Antiangiogenic and cytotoxic effects are considered the principal mechanisms of action of sorafenib, a multitarget kinase inhibitor approved for the treatment of hepatocellular carcinoma (HCC). We report that sorafenib also acts through direct immune modulation, indispensable for its antitumor activity. In vivo cell depletion experiments in two orthotopic HCC mouse models as well as in vitro analysis identified macrophages (MΦ) as the key mediators of the antitumoral effect and demonstrate a strong interdependency of MΦ and natural killer (NK) cells for efficient tumor cell killing. Caspase 1 analysis in sorafenib-treated MΦ revealed an induction of pyroptosis. As a result, cytotoxic NK cells become activated when cocultured with sorafenib-treated MΦ, leading to tumor cell death. In addition, sorafenib was found to down-regulate major histocompatibility complex class I expression of tumor cells, which may reduce the tumor responsiveness to immune checkpoint therapies and favor NK-cell response. In vivo cytokine blocking revealed that sorafenib efficacy is abrogated after inhibition of interleukins 1B and 18. Conclusion:We report an immunomodulatory mechanism of sorafenib involving MΦ pyroptosis and unleashing of an NK-cell response that sets it apart from other spectrum kinase inhibitors as a promising immunotherapy combination partner for the treatment of HCC. (Hepatology 2019;70:1280-1297). L iver cancer is the second leading cause of cancer-related deaths worldwide.(1) Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer, with increasing incidence and dismal prognosis. Hepatitis B and C viral infection, alcoholism, as well as nonalcoholic steatohepatitis are predominant risk factors for HCC development. (2) Sorafenib, a broad-spectrum kinase inhibitor, has been approved since 2007 for treating patients with unresectable HCC. (3) This adenosine triphosphate-competitive kinase inhibitor targets B-Raf, C-Raf, mitogen-activated protein (MAP) kinases, vascular endothelial growth factor (VEGF) receptor, and platelet-derived growth factor

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies. Cancer stem cells (CSCs), which are not targeted by current therapies, may be the reason for pronounced therapy resistance. A new treatment option in phase II trials is cabozantinib that inhibits the pancreatic CSC surface marker and tyrosine kinase receptor c-Met. The purpose of this study was to evaluate the effect of cabozantinib to stem-like features and therapy resistance. Established PDA cell lines, a gemcitabine-resistant subclone, non-malignant pancreatic ductal cells and primary spheroidal cultures from patient tumors were analyzed by MTT-assay, flow cytometry, colony and spheroid formation assays, western blotting, qRT-PCR, antibody protein array, immunohistochemistry and morphological features. Cabozantinib inhibited viability and spheroid formation and induced apoptosis in malignant cells with minor effects in non-malignant cells. After long-term cabozantinib treatment, PDA cells had altered anti- and pro-apoptotic signaling, but still responded to cabozantinib, as apoptosis only slightly decreased and viability only slightly increased suggesting a low resistance-inducing potential of cabozantinib. In parallel, c-Met expression and the pluripotency transcription factor SOX2 were downregulated, which might counteract development of full therapy resistance in long-term treated subclones. In single-treatment studies, cabozantinib increased efficacy of gemcitabine. Most importantly, cabozantinib strongly induced apoptosis and reduced viability in PDA cell lines, which are completely resistant toward gemcitabine. In primary, CSC-enriched spheroidal cultures cabozantinib downregulated CSC markers SOX2, c-Met and CD133 and induced apoptosis. These findings suggest that the clinical use of cabozantinib may be more effective than current chemotherapeutics.

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot-FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumorspecific proteases can be applied to enhance specificity and safety of TCBs.