Reshma Singh

Vaccination strategy determines the emergence and dominance of CD8+ T-cell epitopes in a FVB/N rat HER-2/neu mouse model of breast cancer.

The HER-2/neu oncogene has >25 HLA epitopes, yet only one FVB/N mouse CD8(+) T-cell epitope has been mapped to date. This epitope has been termed the immunodominant epitope for the FVB/N mouse, but we propose that the vaccination strategy determines the dominance of epitopes. Using a series of overlapping peptides, we have mapped another CD8(+) T-cell epitope that emerges in the FVB/N mouse following vaccination with Listeria monocytogenes-based vaccines that express fragments of HER-2/neu. Following the identification of this novel H-2K(q)-restricted epitope, we sought to compare the T-cell response to this epitope with the previously identified PDSLRDLSVF epitope. This newly identified epitope and the previously identified epitope lie within fragments contained in different vaccines, the PDSLRDLSVF epitope in Lm-LLO-EC2 and the newly identified PYNYLSTEV epitope in Lm-LLO-EC1; thus, it has been possible to compare the responses of these epitopes independent of any competing response between the epitopes. CTL analysis of individual peptide-pulsed target cells and intracellular cytokine stain for IFN-gamma produced by splenocytes from Lm-LLO-EC1 compared with Lm-LLO-EC2 vaccinated FVB/N mice shows that there is no difference between the responses generated to either of these epitopes. We also show that the avidity of the CD8(+) T cells for either of these epitopes is similar based on the concentration of peptide necessary to mediate similar levels of lysis of target cells. In addition, HER-2/neu DNA vaccination followed by CTL analysis further showed that both of these peptides can emerge as epitopes.

Lm-LLO-Based Immunotherapies and HPV-Associated Disease

HPV infection is a direct cause of neoplasia and malignancy. Cellular immunologic activity against cells expressing HPV E6 and E7 is sufficient to eliminate the presence of dysplastic or neoplastic tissue driven by HPV infection. Live attenuated Listeria monocytogenes- (Lm-) based immunotherapy (ADXS11-001) has been developed for the treatment of HPV-associated diseases. ADXS11-001 secretes an antigen-adjuvant fusion (Lm-LLO) protein consisting of a truncated fragment of the Lm protein listeriolysin O (LLO) fused to HPV-16 E7. In preclinical models, this construct has been found to stimulate immune responses and affect therapeutic outcome. ADXS11-001 is currently being evaluated in Phase 2 clinical trials for cervical intraepithelial neoplasia, cervical cancer, and HPV-positive head and neck cancer. The use of a live attenuated bacterium is a more complex and complete method of cancer immunotherapy, as over millennia Lm has evolved to infect humans and humans have evolved to prevent and reject this infection over millennia. This evolution has resulted in profound pathogen-associated immune mechanisms which are genetically conserved, highly efficacious, resistant to tolerance, and can be uniquely invoked using this novel platform technology.

Cancer immunotherapy using recombinant Listeria monocytogenes: transition from bench to clinic.

Cancer immunotherapy has developed into a field of intense study as aspects of the immune system involved in the eradication of cancer have become delineated. Listeria monocytogenes is a gram-positive, facultative intracellular bacterium which infects antigen presenting cells (APC), and is being used as a cancer vaccine to deliver tumor antigens directly to the APC. This results in the generation of a strong immune response towards the tumor associated antigen and direct targeting of the tumor by the immune system. Advances in this field have led to the development of a series of L. monocytogenes-based cancer vaccines, which are currently in clinical trials. A phase I study has shown these vaccines can be safely administered and well-tolerated in terminal stage cancer patients and an efficacy signal was observed in patients who did not respond to other therapies. Additional data on the efficacy of these vaccines is expected in the near-term.

