Tara Quinton

Survivin-targeted immunotherapy drives robust polyfunctional T cell generation and differentiation in advanced ovarian cancer patients

DepoVax™ is an innovative and strongly immunogenic vaccine platform. Survivin is highly expressed in many tumor types and has reported prognostic value. To generate tumor-specific immune response, a novel cancer vaccine was formulated in DepoVax platform (DPX-Survivac) using survivin HLA class I peptides. Safety and immune potency of DPX-Survivac was tested in combination with immune-modulator metronomic cyclophosphamide in ovarian cancer patients. All the patients receiving the therapy produced antigen-specific immune responses; higher dose vaccine and cyclophosphamide treatment generating significantly higher magnitude responses. Strong T cell responses were associated with differentiation of naïve T cells into central/effector memory (CM/EM) and late differentiated (LD) polyfunctional antigen-specific CD4+ and CD8+ T cells. This approach enabled rapid de novo activation/expansion of vaccine antigen-specific CD8+ T cells and provided a strong rationale for further testing to determine clinical benefits associated with this immune activation. These data represent vaccine-induced T cell activation in a clinical setting to a self-tumor antigen previously described only in animal models.

A novel breast/ovarian cancer peptide vaccine platform that promotes specific type-1 but not Treg/Tr1-type responses.

In light of lack of efficacy associated with current cancer vaccines, we aimed to develop a novel vaccine platform called DepoVax as a therapeutic vaccine for breast/ovarian cancer. This study was designed to examine the efficacy of this novel platform over conventional emulsion vaccine using human class I MHC transgenic mice. We have developed a water-free depot vaccine formulation (DPX-0907) with high immune activating potential. Naturally processed peptides bound to HLA-A2 molecules isolated from independent breast and ovarian tumor cell lines, but not normal cells, were isolated and used as antigens in DPX-0907 along with a proprietary adjuvant and a T helper peptide epitope. Efficacy of vaccine was tested in immunized HLA-A*0201/H2Dd transgenic mice by measuring the frequency of IFN-γ secreting cells in the draining lymph nodes, and regulatory T-cell frequencies in the spleen. Compared with a water-in-oil emulsion vaccine, DPX-0907 enhanced IFN-γ+CD8+ T cells in vaccine site-draining lymph nodes, as seen by immunofluorescence staining and increased the frequency of IFN-γ+ lymph node cells as seen by enzyme-linked immunosorbent spot assay. Notably, while conventional vaccine formulations elicited elevated levels of splenic Foxp3+CD4+ and IL10-secreting T cells, this was not the case for DPX-0907-based vaccines, with treated animals exhibiting normal levels of regulatory T cells. These data support the unique capabilities of a vaccine formulation containing novel tumor peptides and DPX-0907 to elicit type-1 dominated, specific immunity that may represent a potent clinical therapeutic modality for patients with breast or ovarian carcinoma.

A liposome-based platform, VacciMax, and its modified water-free platform DepoVax enhance efficacy of in vivo nucleic acid delivery.

Nucleic acid vaccines represent a promising alternative to killed bacterial antigen, recombinant protein or peptide vaccines for infectious diseases and cancer immunotherapy. Although significant advances are made with DNA vaccines in animal studies, there are severe limitations to deliver these vaccines effectively and considerable reservations exist about current methods used. In this study, a liposome-based vaccine platform, VacciMax (VM), and its modified water-free version, DepoVax (DPX), were tested for their ability to improve in vivo delivery of plasmid DNA (pDNA), mRNA and siRNA. Subcutaneously injected pDNA for IL12 and pDNA as well as mRNA for green fluorescent protein (GFP) in VM/DPX significantly enhanced their in vivo expression. Enhanced IL12 secretion and GFP expression was restricted to CD11b(+) and CD11c(+) antigen-presenting cells, but not B cells. Further, significant inhibition of plasmid/antigen-induced IL12 secretion was seen after injection of IL12-siRNA in VM. These findings suggest VM and DPX to be promising means of delivering nucleic acid vaccines in vivo, and warrant further studies on their role in inducing effective immune responses.

