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.

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.

Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti-PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

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.

Interferon-γ Production by Peripheral Lymphocytes Predicts Survival of Tumor-Bearing Mice Receiving Dual PD-1/CTLA-4 Blockade.

Immune checkpoint inhibitors are transforming the way cancer is treated. However, these therapies do not benefit all patients and frequently cause significant immune-related adverse events. Biomarkers that identify patients with a favorable early response to therapy are essential for guiding treatment decisions and improving patient outcomes. In this report of our study, we present evidence that shortly after administration of dual PD-1/CTLA-4 blockade, the proinflammatory capacity of peripheral lymphocytes is predictive of tumor progression and survival outcomes in multiple murine models. Specifically, we observed that the quantity of interferon-γ (IFNγ) produced by peripheral lymphocytes in response to CD3/CD28 stimulation was robustly correlated with subsequent survival outcomes. In the tumor models and early time points assessed in this study, this relationship was considerably more predictive than a host of other potential biomarkers, several of which have been previously reported. Overall, these findings suggest that measuring the capacity of peripheral lymphocytes to produce IFNγ may help identify which patients are benefitting from combination anti-PD-1/anti-CTLA-4 immunotherapy. Cancer Immunol Res; 4(8); 650-7. ©2016 AACR.

Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice.

Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti-cytotoxic T-lymphocyte-associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti-DEC-205 (dendritic and epithelial cells, 205 kDa)-HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1ß/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.

Common gamma chain (γc) cytokines differentially potentiate TNFR family signaling in antigen-activated CD8(+) T cells.

BACKGROUND: Several members of the common gamma chain (gc) cytokine family are already approved (IL-2) or actively being developed as vaccine adjuvants and cancer immunotherapies. Studies have indicated that co-administration of gc cytokines may enhance the efficacy of immunotherapies that function via direct activation of co-stimulatory T cell receptors. To define the specific influence of gc cytokines on the co-stimulatory capacity of CD8(+) T cells and identify combinations with synergistic potential, we investigated the direct impact of gc cytokines on the differentiation and transcriptional profile of recently antigen-primed CD8(+) T cells. METHODS: Naïve CD8(+) T cells were activated with peptide-pulsed APCs. After 48 hours, CD8(+) T cells were harvested and re-cultured in media supplemented with IL-2, IL-4, IL-7, IL-15 or IL-21. After 24 hours, cells were analyzed by cytokine bead array, flow cytometry, and mRNA micro-array. Gene networks responsible for specific CD8(+) T cell functions were constructed through literature-meta review and publicly available annotation databases. Gene expression data from the experimental groups was imported into this network to visualize the impact of each gc cytokine on the functional polarization of recently-activated CD8(+) T cells. RESULTS: Among the gc cytokines, IL-2 induced the greatest increase in the expression of co-stimulatory receptors in recently-activated CD8(+) T cells. IL-2 increased significantly expression of 4-1BB, GITR, ICOS and OX40, at both the transcriptional and protein level. IL-2 also drove the greatest increase in cellular proliferation and the most robust shift towards a pro-survival phenotype, compared with the other gc cytokines. Both IL-4 and IL-21 enhanced expression of cytotoxic effector proteins, but drove distinct phenotypic polarizations, Th2/Tc2 and NK-like, respectively. CONCLUSIONS: Overall, these observations suggest that among gc cytokines, IL-2 may be uniquely capable of synergizing with therapeutic strategies that combine immunization with agonists of co-stimulatory T cell receptors. Previous studies have shown that the timing of IL-2 treatment relative to immunization plays a key role in defining the CD8(+) T cell response, and the findings from this study indicate that administration of exogenous IL-2 shortly after the initial antigen-priming event has concluded may augment the receptivity of these cells to subsequent TNFR co-stimulation.

Combined targeting of costimulatory (OX40) and coinhibitory (CTLA-4) pathways elicits potent effector T cells capable of driving robust antitumor immunity.

Ligation of the TNF receptor family costimulatory molecule OX40 (CD134) with an agonist anti-OX40 monoclonal antibody (mAb) enhances antitumor immunity by augmenting T-cell differentiation as well as turning off the suppressive activity of the FoxP3(+)CD4(+) regulatory T cells (Treg). In addition, antibody-mediated blockade of the checkpoint inhibitor CTLA-4 releases the "brakes" on T cells to augment tumor immunotherapy. However, monotherapy with these agents has limited therapeutic benefit particularly against poorly immunogenic murine tumors. Therefore, we examined whether the administration of agonist anti-OX40 therapy in the presence of CTLA-4 blockade would enhance tumor immunotherapy. Combined anti-OX40/anti-CTLA-4 immunotherapy significantly enhanced tumor regression and the survival of tumor-bearing hosts in a CD4 and CD8 T cell-dependent manner. Mechanistic studies revealed that the combination immunotherapy directed the expansion of effector T-bet(high)/Eomes(high) granzyme B(+) CD8 T cells. Dual immunotherapy also induced distinct populations of Th1 [interleukin (IL)-2, IFN-γ], and, surprisingly, Th2 (IL-4, IL-5, and IL-13) CD4 T cells exhibiting increased T-bet and Gata-3 expression. Furthermore, IL-4 blockade inhibited the Th2 response, while maintaining the Th1 CD4 and effector CD8 T cells that enhanced tumor-free survival. These data demonstrate that refining the global T-cell response during combination immunotherapy can further enhance the therapeutic efficacy of these agents.