Low-Dose Apatinib Optimizes Tumor Research Article Low-Dose Apatinib Optimizes Tumor Microenvironment

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  • Research Article

    Low-Dose Apatinib Optimizes Tumor Microenvironment and Potentiates Antitumor Effect of PD-1/PD-L1 Blockade in Lung Cancer Sha Zhao1, Shengxiang Ren1, Tao Jiang1, Bo Zhu2, Xuefei Li3, Chao Zhao3, Yijun Jia1, Jinpeng Shi1, Limin Zhang1, Xiaozhen Liu1, MengQiao1, Xiaoxia Chen1,Chunxia Su1, Hui Yu4, Caicun Zhou1, Jun Zhang5, D. Ross Camidge6, and Fred R. Hirsch4,7


    The lack of response to treatment in most lung cancer patients suggests the value of broadening the benefit of anti–PD-1/PD-L1 monotherapy. Judicious dosing of antiangiogenic agents such as apatinib (VEGFR2-TKI) can modulate the tumor immunosuppressive microenviron- ment, which contributes to resistance to anti–PD-1/ PD-L1 treatment. We therefore hypothesized that inhibit- ing angiogenesis could enhance the therapeutic efficacy of PD-1/PD-L1 blockade. Here, using a syngeneic lung cancer mouse model, we demonstrated that low-dose apatinib alleviated hypoxia, increased infiltration of CD8þ T cells,

    reduced recruitment of tumor-associated macrophages in tumor and decreased TGFb amounts in both tumor and serum. Combining low-dose apatinib with anti–PD-L1 significantly retarded tumor growth, reduced the number of metastases, and prolonged survival in mouse models. Anticancer activity was evident after coadministration of low-dose apatinib and anti–PD-1 in a small cohort of patients with pretreated advanced non–small cell lung cancer. Overall, our work shows the rationale for the treatment of lung cancer with a combination of PD-1/PD-L1 blockade and low-dose apatinib.

    Introduction Lung cancer is the leading cause of cancer-related death

    worldwide, with non–small cell lung cancer (NSCLC) account- ing for approximately 80% of all cases (1, 2). Treatment for advanced NSCLC has been revolutionized with the availability of targeted therapy against driver oncogenes such as epidermal growth factor receptor (EGFR) mutations and anaplastic lym- phoma kinase (ALK) rearrangements (3, 4), immunotherapy

    blocking the interaction between programmed cell death-1 (PD-1) and its ligand (PD-L1; ref. 5), as well as antiangiogenic monoclonal antibodies (mAb) and tyrosine kinase inhibitors (TKI; ref. 6). Clinical trials have demonstrated the superior overall survival (OS) resulting from anti–PD-1/PD-L1 mono- therapies such as nivolumab, pembrolizumab, and atezolizu- mab as compared with docetaxel in the second-line setting in patients with advanced NSCLC. Furthermore, pembrolizumab showed superior efficacy and was received with better tolerance compared with platinum doublet chemotherapy as the first-line treatment in patients with PD-L1 expression in at least 50% of tumor cells (7). However, although predictive biomarkers such as PD-L1 expression and tumor mutational load may allow enrichment of the patient population responsive to PD-1/PD- L1 mAbs, only about 20% of NSCLC patients benefit from monotherapy overall (8, 9). New therapeutic approaches, such as combinations of immunotherapies and conventional treat- ments, are needed to extend the benefits to a larger population of patients with lung cancer.

    Evidence suggests that PD-1/PD-L1mAbs are less efficacious in noninflamed tumors characterized by poor infiltration of lym- phocytes, rare PD-L1 expression, increased immunosuppressive components, and more abnormal angiogenesis in the tumor microenvironment (TME; refs. 8, 10). Combining PD-1/PD-L1 mAbs with agents that can modulate the immunosuppressive milieu of TME may overcome primary treatment resistance in patients with advancedNSCLC (8, 11). Antiangiogenic therapy in NSCLC targets the TME through the proangiogenic factor vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2; ref. 12). Antiangiogenic therapy enhances the effect of cytotoxic chemotherapy or molecular targeted therapy in patients with advanced NSCLC (6, 12, 13). Studies suggest a bidirectional

    1Department of Medical Oncology, Shanghai Pulmonary Hospital and Thoracic Cancer Institute, Tongji University School of Medicine, Shanghai, P.R. China. 2Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongq- ing, P.R. China. 3Department of Lung Cancer and Immunology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, P.R. China. 4Department of Medicine, Division of Medical Oncology and Department of Pathology, University of Colorado Cancer Center, Aurora, Colorado. 5Division of Hematology, Oncology and Blood and Marrow Transplantation, Department of Internal Medicine, Holden Comprehensive Cancer Center, University of Iowa Carver College of Medicine, Iowa City, Iowa. 6Department of Medicine, Division of Medical Oncology, University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, Colorado. 7Clinical Institute for Lung Cancer, Mount Sinai Cancer, Mount Sinai Health System, Tisch Cancer Institute, Icahn School of Medicine, New York, New York.

    Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/).

    S. Zhao, T. Jiang, and S. Ren contributed equally to this article.

    Corresponding Author: Caicun Zhou, Shanghai Pulmonary Hospital, No. 507, Zheng Min Road, Shanghai 200433, P.R. China. Phone: 86-21-65115006; Fax: 86-21-65111298; E-mail: caicunzhou_dr@163.com

    doi: 10.1158/2326-6066.CIR-17-0640

    �2019 American Association for Cancer Research.

    Cancer Immunology Research

    Cancer Immunol Res; 7(4) April 2019630

    on October 19, 2020. © 2019 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

    Published OnlineFirst February 12, 2019; DOI: 10.1158/2326-6066.CIR-17-0640

    http://crossmark.crossref.org/dialog/?doi=10.1158/2326-6066.CIR-17-0640&domain=pdf&date_stamp=2019-3-19 http://cancerimmunolres.aacrjournals.org/

  • relationship between angiogenesis and antitumor immunity in the TME (13–15). Antiangiogenic agents could eliminate immu- nosuppression by the TME (16–18) andmay enhance therapeutic efficacy when given in combination with immunotherapy. Indeed, several preclinical studies have observed promising anti- tumor effects from antiangiogenic agents combined with immu- notherapies inmultiple tumor types such asmelanoma, renal cell carcinoma, and colon adenocarcinoma (17, 19–21). Trials inves- tigating these combinations in patients with advancedNSCLC are also ongoing; the existing data are preliminary but promising (13, 22). However, challenges remain to be overcome before the full potential of antiangiogenesis and immunotherapy combination can be realized.

    In this study, we analyzed the impact of apatinib, a small- molecule VEGFR2-TKI, on the TME in murine Lewis lung carcinoma (LLC) models at different doses alone and in com- bination with an anti–PD-L1. Apatinib has been approved as the third line therapy for patients with advanced gastric cancer by the CFDA (23) and is undergoing evaluation for patients with advanced nonsquamous NSCLC in a phase III trial (NCT02515435). We found that low-dose apatinib alleviated hypoxia and remodeled the immunosuppressive TME to one more permissive for antitumor immunity. In addition, using optimized dosing of apatinib, we observed increased antitumor efficacy when it was combined with anti–PD-L1 in the in vivo experiment. Moreover, in preliminary results from a phase IB clinical trial, the combination of SHR-1210 (a clinically avail- able PD-1 mAb (24) with low-dose apatinib achieved an objective response rate of 55.6% (5/9) in patients with pre- treated NSCLC (ClinicalTrials.gov: NCT03 083041).

    Materials and Methods Cell line, tumor models, and agents

    LLC cells were obtained from ATCC at 2014 and cultured at 37�C in a humidified incubator with 5% CO2 in Dulbecco's modified Eaglemedium (DMEM;HyClone) containing 10% fetal bovine serum (Life Technologies) with 100 units/mL penicillin and 100 mg/mL streptomycin. This cell line was not reauthenti- cated using specific assays in the past years, and only identified by morphology and growth characteristic. Cells were routinely tested to confirm the absence of Mycoplasma contamination and were cultured for less than 20 generations.

    Male C57BL/6 mice (6–8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. Animals were housed and maintained under optimal conditions of light, temperature, and humidity with free access to food and water. For a subcutaneous tumor model, a total of 1 � 106 LLC cells were resuspended in 200 mL PBS and inoculated subcutane- ously into the right flank of mice. Tumor dimensions were measured by caliper every other day, and tumor volume (mm3) was estimated using this formula: tumor volume ¼ (long axis) � (short axis)2 � p/6. For the metastasis tumor model, 5 � 105 LLC cells in 100 mL PBS were intravenously injected into the tail vein of C57BL/6J mice, and microcomputerized tomography (micro-CT, the eXplore Locus in vivo MicroCT scanner, GE Healthcare) was used to monitor tumor burden in the lung. Apatinib (kindly provided by Hengrui Medicine Co. Ltd) treat- ment at various doses was initiated 3 days after tumor cell inoculation and administered by oral gavage every day. Anti– PD-L1 (anti-mouse CD274, clone: MIH5, eBioscience) was

    administered at 200 mg/mouse by intraperitoneal injection every 3 days. At indicated days, tumor-bearing mice were anesthetized and tissues were harvested for analysis and mea- surement. For comparing the survival time of the mice in each group, additional independent experiments were conducted for observation.

    All the animal studies were approved by the Institutional Committee for Animal Care and Use, Tongji University School of Medicine, and were performed in accordance with the institu