Immunomodulatory Effects of Lenvatinib Plus Anti-Programmed Cell Death Protein 1 in Mice and Rationale for Patient Enrichment in Hepatocellular Carcinoma.

Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P < 0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Torrens L ，Montironi C ，Puigvehí M ，Mesropian A ，Leslie J ，Haber PK ，Maeda M ，Balaseviciute U ，Willoughby CE ，Abril-Fornaguera J ，Piqué-Gili M ，Torres-Martín M ，Peix J ，Geh D ，Ramon-Gil E ，Saberi B ，Friedman SL ，Mann DA ，Sia D ，Llovet JM ... -  《-》

Lenvatinib Targets FGF Receptor 4 to Enhance Antitumor Immune Response of Anti-Programmed Cell Death-1 in HCC.

Recently, clinical trials of lenvatinib plus pembrolizumab in HCC have displayed an impressive objective response rate. This study aimed to clarify the mechanism for optimal patient selection. First, in patients with HCC, lenvatinib-treated recurrent tumors had lower programmed death ligand 1 (PD-L1) expression and regulatory T cell (Treg) infiltration compared with matched primary tumors. Consistently, in C57BL/6 wild-type mice receiving anti-programmed cell death 1 (PD-1) therapy, PD-L1 expression and Treg infiltration in s.c. tumors were reduced when adding lenvatinib to the scheme. Mechanistically, on the one hand, FGF receptor 4 (FGFR4) was the most pivotal target in PD-L1 down-regulation by lenvatinib in vitro. Furthermore, lenvatinib reinforced the proteasomal degradation of PD-L1 by blocking the FGFR4-glycogen synthase kinase 3β axis and rescued the sensitivity of interferon-γ-pretreated HCC cells to T-cell killing by targeting FGFR4. On the other hand, the level of IL-2 increased after anti-PD-1 treatment, but IL-2-mediated Treg differentiation was blocked by lenvatinib through targeting FGFR4 to restrain signal transducer and activator of transcription 5 (STAT5) phosphorylation. By regulating the variations in the number of Tregs and the tumor FGFR4 level in C57BL/6-forkhead box protein P3 (Foxp3DTR ) mice, we found that high levels of FGFR4 and Treg infiltration sensitized tumors to the combination treatment. Finally, high levels of FGFR4 and Foxp3 conferred immune tolerance but better response to the combined therapy in patient cohorts. Lenvatinib reduced tumor PD-L1 level and Treg differentiation to improve anti-PD-1 efficacy by blocking FGFR4. Levels of FGFR4 expression and Treg infiltration in tumor could serve as biomarkers for screening patients with HCC using lenvatinib plus anti-PD-1 combination therapy.

Yi C ，Chen L ，Lin Z ，Liu L ，Shao W ，Zhang R ，Lin J ，Zhang J ，Zhu W ，Jia H ，Qin L ，Lu L ，Chen J ... -  《-》

Immunomodulatory activity of lenvatinib contributes to antitumor activity in the Hepa1-6 hepatocellular carcinoma model.

Angiogenesis inhibitors such as lenvatinib and sorafenib, and an immune checkpoint inhibitor (ICI), nivolumab, are used for anticancer therapies against advanced hepatocellular carcinoma (HCC). Combination treatments comprising angiogenesis inhibitors plus ICIs are promising options for improving clinical benefits in HCC patients, and clinical trials are ongoing. Here, we investigated the antitumor and immunomodulatory activities of lenvatinib (a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor 1-3, fibroblast growth factor receptor 1-4, platelet-derived growth factor receptor α, KIT and RET) and the combined antitumor activity of lenvatinib plus anti-programmed cell death 1 (PD-1) antibody in the Hepa1-6 mouse HCC syngeneic model. We found that the antitumor activities of lenvatinib and sorafenib were not different in immunodeficient mice, but lenvatinib showed more potent antitumor activity than sorafenib in immunocompetent mice. The antitumor activity of lenvatinib was greater in immunocompetent mice than in immunodeficient mice and was attenuated by CD8+ T cell depletion. Treatment with lenvatinib plus anti-PD-1 antibody resulted in more tumor regression and a higher response rate compared with either treatment alone in immunocompetent mice. Single-cell RNA sequencing analysis demonstrated that treatment with lenvatinib with or without anti-PD-1 antibody decreased the proportion of monocytes and macrophages population and increased that of CD8+ T cell populations. These data suggest that lenvatinib has immunomodulatory activity that contributes to the antitumor activity of lenvatinib and enhances the antitumor activity in combination treatment with anti-PD-1 antibody. Combination treatment of lenvatinib plus anti-PD-1 antibody therefore warrants further investigation against advanced HCC.