Tumor Inhibition by DepoVax-Based Cancer Vaccine Is Accompanied by Reduced Regulatory/Suppressor Cell Proliferation and Tumor Infiltration

A successful cancer vaccine needs to overcome the effects of immune-suppressor cells such as Treg lymphocytes, suppressive cytokine-secreting Tr1 cells, and myeloid-derived suppressor cells (MDSCs), while enhancing tumor-specific immune responses. Given the relative poor efficacy associated with current cancer vaccines, a novel vaccine platform called DepoVaxTM (DPX) was developed. C3 tumor-challenged mice were immunized with HPV-E7 peptide in DPX- or conventional-emulsion- (CE-) based vaccine. While control mice showed marked increase in Treg/MDSCs in spleen and blood, in mice treated with DPX-E7 the levels remained similar to tumor-free naive mice. Such differences were also seen within the tumor. Antigen-specific IL10-secreting CD4/CD8 T cells and TGF-β +CD8+ T cell frequencies were increased significantly in CE-treated and control mice in contrast to DPX-E7-immunized mice. Analysis of tumor-infiltrating cells revealed higher frequency of suppressor cells in untreated controls than in DPX-E7 group while the converse was true for tumor-infiltrating CD8 T cells. Immunization of tumor-bearing HLA-A2 transgenic mice with human vaccine DPX-0907, a peptide-based vaccine for breast/ovarian/prostate cancers, showed efficient induction of immune response to cancer peptides despite the presence of suppressor cells. Thus, this study provides the rationale for using DPX-based cancer vaccines in immune-suppressed cancer patients, to induce effective anticancer immunity.

Abstract 3657: Phosphatidylserine-targeting antibodies enhance anti-tumor activity of a tumor vaccine in a HPV-induced tumor model

Antibodies targeting phosphatidylserine (PS) have been shown to induce anti-tumor responses by induction of tumor-specific T cells. Based on this observation, we evaluated the responses of PS and PD-1 targeting antibody therapy to enhance anti-tumor responses of a HPV16 peptide vaccine formulated in DepoVax™ (DPX) in mice bearing HPV-transformed C3 mouse tumors. The addition of PS-targeting antibody (mch1N11) to DPX/metronomic cyclophosphamide (mCPA) immunotherapy prolonged survival in comparison to mice receiving an isotype control in combination with DPX/mCPA. When anti-PD-1 was added to mch1N11 + mCPA, there was no increase in survival. The addition of mch1N11 to DPX/mCPA immunotherapy had no effect on tumor growth or survival in the aggressive B16-F10 model. TIL analysis revealed an increase in CD8+ T cells, antigen specific CD8+ T cells and PD-1+ T cells in the tumor with mch1N11 treatment. The expression of surface markers for macrophages (CD68high, F4/80) and dendritic cells (CD11c) were also increased in the tumors of mice treated with mch1N11. RT-qPCR analysis of the tumor confirmed higher mRNA expression of T cells markers (CD8, Granzyme B, PD-1) and antigen presenting cell markers (F4/80, CD74). In the spleen, expression of cell surface markers for monocytes (CD11b) and PD-1+ T cells (CD8) were elevated in groups treated with mch1N11 in combination with anti-PD-1. Combined, these findings indicate that in this model, PS-targeting antibodies can enhance the activity of phagocytic cells involved in antigen presentation. We have found that PD-1 expression increases as anti-tumor activity increases, therefore these results also provide an indication that antibodies targeting PS enhance the anti-tumor immune response induced by DPX/mCPA therapy. The observations suggest that PS-targeting antibodies may enhance therapeutic vaccines for the treatment of cancer. Citation Format: Genevieve Weir, Tara Quinton, Jeff T. Hutchins, Bruce D. Freimark, Marianne Stanford. Phosphatidylserine-targeting antibodies enhance anti-tumor activity of a tumor vaccine in a HPV-induced tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3657. doi:10.1158/1538-7445.AM2017-3657

Abstract B113: Translational studies demonstrate that treatment with anti-PD-1 in unresponsive tumors can be improved by enhancing T cell activation in the tumor microenvironment with vaccine based immune therapy

