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      Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8 + T cells through reduction of tumor-associated macrophage and activation of the interferon pathway

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          Abstract

          Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting mainly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors. We investigated the immunomodulatory activities of lenvatinib in the tumor microenvironment and its mechanisms of enhanced antitumor activity when combined with a programmed cell death-1 (PD-1) blockade. Antitumor activity was examined in immunodeficient and immunocompetent mouse tumor models. Single-cell analysis, flow cytometric analysis, and immunohistochemistry were used to analyze immune cell populations and their activation. Gene co-expression network analysis and pathway analysis using RNA sequencing data were used to identify lenvatinib-driven combined activity with anti-PD-1 antibody (anti-PD-1). Lenvatinib showed potent antitumor activity in the immunocompetent tumor microenvironment compared with the immunodeficient tumor microenvironment. Antitumor activity of lenvatinib plus anti-PD-1 was greater than that of either single treatment. Flow cytometric analysis revealed that lenvatinib reduced tumor-associated macrophages (TAMs) and increased the percentage of activated CD8 + T cells secreting interferon (IFN)-γ + and granzyme B (GzmB). Combination treatment further increased the percentage of T cells, especially CD8 + T cells, among CD45 + cells and increased IFN-γ + and GzmB + CD8 + T cells. Transcriptome analyses of tumors resected from treated mice showed that genes specifically regulated by the combination were significantly enriched for type-I IFN signaling. Pretreatment with lenvatinib followed by anti-PD-1 treatment induced significant antitumor activity compared with anti-PD-1 treatment alone. Our findings show that lenvatinib modulates cancer immunity in the tumor microenvironment by reducing TAMs and, when combined with PD-1 blockade, shows enhanced antitumor activity via the IFN signaling pathway. These findings provide a scientific rationale for combination therapy of lenvatinib with PD-1 blockade to improve cancer immunotherapy.

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          Visualizing data using t‐SNE

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            Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage

            Background Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study. Method We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors. Result Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score. Conclusion These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.
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              Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1.

              Treatment of human monocytes with vascular endothelial growth factor (VEGF) isolated from tumor cell supernatants was reported to induce monocyte activation and migration. In this study we show that recombinant human VEGF165, and VEGF121 had a maximal effect on human monocyte migration at 65 to 250 pmol/L. Chemotactic activity of VEGF165 was inhibited by a specific antiserum against VEGF, by heat treatment of VEGF165, and by protein kinase inhibitors. In addition, we could show that VEGF-stimulated monocyte migration is mediated by a pertussis toxin-sensitive GTP-binding protein. Placenta growth factor (PlGF152), a heparin-binding growth factor related to VEGF, was also chemotactic for monocytes at concentrations between 2.5 and 25 pmol/L. In accordance with these findings, human monocytes showed specific and saturable binding for 125I-VEGF165 (half-maximal binding at 1 to 1.5 nmol/L). Using Northern blot analysis, we further could show that human monocytes express only the gene for the VEGF receptor type, flt-1, but not for the second known VEGF receptor, KDR. Resting monocytes expressed low levels of flt-1 gene only. Brief exposure (2 to 4 hours) of human monocytes to lipopolysaccharide, a prototypic monocyte activator, led to a significant upregulation of the flt-1 mRNA level. The results presented here suggest that monocyte chemotaxis in response to VEGF and most likely to PlGF152 is mediated by flt-1 and thus show a possible function for the VEGF-receptor flt-1.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: Project administrationRole: Writing – original draft
                Role: Investigation
                Role: InvestigationRole: Project administration
                Role: InvestigationRole: Methodology
                Role: Investigation
                Role: Investigation
                Role: InvestigationRole: Methodology
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Supervision
                Role: Supervision
                Role: Supervision
                Role: SupervisionRole: Writing – review & editing
                Role: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 February 2019
                2019
                : 14
                : 2
                : e0212513
                Affiliations
                [1 ] Tsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan
                [2 ] The Systems Biology Institute, Shinagawa, Tokyo, Japan
                [3 ] Oncology Business Group, Eisai Inc., Woodcliff Lake, New Jersey, United States of America
                Istituto Superiore di Sanità, ITALY
                Author notes

                Competing Interests: Lenvatinib is the marketed product. YK, KT, and YH obtained the patent entitled “Combination of a PD-1 antagonist and a VEGFR/FGFR/RET tyrosine kinase inhibitor for treating cancer” (WO2016140717A1). These do not interfere with our adherence to PLOS ONE policies regarding sharing data and materials.

                Author information
                http://orcid.org/0000-0003-1662-6983
                Article
                PONE-D-18-29515
                10.1371/journal.pone.0212513
                6392299
                30811474
                a40e739e-43ef-4ad1-b4d0-9573f2216cb4
                © 2019 Kato et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 11 October 2018
                : 4 February 2019
                Page count
                Figures: 6, Tables: 0, Pages: 18
                Funding
                Eisai Co., Ltd. or Eisai Inc. provided support in the form of salaries for authors (YK, KT, TK, JI, YO, KY, ST, YH, MM, JM, KN and YF), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
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