Combination of a T cell activating therapy and anti-phosphatidylserine enhances anti-tumour immune responses in a HPV16 E7-expressing C3 tumour model.

DPX is a novel delivery platform that generates targeted CD8 + T cells and drives antigen-specific cytotoxic T cells into tumours. Cancer cells upregulate phosphatidylserine (PS) on the cell surface as a mechanism to induce an immunosuppressive microenvironment. Development of anti-PS targeting antibodies have highlighted the ability of a PS-blockade to enhance tumour control by T cells by releasing immunosuppression. Here, C57BL/6 mice were implanted with HPV16 E7 target-expressing C3 tumours and subjected to low dose intermittent cyclophosphamide (CPA) in combination with DPX-R9F treatment targeting an E7 antigen with and without anti-PS and/or anti-PD-1 targeting antibodies. Immune responses were assessed via IFN-γ ELISPOT assay and the tumour microenvironment was further analyzed using RT-qPCR. We show that the combination of DPX-R9F and PS-targeting antibodies with and without anti-PD-1 demonstrated increased efficacy compared to untreated controls. All treatments containing DPX-R9F led to T cell activation as assessed by IFN-γ ELISPOT. Furthermore, DPX-R9F/anti-PS treatment significantly elevated cytotoxic T cells, macrophages and dendritic cells based on RT-qPCR analysis. Overall, our data indicates that anti-tumour responses are driven through a variety of immune cells within this model and highlights the need to investigate combination therapies which increase tumour immune infiltration, such as anti-phosphotidylserine.

Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide.

BACKGROUND: Future cancer immunotherapies will combine multiple treatments to generate functional immune responses to cancer antigens through synergistic, multi-modal mechanisms. In this study we explored the combination of three distinct immunotherapies: a class I restricted peptide-based cancer vaccine, metronomic cyclophosphamide (mCPA) and anti-PD-1 treatment in a murine tumor model expressing HPV16 E7 (C3). METHODS: Mice were implanted with C3 tumors subcutaneously. Tumor bearing mice were treated with mCPA (20 mg/kg/day PO) for seven continuous days on alternating weeks, vaccinated with HPV16 E749-57 peptide antigen formulated in the DepoVax (DPX) adjuvanting platform every second week, and administered anti-PD-1 (200 µg/dose IP) after each vaccination. Efficacy was measured by following tumor growth and survival. Immunogenicity was measured by IFN-γ ELISpot of spleen, vaccine draining lymph nodes and tumor draining lymph nodes. Tumor infiltration was measured by flow cytometry for CD8α+ peptide-specific T cells and RT-qPCR for cytotoxic proteins. The clonality of tumor infiltrating T cells was measured by TCRß sequencing using genomic DNA. RESULTS: Untreated C3 tumors had low expression of PD-L1 in vivo and anti-PD-1 therapy alone provided no protection from tumor growth. Treatment with DPX/mCPA could delay tumor growth, and tri-therapy with DPX/mCPA/anti-PD-1 provided long-term control of tumors. We found that treatment with DPX/mCPA/anti-PD-1 enhanced systemic antigen-specific immune responses detected in the spleen as determined by IFN-γ ELISpot compared to those in the DPX/mCPA group, but immune responses in tumor-draining lymph nodes were not increased. Although no increases in antigen-specific CD8α+ TILs could be detected, there was a trend for increased expression of cytotoxic genes within the tumor microenvironment as well as an increase in clonality in mice treated with DPX/mCPA/anti-PD-1 compared to those with anti-PD-1 alone or DPX/mCPA. Using a library of antigen-specific CD8α+ T cell clones, we found that antigen-specific clones were more frequently expanded in the DPX/mCPA/anti-PD-1 treated group. CONCLUSIONS: These results demonstrate how the efficacy of anti-PD-1 may be improved by combination with a potent and targeted T cell activating immune therapy.

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.

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 DepoVax(TM) (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- ß (+)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.

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.

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-gamma 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-gamma+CD8+ T cells in vaccine site-draining lymph nodes, as seen by immunofluorescence staining and increased the frequency of IFN-gamma+ 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.