Kimura T ，Kato Y ，Ozawa Y ，Kodama K ，Ito J ，Ichikawa K ，Yamada K ，Hori Y ，Tabata K ，Takase K ，Matsui J ，Funahashi Y ，Nomoto K ... -  《-》

Regorafenib combined with PD1 blockade increases CD8 T-cell infiltration by inducing CXCL10 expression in hepatocellular carcinoma.

Combining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms of benefit remain unclear. We examined the efficacy and defined the mechanisms of benefit when combining regorafenib (a multikinase antivascular endothelial growth factor receptor inhibitor) with PD1 blockade in murine hepatocellular carcinoma (HCC) models. We used orthotopic models of HCC in mice with liver damage to test the effects of regorafenib-dosed orally at 5, 10 or 20 mg/kg daily-combined with anti-PD1 antibodies (10 mg/kg intraperitoneally thrice weekly). We evaluated the effects of therapy on tumor vasculature and immune microenvironment using immunofluorescence, flow cytometry, RNA-sequencing, ELISA and pharmacokinetic/pharmacodynamic studies in mice and in tissue and blood samples from patients with cancer. Regorafenib/anti-PD1 combination therapy increased survival compared with regofarenib or anti-PD1 alone in a regorafenib dose-dependent manner. Combination therapy increased regorafenib uptake into the tumor tissues by normalizing the HCC vasculature and increasing CD8 T-cell infiltration and activation at an intermediate regorafenib dose. The efficacy of regorafenib/anti-PD1 therapy was compromised in mice lacking functional T cells (Rag1-deficient mice). Regorafenib treatment increased the transcription and protein expression of CXCL10-a ligand for CXCR3 expressed on tumor-infiltrating lymphocytes-in murine HCC and in blood of patients with HCC. Using Cxcr3-deficient mice, we demonstrate that CXCR3 mediated the increased intratumoral CD8 T-cell infiltration and the added survival benefit when regorafenib was combined with anti-PD1 therapy. Judicious regorafenib/anti-PD1 combination therapy can inhibit tumor growth and increase survival by normalizing tumor vasculature and increasing intratumoral CXCR3+CD8 T-cell infiltration through elevated CXCL10 expression in HCC cells.

Shigeta K ，Matsui A ，Kikuchi H ，Klein S ，Mamessier E ，Chen IX ，Aoki S ，Kitahara S ，Inoue K ，Shigeta A ，Hato T ，Ramjiawan RR ，Staiculescu D ，Zopf D ，Fiebig L ，Hobbs GS ，Quaas A ，Dima S ，Popescu I ，Huang P ，Munn LL ，Cobbold M ，Goyal L ，Zhu AX ，Jain RK ，Duda DG ... -  《Journal for ImmunoTherapy of Cancer》

CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice.

Sorafenib, a broad tyrosine kinase inhibitor, is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC) but provides limited survival benefits. Recently, immunotherapy has emerged as a promising treatment strategy, but its role remains unclear in HCCs, which are associated with decreased cytotoxic CD8(+) T-lymphocyte infiltration in both murine and human tumors. Moreover, in mouse models after sorafenib treatment intratumoral hypoxia is increased and may fuel evasive resistance. Using orthotopic HCC models, we now show that increased hypoxia after sorafenib treatment promotes immunosuppression, characterized by increased intratumoral expression of the immune checkpoint inhibitor programmed death ligand-1 and accumulation of T-regulatory cells and M2-type macrophages. We also show that the recruitment of immunosuppressive cells is mediated in part by hypoxia-induced up-regulation of stromal cell-derived 1 alpha. Inhibition of the stromal cell-derived 1 alpha receptor (C-X-C receptor type 4 or CXCR4) using AMD3100 prevented the polarization toward an immunosuppressive microenvironment after sorafenib treatment, inhibited tumor growth, reduced lung metastasis, and improved survival. However, the combination of AMD3100 and sorafenib did not significantly change cytotoxic CD8(+) T-lymphocyte infiltration into HCC tumors and did not modify their activation status. In separate experiments, antibody blockade of the programmed death ligand-1 receptor programmed death receptor-1 (PD-1) showed antitumor effects in treatment-naive tumors in orthotopic (grafted and genetically engineered) models of HCC. However, anti-PD-1 antibody treatment had additional antitumor activity only when combined with sorafenib and AMD3100 and not when combined with sorafenib alone. Anti-PD-1 treatment can boost antitumor immune responses in HCC models; when used in combination with sorafenib, anti-PD-1 immunotherapy shows efficacy only with concomitant targeting of the hypoxic and immunosuppressive microenvironment with agents such as CXCR4 inhibitors.

Chen Y ，Ramjiawan RR ，Reiberger T ，Ng MR ，Hato T ，Huang Y ，Ochiai H ，Kitahara S ，Unan EC ，Reddy TP ，Fan C ，Huang P ，Bardeesy N ，Zhu AX ，Jain RK ，Duda DG ... -  《-》