Anti-PD-1 antibody significantly increases therapeutic efficacy of Listeria monocytogenes (Lm)- LLO immunotherapy

BackgroundOne of the significant tumor immune escape mechanisms and substantial barrier for successful immunotherapy is tumor-mediated inhibition of immune response through cell-to-cell or receptor/ligand interactions. Programmed death receptor-1 (PD-1) interaction with its ligands, PD-L1 and PD-L2, is one of the important strategies that many tumors employ to escape immune surveillance. Upon PD-Ls binding to PD-1, T cell receptor (TCR) signaling is dampened, causing inhibition of proliferation, decreased cytokine production, anergy and/or apoptosis. Thus PD-Ls expression by tumor cells serves as a protective mechanism, leading to suppression of tumor-infiltrating lymphocytes in the tumor microenvironment. Lm-LLO immunotherapies have been shown to be therapeutically effective due to their ability to induce potent antigen-specific immune responses. However, it has been demonstrated that infection with Lm leads to up-regulation of PD-L1 on mouse immune cells that can inhibit effector T cells through PD-1/PD-L1 pathway.MethodsTherapeutic and immune efficacy of Listeria-based vaccine (Lm-LLO-E7) in combination with anti-PD-1 antibody was tested in E7 antigen expressing TC-1 mouse tumor model. Tumor growth, survival, as well as peripheral and tumor-infiltrating immune cell profiles after immunotherapy were assessed.ResultsHere we demonstrate that the combination of an Lm-LLO immunotherapy with anti-PD-1 antibody that blocks PD-1/PD-L1 interaction, significantly improves immune and therapeutic efficacy of treatment in TC-1 mouse tumor model. Importantly, we show that in addition to significant reduction of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) in both spleen and tumor microenvironment that are mediated solely by the Lm-LLO immunotherapy, the addition of anti-PD-1 antibody to the treatment results in significant increase of antigen-specific immune responses in periphery and CD8 T cell infiltration into the tumor. As a result, this combinational treatment leads to significant inhibition of tumor growth and prolonged survival/complete regression of tumors in treated animals.We also demonstrate that in vitro infection with Lm results in significant upregulation of surface PD-L1 expression on human monocyte-derived dendritic cells suggesting the translational capacity of this finding.ConclusionsOur findings demonstrate that combination of Lm-LLO-based vaccine with blocking of PD-1/PD-L1 interaction is a feasible approach with clinical translation potential that can lead to overall enhancement of the efficacy of anti-tumor immunotherapy.

Combined immunotherapy with Listeria monocytogenes-based PSA vaccine and radiation therapy leads to a therapeutic response in a murine model of prostate cancer

Radiation therapy (RT) is an integral part of prostate cancer treatment across all stages and risk groups. Immunotherapy using a live, attenuated, Listeria monocytogenes-based vaccines have been shown previously to be highly efficient in stimulating anti-tumor responses to impact on the growth of established tumors in different tumor models. Here, we evaluated the combination of RT and immunotherapy using Listeria monocytogenes-based vaccine (ADXS31-142) in a mouse model of prostate cancer. Mice bearing PSA-expressing TPSA23 tumor were divided to 5 groups receiving no treatment, ADXS31-142, RT (10xa0Gy), control Listeria vector and combination of ADXS31-142 and RT. Tumor growth curve was generated by measuring the tumor volume biweekly. Tumor tissue, spleen, and sera were harvested from each group for IFN-γ ELISpot, intracellular cytokine assay, tetramer analysis, and immunofluorescence staining. There was a significant tumor growth delay in mice that received combined ADXS31-142 and RT treatment as compared with mice of other cohorts and this combined treatment causes complete regression of their established tumors in 60xa0% of the mice. ELISpot and immunohistochemistry of CD8+xa0cytotoxic T Lymphocytes (CTL) showed a significant increase in IFN-γ production in mice with combined treatment. Tetramer analysis showed a fourfold and a greater than 16-fold increase in PSA-specific CTLs in animals receiving ADXS31-142 alone and combination treatment, respectively. A similar increase in infiltration of CTLs was observed in the tumor tissues. Combination therapy with RT and Listeria PSA vaccine causes significant tumor regression by augmenting PSA-specific immune response and it could serve as a potential treatment regimen for prostate cancer.

