Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer

RESEARCH ARTICLE Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer Minsung Kim1,2, Suhkneung Pyo2, Chung Hyo Kang1,3, Chong Ock Lee1, Heung Kyoung Lee1, Sang Un Choi1*, Chi Hoon Park1,4* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea, 2 School of Pharmacy, Sungkyunkwan University, Suwon City, Kyunggi-do, Republic of Korea, 3 College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea, 4 Department of Medicinal Chemistry and Pharmacology, Korea University of Science and Technology, Daejeon, Republic of Korea * (SUC); (CHP) Abstract OPEN ACCESS Citation: Kim M, Pyo S, Kang CH, Lee CO, Lee HK, Choi SU, et al. (2018) Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer. PLoS ONE 13(6): e0198347. https://doi.org/10.1371/journal. pone.0198347 Editor: Ryan M. Teague, Saint Louis University School of Medicine, UNITED STATES Received: February 21, 2018 Accepted: May 17, 2018 Published: June 6, 2018 Copyright: © 2018 Kim 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. Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over onethird of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: This work was supported by the KOREA RESEARCH INSTITUTE of CHEMICAL TECHNOLOGY (SI1706) and the National Research Foundation of Korea (https://www.nrf.re.kr/index) grant number: NRF-2017M2A2A6A01071250 to Chi Hoon Park. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Immunotherapy for cancer has made considerable progress due to improved efficacy in chemotherapy-refractory blood and solid tumors from patients. Clinical trials using immunotherapy have been successful in the treatment of malignant tumors by blocking immune cell inhibitory signals or by redirecting T cells to target cancer cells [1]. In adoptive T cell immunotherapy for cancer, T cells isolated from a patient are manipulated and expanded in vitro, and then reinfused into the patient [2]. One of the main types of adoptive T cell immunotherapy is the use of chimeric antigen receptor (CAR) T cells. T cells are reintroduced into a patient after PLOS ONE | https://doi.org/10.1371/journal.pone.0198347 June 6, 2018 1 / 20 FOLR1-positive gastric cancer treatment using CAR T cells Competing interests: The authors have declared that no competing interests exist. Abbreviations: CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; EpCAM, epithelial cell adhesion molecule; FACS, fluorescence-activated cell sorting; FOLR1, folate receptor 1; GC, gastric cancer; GM-CSF, granulocyte-macrophage colonystimulating factor; HER2, human epidermal growth factor receptor 2; IFN-γ, interferon- γ; IL-2, interleukin-2; KB cell line, a subline of the ubiquitous keratin forming tumor cell line HeLa; MHC, major histocompatibility complex; MSLN, mesothelin; NK, natural killer; PBMC, peripheral blood mononuclear cells; scFv, single-chain variable fragment; TCR, T cell receptor; TNF-α, tumor necrosis factor- α; ZAP70, zeta-chainassociated protein kinase 70. conversion from the patient’s T cells to CAR T cells that express the engineered receptor specific for a cancer target through a retrovirus or lentivirus, leading to effective anticancer activity [3]. CAR consist of a combination of target recognition and T cell activation regions. The target recognition region is typically derived from a single-chain variable fragment (scFv) of an antibody and T cell activation regions are composed of one or more intracellular signaling domains that induce persistence and effector functions in T cells [4]. CAR T cells exhibit cytotoxic effects against target cells by recognizing specific antigens on the surface of target cells in a major histocompatibility complex (MHC) independent manner. CAR T cell immunotherapy has been developed for two decades, beginning with first-generation CARs that combined scFv of antibodies with FcR γ or CD3z chains. Second and third-generation CARs were developed to have one or more costimulatory domains, such as CD28, CD137 (4-1BB), ICOS, and OX40 [5]. In addition, several types of CARs targeting different antigens have been constructed and their effectiveness has been verified in clinical trials [6]. While this strategy is highly effective against blood cancers, clinical application for solid cancer has lacked efficacy. Additional factors for solid tumors require consideration, including disease status, tumor burden, CAR T cell infiltration, and the recruitment and activation of other immune responses, such as inflammation and immunosuppression [7]. Although the therapeutic efficacy of all types of CAR T cells has not been elucidated, an important issue is the choice of a target antigen. These targets include epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN) all of which are currently being investigated in clinical trials [8]. Folate receptor 1 (FOLR1), also known as folate receptor alpha and folate binding protein, is a glycosylphosphatidylinositol-linked protein. Although the function of FOLR1 is unclear, FOLR1 has a high affinity for folate and is capable of internalizing folate [9]. FOLR1 (...truncated)