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

Yu Kato,Junichi Ito,Hiroaki Kitano,Junji Matsui,Kazuhiko Yamada,Yukiko Matsuoka,Masahiro Matsuki,Sho Tachino,Yusaku Hori,Kimiyo Tabata,Kenichi Nomoto,Ayako Yachie-Kinoshita,Samik Ghosh,Takayuki Kimura,Yasuhiro Funahashi,Yoichi Ozawa,Fabrizio Mattei

doi:10.1371/journal.pone.0212513

Yu Kato, Junichi Ito + Show 15 more

Open Access

https://doi.org/10.1371/journal.pone.0212513

Copy DOI

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.

Highlights

Immune escape of cancer cells is a major mechanism of cancer malignancy, mainly due to exhaustion of CD8+ T cells that recognize tumor antigens [1]
Lenvatinib at 3 and 10 mg/ kg inhibited tumor growth of the BNL model in Balb/cnu/nu mice, but it caused shrinkage of BNL tumors in Balb/cwt/wt mice only (S2 Fig). These findings indicate that lenvatinib has more potent antitumor activity in the immunocompetent tumor microenvironment
We collected and sequenced RNA from 301 and 220 cells of non-treated and lenvatinib-treated tumors, respectively. tSNE analysis showed that the total tumor-infiltrating lymphocytes (TILs) (521 cells) from the lenvatinib-treated and vehicle groups could be divided into three immune cell populations

Summary

Introduction

Immune escape of cancer cells is a major mechanism of cancer malignancy, mainly due to exhaustion of CD8+ T cells that recognize tumor antigens [1]. PD-1 blockade was first approved for use in malignant melanoma patients and in several other tumor types, such as renal cell carcinoma and non-small cell lung cancer [3,4,5,6], and further clinical trials are ongoing. Lenvatinib is currently used as monotherapy in patients with radio-iodine-refractory differentiated thyroid cancer (RAI-DTC) [11] and in combination with everolimus to treat advanced renal cell carcinoma after VEGF therapy in the United States and the European Union [12]. Lenvatinib was approved for first-line treatment of unresectable hepatocellular carcinoma (HCC) [13] in the United States, the European Union, Japan, China and other countries. Exploratory biomarker analysis of lenvatinib in a Phase 3 study of patients with RAI-DTC revealed that lenvatinib increased VEGF and FGF23 levels in serum and that the increased FGF23 levels were associated with progression-free survival, demonstrating the target engagement of lenvatinib to inhibit VEGFR and FGFR signaling [14]

Methods

Results

Discussion

Conclusion