Enhancement of anti-tumor efficacy of immune checkpoint blockade by alpha-tocopheryloxacetic acid-lysine

Background While much success has been achieved with immune checkpoint blockade (ICB)-targeted therapies, curative responses occur in only a fraction of patients. Adjuvant chemotherapy, which frequently consists of DNA targeting agents, while necessary, can contribute to additional acute toxicity and side effects to the immune-related toxicictes inherent in the use of immune checkpoint. These limitations can negatively impact the quality of life of patients during and after treatment and highlight the need for safer, less toxic anti-cancer agents that can be used in combination with ICB to improve patient outcomes. Alpha-Tocopheryloxy acetic acid-Lysine (a-TEA-LS) is a scalable salt form of alpha-TEA that exhibits tumor cytotoxicity by preferentially targeting dysregulated tumor cell mitochondria to generate toxic reactive oxygen species that trigger immunogenic cell death (ICD). This activity causes release of ”danger signals” including heat shock proteins, ATP, Calreticulin and HMGB-1 and generates antigen-containing autophagosomes which stimulate cross-presentation within dendritic cells leading to antigen-specific T cell priming. Methods Mice bearing establsihed 4T1 mammary tumors were treated with alpha-TEA-lysine salt in combination with the immune checkpoint blockade antibodies anti-PD-1, anti-PD-L1, and anti-CTLA4 and monitored for tumor growth suppression, and overall survival. The combination treatment group that was not effective in controlling 4T1 tumor growth was evaluated for anti-tumor activity against MMTV-PyMT and Eph4 1424 mammary tumors. Results Combination therapy consisting of alpha-TEA-Lys+anti- PD-1 or alpha-TEA-Lys+anti-CTLA4 but not of alpha-TEA-LS+anti- PD-L1 controlled tumor growth in the 4T1 tumor model. Anti-PD-L1 which had no impact on growth of 4T1 tumors inhibited tumor growth and increased overall survival in MMTV-PyMT and Eph4 1424 tumor models. When compared with an immunogenic cell death inducing agent, Doxorubicin, a-TEA-LS+anti-PD-L1 treatment, both treatments were effective in controlling tumor growth but the alpha-TEA-LS+anti-PD-L1 treatment was better tolerated than Doxorubicin+anti-PD-L1 treatment. Mice on the alpha-TEA/anti-PD-L1 therapy demonstrated no weight loss whereas mice in the Doxorubin/aanti-PD-L1 treatment group demonstrated ruffling, were hunched and experienced significant weight loss. Conclusions The data demonstrate that the efficacy of ICB therapy is dependent on the mammary tumor model and on the type of ICB antibody used. Based on the safety profile of a-TEA-LS and the enhanced anti-tumor efficacy demonstrated by a-TEA-LS+ICB in these pre-clinical mammary tumor models, a-TEA-LS has the potential to be used as an adjuvant therapeutic to improve the effectiveness of ICB in human breast cancer. Experiments are underway to interrogate the immune tumor microenvironment to find out what role they may play in the different responses observed. Acknowledgements Research contract services were provided by the University of Arizona Cancer Center Shared Resources with support from the National Cancer Institute of the National Institutes of Health under award number P30 CA02307

Enhancement of anti-tumor efficacy of immune checkpoint blockade by alpha-TEA.

Cancer immunotherapy such as anti-PD-1/anti-PD-L1 immune checkpoint blockade (ICB) can provide significant clinical benefit in patients with advanced malignancies. However, most patients eventually develop progressive disease, thus necessitating additional therapeutic options. We have developed a novel agent, a-TEA-LS, that selectively induces tumor cell death while sparing healthy tissues, leading to increased activation of tumor-reactive T cells and tumor regression. In the current study, we explored the impact of combined a-TEA-LS + ICB in orthotopic and spontaneously arising murine models of mammary carcinoma. We found that a-TEA-LS + ICB led to increased production of pro-inflammatory cytokines that were associated with a reduction in tumor growth and prolonged survival. Together, these data demonstrate the potential utility of a-TEA-LS + ICB for the treatment of breast cancer and provide the rationale for clinical translation of this novel approach

NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8+ T cell responses capable of curing multi-focal cancer

Background High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8+ T cell and natural killer cell stimulation compared with IL-2.Methods Animals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray.Results We demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8+ T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors.Conclusions Given the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors

Galectin-3 inhibition with belapectin combined with anti-OX40 therapy reprograms the tumor microenvironment to favor anti-tumor immunity.

Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis. A wide-variety of tumors also upregulate Gal-3, which is associated with poor prognosis. Tumor-bearing (MCA-205 sarcoma, 4T1 mammary carcinoma, TRAMP-C1 prostate adenocarcinoma) mice were treated with a Gal-3 inhibitor (belapectin; GR-MD-02), aOX40, or combination therapy and the extent of tumor growth was determined. The phenotype and function of tumor-infiltrating lymphocytes was determined by flow cytometry, multiplex cytokine assay, and multiplex immunohistochemistry. Gal-3 inhibition synergized with aOX40 to promote tumor regression and increase survival. Specifically, aOX40/belapectin therapy significantly improved survival of tumor-bearing mice through a CD8+ T cell-dependent mechanism. Combination aOX40/belapectin therapy enhanced CD8+ T cell density within the tumor and reduced the frequency and proliferation of regulatory Foxp3+CD4+ T cells. Further, aOX40/belapectin therapy significantly reduced monocytic MDSC (M-MDSCs) and MHC-IIhi macrophage populations, both of which displayed reduced arginase 1 and increased iNOS. Combination aOX40/belapectin therapy alleviated M-MDSC-specific functional suppression compared to M-MDSCs isolated from untreated tumors. Our data suggests that Gal-3 inhibition plus aOX40 therapy reduces M-MDSC-meditated immune suppression thereby increasing CD8+ T cell recruitment leading to increased tumor regression and survival

Galectin-3 inhibition with belapectin combined with anti-OX40 therapy reprograms the tumor microenvironment to favor anti-tumor immunity

Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis. A wide-variety of tumors also upregulate Gal-3, which is associated with poor prognosis. Tumor-bearing (MCA-205 sarcoma, 4T1 mammary carcinoma, TRAMP-C1 prostate adenocarcinoma) mice were treated with a Gal-3 inhibitor (belapectin; GR-MD-02), aOX40, or combination therapy and the extent of tumor growth was determined. The phenotype and function of tumor-infiltrating lymphocytes was determined by flow cytometry, multiplex cytokine assay, and multiplex immunohistochemistry. Gal-3 inhibition synergized with aOX40 to promote tumor regression and increase survival. Specifically, aOX40/belapectin therapy significantly improved survival of tumor-bearing mice through a CD8+ T cell-dependent mechanism. Combination aOX40/belapectin therapy enhanced CD8+ T cell density within the tumor and reduced the frequency and proliferation of regulatory Foxp3+CD4+ T cells. Further, aOX40/belapectin therapy significantly reduced monocytic MDSC (M-MDSCs) and MHC-IIhi macrophage populations, both of which displayed reduced arginase 1 and increased iNOS. Combination aOX40/belapectin therapy alleviated M-MDSC-specific functional suppression compared to M-MDSCs isolated from untreated tumors. Our data suggests that Gal-3 inhibition plus aOX40 therapy reduces M-MDSC-meditated immune suppression thereby increasing CD8+ T cell recruitment leading to increased tumor regression and survival