Galectin-9 Triggers Neutrophil-Mediated Anticancer Immunity

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

In Vivo Imaging With Two-Photon Microscope For Assessing Tumor Selective Binding Of Anti-CD137 Switch Antibody

STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP (exATP) concentration dependent manner. Although STA551 was assumed to show higher target binding in tumor than normal tissues, quantitative detection of the target binding of switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered 1 mg/kg of fluorescent-labeled antibodies at day 0 and 3. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy was conducted at day4. Higher CD137 expression in tumor than spleen was detected by flow cytometry analysis, and T cells and NK cells were major CD137 expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibody showed binding in tumor. However, in spleen, the fluorescence of switch antibody was much weaker than conventional anti-CD137 antibody and comparable with isotype control. In conclusion, we could assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggested that the tumor selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.

Safety and efficacy of the tumor-selective adenovirus enadenotucirev with or without paclitaxel in platinum-resistant ovarian cancer: a phase 1 clinical trial

BackgroundTreatment outcomes remain poor in recurrent platinum-resistant ovarian cancer. Enadenotucirev, a tumor-selective and blood stable adenoviral vector, has demonstrated a manageable safety profile in phase 1 studies in epithelial solid tumors.MethodsWe conducted a multicenter, open-label, phase 1 dose-escalation and dose-expansion study (OCTAVE) to assess enadenotucirev plus paclitaxel in patients with platinum-resistant epithelial ovarian cancer. During phase 1a, the maximum tolerated dose of intraperitoneally administered enadenotucirev monotherapy (three doses; days 1, 8 and 15) was assessed using a 3+3 dose-escalation model. Phase 1b included a dose-escalation and an intravenous dosing dose-expansion phase assessing enadenotucirev plus paclitaxel. For phase 1a/b, the primary objective was to determine the maximum tolerated dose of enadenotucirev (with paclitaxel in phase 1b). In the dose-expansion phase, the primary endpoint was progression-free survival (PFS). Additional endpoints included response rate and T-cell infiltration.ResultsOverall, 38 heavily pretreated patients were enrolled and treated. No dose-limiting toxicities were observed at any doses. However, frequent catheter complications led to the discontinuation of intraperitoneal dosing during phase 1b. Intravenous enadenotucirev (1×1012 viral particles; days 1, 3 and 5 every 28-days for two cycles) plus paclitaxel (80 mg/m2; days 9, 16 and 23 of each cycle) was thus selected for dose-expansion. Overall, 24/38 (63%) patients experienced at least 1 Grade ≥3 treatment-emergent adverse event (TEAE); most frequently neutropenia (21%). Six patients discontinued treatment due to TEAEs, including one patient due to a grade 2 treatment-emergent serious AE of catheter site infection (intraperitoneal enadenotucirev monotherapy). Among the 20 patients who received intravenous enadenotucirev plus paclitaxel, 4-month PFS rate was 64% (median 6.2 months), objective response rate was 10%, 35% of patients achieved stable disease and 65% of patients had a reduction in target lesion burden at ≥1 time point. Five out of six patients with matched pre-treatment and post-treatment biopsies treated with intravenous enadenotucirev plus paclitaxel had increased (mean 3.1-fold) infiltration of CD8 +T cells in post-treatment biopsies.ConclusionsIntravenously dosed enadenotucirev plus paclitaxel demonstrated manageable tolerability, an encouraging median PFS and increased tumor immune-cell infiltration in platinum-resistant ovarian cancer.Trial registration numberNCT02028117.

719 XTX301, a protein-engineered IL-12, exhibits tumor-selective activity in mice without peripheral toxicities and is well tolerated in non-human primates

BackgroundInterleukin-12 (IL-12) is a proinflammatory cytokine which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promoting cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12 has been limited by severe systemic toxicities. To overcome toxicity and improve the therapeutic index of IL-12, we employed protein engineering to generate XTX301, a highly potent, half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 systemically and render an active IL-12 moiety upon cleavage by proteases that are enriched in the tumor microenvironment.MethodsWe conducted experiments to assess the binding, bioactivity, safety, and anti-tumor efficacy of XTX301. Binding interactions were measured via SPR, bioactivity was measured using STAT-4 phosphorylation in a reporter cell line, and IFN-g production was assessed in human PBMCs via ELISA. Anti-tumor efficacy and pharmacodynamics were assessed in MC38 and B16F10 syngeneic tumor mouse models using a XTX301 murine surrogate, mXTX301. Safety and pharmacokinetics of XTX301 were evaluated in non-human primates (NHP).ResultsXTX301 showed no detectable binding to the high affinity IL12RB2 demonstrating that the masking domain indeed prevents interaction with the receptor. Upon cleavage of the masking domain by relevant proteases, binding was observed and was comparable to XTX300 unmasked control. Likewise, restoration of activity upon proteolytic cleavage was observed in an IL-12-dependent reporter gene assay and in primary human PBMCs. Human IL-12 does not cross react with mouse IL-12 receptors; hence a murine surrogate (mXTX301) was created for in vivo anti-tumor efficacy evaluation. A single dose of mXTX301 demonstrated up to 90% tumor growth inhibition in an inflamed MC38 and non-inflamed B16F10 syngeneic mouse models. mXTX301 induced a ~3 fold increase in IFN-g in tumors compared to vehicle control and ~150 fold less peripheral IFN-g compared to mXTX300. XTX301 exhibits minimal elevation in liver enzymes and a 50-fold improvement in tolerability compared to XTX300, in a repeat dose NHP safety study.ConclusionsOur data demonstrates that both XTX301 and mXTX301 are inactive when in masked form and become activated upon proteolytic cleavage to exert bioactivity comparable to recombinant IL-12. For efficacy, mXTX301 demonstrated tumor selective activity in syngeneic mouse models. XTX301 was well tolerated in repeat dose NHP safety study. In conclusion, XTX301 has potential for exerting potent anti-tumor activity with a favorable tolerability profile.