defects of the immune system are responsible for T cell unresponsiveness to cancer vaccination at older age. Major immune defects at older age are lack of naive T cells, impaired activation pathways of T cells and antigen-presenting cells (APCs), and age-related changes in the tumor microenvironment (TME). This raises the question whether cancer vaccination is feasible at older age. We compared various cancer vaccine studies at young and old age, thereby focusing on the importance of both innate and adaptive immune responses for cancer immunotherapy. These analyses suggest that creating an immune-stimulating environment with help of the innate immune system may improve T cell responses in cancer vaccination at older age.

Cryoablation and Meriva have strong therapeutic effect on triple-negative breast cancer

Interleukin-6, a cytokine produced particularly by triple-negative breast cancers, strongly inhibits T cell responses in the tumor microenvironment. Here we tested cryoablation combined with Meriva (a lecithin delivery system of curcumin with improved bioavailability) in mice with metastatic breast cancer (4T1). Cryoablation involves killing of tumor cells through freezing and thawing, resulting in recruitment of tumor-specific T cells, while curcumin stimulates T cells through the reduction of IL-6 in the TME. Cryoablation plus Meriva accumulated and activated CD8+ T cells to multiple tumor-associated antigens such as Mage-b and Survivin (both expressed by 4T1 tumors). This correlated with a nearly complete reduction of 4T1 primary tumors and lung metastases while little effect was observed from saline or Meriva alone (28 d after tumor cell injection). The survival rate in the group of cryoablation plus Meriva was significantly improved compared to all control groups. Using a less aggressive 4T1 model expressing luciferase (4T1.2luc3), we demonstrated that all mice receiving saline or Meriva developed metastases in the lungs and a primary tumor (38 d after tumor cell injection; and died soon after that), but not the mice receiving cryoablation or cryoablation plus Meriva. However, on day 58 the mice receiving cryoablation developed 4T1.2luc3 metastases in the lungs, while mice receiving cryoablation plus Meriva were free of metastases. These results strongly suggest that cryoablation delayed the development of lung metastases on the short-term, but Meriva administered after cryoablation was significantly better in delaying the development of lung metastases and survival on the long-term.

Immunotherapy with Listeria reduces metastatic breast cancer in young and old mice through different mechanisms

ABSTRACT Cancer immunotherapy is one of the most promising and benign therapies against metastatic cancer. However, most cancer patients are old and elderly react less efficient to cancer vaccines than young adults, due to T cell unresponsiveness. Here we present data of cancer vaccination in young and old mice with metastatic breast cancer (4T1 model). We tested adaptive and innate immune responses to foreign antigens (Listeria-derived) and self-antigens (tumor-associated antigens (TAA)) and their contribution to elimination of metastases at young and old age. Three different protocols were tested with Listeria: a semi- and exclusive-therapeutic protocol both one-week apart, and an exclusive therapeutic protocol frequently administered. Adaptive and innate immune responses were measured by ELISPOT in correlation with efficacy in the 4T1 model. We found that Listeria induced immunogenic tumor cell death, resulting in CD8 T cell responses to multiple TAA expressed by the 4T1 tumors. Only exclusive therapeutic frequent immunizations were able to overcome immune suppression and to activate TAA- and Listeria-specific CD8 T cells, in correlation with a strong reduction in metastases at both ages. However, MHC class Ia antibodies showed inhibition of CD8 T cell responses to TAA at young but not at old age, and CD8 T cell depletions in vivo demonstrated that the T cells contributed to reduction in metastases at young age only. These results indicate that CD8 T cells activated by Listeria has an antitumor effect at young but not at old age, and that metastases at old age have been eliminated through different mechanism(s).

Abstract B076: Novel use of Listeria and gemcitabine to improve immunotherapy for pancreatic cancer

Introduction: Pancreatic ductal adenocarcinoma (pancreatic cancer), is the 4th leading cause of cancer deaths. This “silent killer” is characterized by its development of metastases often before the primary tumor can be detected, resulting in a five-year survival rate of only 4%. Gemcitabine and erlotinib, FDA-approved drugs for pancreatic cancer, improve median survival by less than six months in advanced stage patients, underscoring the need for additional and/or new alternative approaches. Cancer immunotherapy could be such new approach because of its great promise for metastatic cancer, however hurdles need to be overcome. The first hurdle is that tumor-associated antigens (TAA) used in most cancer vaccines are poorly immunogenic, and the second hurdle is immune suppression, particularly by myeloid-derived suppressor cells (MDSC) that prevent T cell activation in the tumor microenvironment (TME). To address these problems we have developed a novel strategy using an attenuated bacterium Listeria monocytogenes that delivers highly immunogenic antigens selectively into tumor cells of metastases and tumors combined with Gemcitabine (GEM) to reduce immune suppression through MDSC. We have cloned highly immunogenic recall antigens (RA) such as tetanus toxoid (TT), poliovirus (PV), and measlevirus (MV) antigens into the Listeria to activate existing memory T cells to foreign highly immunogenic antigens, which most individuals have been exposed to during childhood through vaccinations, and to deliver these antigens selectively into tumor cells. Memory T cells generated to the RA following childhood vaccinations circulate in their blood for life and can upon reactivation kill the infected tumor cells with high efficiency. We have previously demonstrated this with Listeria-TT in a triple-negative breast cancer model 4T1. Listeria and GEM also kill tumor cells through reactive oxygen species (ROS). In the current study we analyzed whether GEM could improve the efficacy of Listeria-TT immunotherapy in mice with pancreatic cancer (Panc-02 model). Experimental procedures: Tumor-bearing mice (pre-immunized with the human TT vaccine) were treated with GEM and Listeria-TT, and effects were analyzed on metastases, tumors, and MDCS. Results: We found that Listeria-TT combined with GEM resulted in a near complete elimination of metastatic pancreatic cancer (metastases and tumors), which was not possible with Listeria-TT or GEM alone. We also found that GEM reduced the MDSC population by 50% in the blood. Currently, we are analyzing CD8 T cell responses of treated and control mice with pancreatic cancer to immunodominant epitopes within the TT protein (TT1162-1169:SNWYFNHL and TT12-86-1294:LNIYYRRL) matching the H2-b haplotype. In conclusion, the combination of GEM and Listeria-TT eliminated metastatic pancreatic cancer in the Panc-02 model. We expect that this dramatic effect is a combination of immune-based and non-immune-based mechanisms. Future prospects: Since the cloned recall antigen fragments also contain many T cell epitopes recognized in humans, we expect that these constructs are directly translatable to human clinical trials in the near future. Finally, cancer patients could be tested in advance by revaccination with human childhood vaccines to determine to which antigens they react to best before starting the Listeria-recall antigen vaccine therapy. Based on patient9s best response (most likely determined by MHC haplotype and the TME), a personalized therapy with the best combination of recall antigen(s) could be designed. Citation Format: Dinesh Chandra, Wilber Quispe-Tintaya, Arthee Jahangir, Alice Kwon, Rodrigo Alves da Silva, Lukman Solola, Ziqiang Yuan, Steven K. Libutti, Claudia Gravekamp. Novel use of Listeria and gemcitabine to improve immunotherapy for pancreatic cancer. [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 B076.