Checkpoint blockade using monoclonal antibodies has been a significant breakthrough for patients that are non-responsive to conventional therapy. Anti-PD-1 therapy can reactivate tumor infiltrating leukocytes (TILs) that were rendered exhausted due to suppression mediated by PD-L1-upregulation on tumor or antigen presenting cells. However if there are no pre-existing TILs, anti-PD-1 therapy is believed to be less effective. DPX-Survivac is a potent, peptide-based T cell activation therapy that contains multiple HLA class 1 restricted antigens from the oncoprotein survivin and is formulated in the DepoVaxTM platform, a unique lipid-in-oil formulation. In Phase 1/1b clinical trials, DPX-Survivac in combination with metronomic cyclophosphamide (mCPA; 50 mg BID) could enhance systemic immune responses detected by IFN-γ ELISPOT and tetramer analysis of PBMCs. We evaluated if this combination could potentially facilitate treatment of anti-PD-1 non-responsive tumors in a HPV-expressing murine tumor model (C3). Untreated C3 tumors had low expression of PD-L1 in vivo and low TILs. Anti-PD-1 treatment alone provided no protection from tumor growth. Treatment of mice bearing advanced tumors with DPX peptide vaccine and mCPA (20 mg/kg PO) increased the levels of antigen-specific CD8a+ T cells within the tumor microenvironment, detected using flow cytometry. We also detected increased expression of PD-1 on the T cells and PD-L1 on the tumor cells, suggesting that the tumor microenvironment (TME) was mediating immune suppression through increased PD-1:PD-L1 signaling. Treatment of tumor bearing mice with vaccine, mCPA and anti-PD-1 resulted in better tumor control of established tumors. Analysis of tumor infiltrating leukocytes by flow cytometry demonstrated that anti-PD-1 treatment did not further enhance tumor infiltration with antigen-specific CD8+ T cells induced by the vaccine/ mCPA treatment. However, RT-qPCR analysis of the tumor detected an increase in expression of cytotoxic T cell gene signatures within the tumor in combination with anti-PD-1 treatment. Clonal analysis was performed of the total TCRβ sequences using gDNA extracted from the tumors. Vaccine and mCPA treatment resulted in selective expansion of clones, as the top 10 clones accounted for 35% of the total TCRβ sequences; tri-therapy including anti-PD-1 significantly enhanced the expansion of T cells within the TME so that the top 10 clones accounted for 46% of the total TCRβ sequences (p Citation Format: Genevieve Weir, Olga Hrytsenko, Richard van der Jagt, Matthew Cheung, Rena Buckstein, Tara Quinton, Lisa MacDonald, Rita Nigam, Marc Mansour, Neil L. Berinstein, Marianne Stanford. Translational studies demonstrate that treatment with anti-PD-1 in unresponsive tumors can be improved by enhancing T cell activation in the tumor microenvironment with vaccine based immune therapy [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 B113.

Abstract 4903: Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. Using a HPV-expressing murine tumor model (C3), we found that treatment with mCPA (20 mg/kg/day PO) in combination with a DPX peptide vaccine caused selective enrichment of antigen-specific CD8+ T cells, resulting in increased immune responses detected by IFN-a ELISPOT and in vivo cytotoxicity assay. The combination provided long term-control of tumors when initiated within one week of tumor implantation, however efficacy was limited in mice bearing advanced tumors. Antigen-specific CD8+ T cells could be detected infiltrating advanced tumors by flow cytometry, along with increased expression of PD-1 on the T cells and PD-L1 on the tumor cells, suggesting that the tumor microenvironment (TME) was mediating immune suppression through increased PD-1:PD-L1 signaling. Treatment of tumor bearing mice with vaccine, mCPA and PD-1 blockade (with anti-PD-1 or anti-PD-L1) resulted in tumor control of established tumors which were not successfully treated with antibody monotherapy. Analysis of tumor infiltrating leukocytes by flow cytometry demonstrated that anti-PD-1 treatment did not further enhance tumor infiltration with antigen-specific CD8+ T cells induced by the vaccine/ mCPA treatment. However, RT-qPCR analysis of the tumor detected an increase in expression of cytotoxic T cell gene signatures within the tumor in combination with anti-PD-1 treatment. Clonal analysis was performed of the total TCRâ sequences using gDNA extracted from the tumors. Vaccine and mCPA treatment resulted in selective expansion of clones, as the top 10 clones accounted for 35% of the total TCRâ sequences; tri-therapy including anti-PD-1 significantly enhanced the expansion of T cells within the TME so that the top 10 clones accounted for 46% of the total TCRâ sequences (p Citation Format: Genevieve Weir, Olga Hrytsenko, Tara Quinton, Mohan Karkada, Neil L. Berinstein, Marianne Stanford, Marc Mansour. Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4903.

Plasma biomarker analysis of ovarian cancer patients undergoing immunotherapy with therapeutic cancer vaccine DPX-Survivac

Meeting abstracts Immune based therapies for cancer are emerging as promising strategies to treat a number of malignancies. A more thorough biomarker assessment of the patients may provide insights into the variability in response to such immune therapies. In a Phase I/Ib clinical trial with the