Listeria monocytogenes (Lm)-LLO immunotherapies reduce the immunosuppressive activity of myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment.

Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) are major components of the immune suppressive cells that potentially limit the effectiveness of an immunotherapy-based treatment. Both of these suppressive cell types have been shown to expand in tumor models and promote T-cell dysfunction that in turn favors tumor progression. This study demonstrates that Listeria monocytogenes (Lm)-LLO immunotherapies effect on the suppressive ability of MDSC and Treg in the tumor microenvironment (TME), resulting in a loss in the ability of these cells to suppress T cells. This alteration of immunosuppression in the TME was an inherent property of all Lm-LLO immunotherapies tested and was independent of the tumor model. The virtually total loss in the suppressive ability of these cells in the TME was linked to the reduction in the expression of arginase I in MDSC and IL-10 in Treg. The results presented here provide insight into a novel mechanism of Lm-LLO immunotherapies that potentially contributes to therapeutic antitumor responses.

Abstract 5241: Development of a single Listeria monocytogene-based vaccine simultaneously targeting two antigens

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnPreviously published studies reported that recombinant Listeria monocytogenes (Lm-LLO)-based vaccines expressing either tumor associated antigens (TAA) or angiogenesis associated antigens fused to an immunogenic fragment of listeriolysin O (LLO) have demonstrated efficacy in mouse models with several types of tumors such as lung, breast, prostate or melanoma. Therefore, we hypothesized that an Lm-LLO vaccine capable of delivering two disparate antigens with two different therapeutic mechanisms would likely have a synergistic effect on decreasing tumor growth by 1) targeting blood vessels, which may enhance effector T cell infiltration into the tumor and 2) improving the therapeutic efficacy of the vaccine. In addition, a dual vaccine construct creates a flexible platform for future use. The Lm-LLO-cHER2 vaccine was developed using a chimeric HER2/neu (cHER2) construct, and was found to regress tumors, elicit a strong T cell immune response and break immune tolerance towards the HER2/neu self-antigen in experimental animals. The Lm-LLO-VEGR2 vaccine has been shown to eradicate established breast tumors, reduce microvascular density, protect against tumor rechallange and metastases, and induce epitope spreading to various regions of the TAA such as HER2/neu. A dual Lm-LLO vaccine was developed that expresses and secretes both the VEGFR2 and cHER2 antigens as LLO-based proteins and is based on a highly attenuated Listeria monocytogenes backbone Lm Δ dal dat actA, which is cleared 48 hours post-injection in interferon gamma knock-out mice. Initial characterization of the dual vaccine indicates that the two antigens cHER2 and VEGFR2 are stably expressed and secreted after two in vivo mouse passages. Currently, we are evaluating the anti-tumor effects and antigen specific immune responses generated by the dual vaccine in mouse models. If successful, this dual vaccine may offer a new immunotherapy for the treatment of cancer in humans.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5241. doi:1538-7445.AM2012-5241

