Anti-PD-L1/TGFßR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis.

BACKGROUND: Avelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFß) receptor 2, which functions as a TGFß "trap." Advanced urothelial tumors have been shown to express TGFß, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFß from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines. METHODS: Human urothelial (transitional cell) carcinoma cell lines HTB-4, HTB-1, and HTB-5 were treated with M7824, M7824mut (M7824 that is mutated in the anti-PD-L1 portion of the molecule and thus does not bind PD-L1), anti-PD-L1 (avelumab), or IgG1 isotype control monoclonal antibody, and were assessed for gene expression, cell-surface phenotype, and sensitivity to lysis by TRAIL, antigen-specific cytotoxic T lymphocytes and natural killer cells. RESULTS: M7824 retains the ability to mediate antibody-dependent cellular cytotoxicity of tumor cells, although in some cases to a lesser extent than anti-PD-L1. However, compared to anti-PD-L1, M7824 increases (A) gene expression of molecules involved in T-cell trafficking in the tumor (e.g., CXCL11), (B) TRAIL-mediated tumor cell lysis, and (C) antigen-specific CD8+ T-cell-mediated lysis of tumor cells. CONCLUSIONS: These studies demonstrate the immunomodulatory properties of M7824 on both tumor cell phenotype and immune-mediated lysis. Compared to anti-PD-L1 or M7824mut, M7824 induces immunogenic modulation of urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. These findings show the relevance of the dual blockade of PD-L1 and TGFß in urothelial carcinoma cell lines and thus support the rationale for future clinical studies of M7824 in patients with urothelial cancer.

The differential immunological activities of Ganoderma lucidum on human pre-cancerous uroepithelial cells.

AIMS OF THE STUDY: Ganoderma lucidum is active to stimulate immunological effector cells, but the effects on uroepithelial cells have never been explored. The present study compared the expression of major cytokines induced by the water (GLw) and ethanol (GLe) extracts of G. lucidum. MATERIALS AND METHODS: The pre-cancerous human uroepithelial cell (HUC-PC) line was employed. A total of 15 cytokines, including major Th1/Th2 cytokines and chemokines, were measured in the complete media after 24h incubation with GLw and GLe. Additionally, the following assays were performed: cytotoxicity, apoptosis, migration of neutrophils, and nuclear factor-kappaB (NF-κB) DNA binding activity. RESULTS: GLe inhibited the growth of HUC-PC cells through apoptosis. Interleukins IL-2, IL-6, and IL-8 were significantly up-regulated by GLe in dose-dependent manners, but not by GLw. However, MCP-1 level was significantly increased by GLw but was oppositely reduced by GLe. Furthermore, the elevation of cytokine expression was correlated with the enhancement of p50/p65 NF-κB activity induced by GLe. The elevated IL-8 levels in GLe-treated cells were also correlated with the migration of neutrophils. CONCLUSIONS: GLe and GLw exhibited different immunological activities on the HUC-PC cells. In particular, the activities of GLe may favor the clearance of high risk urothelial cells, suggesting potent chemopreventive ingredients are extractable by ethanol from G. lucidum.

Chinese medicinal herbs reverse macrophage suppression induced by urological tumors.

Tumors are known to produce factors suppressing macrophage function. In this study we demonstrated in vitro suppression of macrophage chemiluminescent oxidative burst associated with viable cells and cell-free extracts of two urological neoplasms--murine renal cell carcinoma (Renca) and murine bladder tumor (MBT). Suppression was reversed by extracts of two Chinese medicinal herbs, Astragalus membranaceus (AM) and Ligustrum lucidum (LL). Murine macrophage cell line J774 was incubated with either the viable tumor cells or the cell-free tumor extract for 18 hours at 37C and 5% CO2. Chemiluminescent oxidative burst as an indicator of macrophage function was triggered by adding zymosan A suspension containing luminol and assayed in an automated luminometer. Photon emission over time was counted and the results were expressed as integrated photon emission. Significant dose-related depression of oxidative burst occurred with either the viable tumor cells or the cell-free tumor extracts. Depression was partially or completely reversed by the presence of 50-100 micrograms./ml. of either the AM or the LL extract. AM and LL have previously been shown to modulate immune response. Data from this study suggest that they may also exert their antitumor activity via abolition of tumor-associated macrophage suppression.