Abstract 5243: Reductions in hypoxia in the tumor microenvironment after Listeria monocytogenes-LLO based immunotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnAs a consequence of increasing growth and outpacing blood supply, tumors become hypoxic as they get larger, with the interior of the tumor becoming extremely hypoxic. This makes therapeutic immune responses difficult, as lymphocytes do not typically infiltrate the interior regions of large tumors due to the hypoxic nature of these tumors. Initial observations in preclinical studies after Listeria monocytogenes (Lm)-LLO based immunotherapy specifically targeted to the hypoxia marker CA9 show the hypoxic nature of the interior of the tumor microenvironment is altered. Initially the tumors are extremely hypoxic (as evidenced by HIF-1α and HIF-2α staining) however, after Lm-LLO-CA9 vaccination they show reduced expression of these hypoxic markers. This change in hypoxia is seen with irrelevant Lm vaccines that overexpress listeriolysin O (LLO) and also with Lm vaccines targeting CA9 fused to LLO: however the change is much more dramatic and complete in the case of the Lm-LLO-CA9 immunotherapy. The irrelevant Lm-LLO effect may be seen due to the fact that the irrelevant vaccine is actually specific for a tumor associated antigen being expressed by the tumor. This does limit the growth of the tumor and may not allow it to reach the same level of hypoxia as an untreated tumor. In current studies, an additional truly irrelevant Lm-LLO vaccine is being added to see if there is a truly Listeria-driven component to the hypoxic changes being observed. As compared to the naive untreated tumors or an irrelevant Lm-LLO control, tumors treated with the CA9 Lm-LLO immunotherapy show an increase in lymphocyte infiltration into the interior areas of the tumor. Studies are underway to determine if this increase in infiltration has an impact on the regression of these tumors by combining the Lm-LLO-CA9 vaccine with a vaccine that is specific for a tumor associated antigen. In addition, the function of these infiltrating lymphocytes are being studied under normoxic and hypoxic conditions, to determine if the infiltrating cells are functional CTLs after the tumors hypoxic levels change. Finally, studies are being conducted to determine the exact signaling mechanism involved in the hypoxic changes of the tumor microenvironment.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5243. doi:1538-7445.AM2012-5243

Abstract P4-04-05: Listeria monocytogenes-based bivalent Lm-LLO immunotherapy for the treatment of HER2/neu positive and triple negative breast cancer and its impact on immunosuppression

Overexpression of tumor associated antigens (TAA) such as HER2/neu and high molecular weight melanoma associated antigen (HMW-MAA) are associated with aggressive high-grade tumors leading to disease progression and reduced survival. HMW-MAA has been reported as a TAA in triple negative breast tumors and is also expressed at high levels by activated pericytes and pericytes in tumor angiogenic vasculature that are associated with neovascularization in vivo . Previously published reports have shown that bioengineered Listeria monocytogenes (Lm) secreting either a chimeric HER2/neu antigen (cHER2), or HMW-MAA fragment, fused to a truncated highly immunogenic fragment of the adjuvant protein listeriolysin O (LLO) were found to impact the growth of both transplantable and transgenic mouse breast tumor models. Therefore, we hypothesized that an Lm -LLO immunotherapy capable of delivering two different antigens would likely have a synergistic effect of decreasing tumor growth by targeting two independent mechanisms that support tumor growth; 1) tumor angiogenesis, and 2) tumor cell surface marker, thus improving the therapeutic efficacy of the agent. Here we report the construction of a bivalent recombinant Lm -LLO immunotherapy that expresses and secretes both the cHER2 and HMW-MAA antigens concomitantly as fusion proteins. Initial characterization of the bivalent immunotherapy indicates that both the antigens cHER2 and HMW-MAA are stably expressed and secreted after two in vivo mouse passages. Preliminary studies support the hypothesis that bivalent immunotherapy causes efficient reduction of tumor growth in both HER2 expressing and triple negative breast tumors. Further, mechanistic studies demonstrate that treatment with Lm -LLO immunotherapy resulted in a decreased proportion of regulatory T cells (Tregs) as well as Myeloid Derived Suppressor Cells (MDSC) particularly in the tumor microenvironment and not in spleen, tumor draining lymph nodes, or peripheral blood. Interestingly, both residual Tregs and MDSCs isolated from the tumor microenvironment were found to have a decreased ability to suppress the division of T cells after Lm -LLO immunotherapy, a finding which warrants further investigation. We will present data on the efficacy of bivalent Lm -LLO immunotherapy and the mechanisms that are likely responsible for the observed tumor regression. Currently Lm -LLO immunotherapies are being evaluated in Phase 2 clinical trials for HPV-associated dysplasia and malignancies such as CIN2/3, cervix cancer and head & neck cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-05.