T Cells Expressing NKG2D Chimeric Antigen Receptors Efficiently
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Yang et al. Journal for ImmunoTherapy of Cancer (2019) 7:171 J Immunother Cancer: first published as 10.1186/s40425-019-0642-9 on 9 July 2019. Downloaded from https://doi.org/10.1186/s40425-019-0642-9 RESEARCHARTICLE Open Access T cells expressing NKG2D chimeric antigen receptors efficiently eliminate glioblastoma and cancer stem cells Dong Yang1,3†, Bin Sun1,3†, Hongjiu Dai2*†, Wenxuan Li4, Lan Shi5, Peixian Zhang5, Shirong Li1 and Xudong Zhao1,3,6* Abstract Background: Traditional therapies fail to cure most glioblastoma patients and the 5-year survival rate is less than 10%, highlighting need for new therapeutic approaches. The natural killer group 2 member D ligands (NKG2DLs) are highly expressed in glioblastomas and are considered promising targets for chimeric antigen receptor (CAR) T-cell therapy. The aim of this study was to investigate the effect of NKG2D-expressing CAR-T cells on glioblastomas and glioblastoma stem cells. Methods: The expression of NKG2DLs was analyzed by flow cytometry and immunohistochemistry. NKG2D-BBz CAR, containing the extracellular domain of NKG2D, was constructed and delivered into T cells by lentiviral particles. In vitro cytotoxicity of the CAR-T cells was assessed by flow cytometry. Release of cytokine, perforin and granzyme B was quantified using enzyme-linked immunosorbent assay kits. The therapeutic efficacy of NKG2D-BBz CAR-T cells in vivo was evaluated using subcutaneous tumor models. The safety of the CAR was analyzed by investigating the effects on proliferation, apoptosis, and karyotype. Results: Our data confirmed the high expression of NKG2DLs in human glioblastoma cells, cancer stem cells, and tumor samples. Further, the NKG2D-BBz CAR-T cells efficiently lysed glioblastoma cells and cancer stem cells in vitro and produced high levels of cytokines, perforin, and granzyme B. The CAR-T cells markedly eliminated xenograft http://jitc.bmj.com/ tumors in vivo and did not exhibit significant treatment-related toxicity in the treated mice. The CAR expression also did not exert any obvious effects on cell proliferation, apoptosis, and genomic stability. Conclusion: Our findings demonstrated that NKG2D CAR-T cells targeted glioblastoma cells and cancer stem cells in an NKG2D-dependent manner, supporting the use of CAR-T therapy in glioblastoma therapeutic strategies. Keywords: Chimeric antigen receptor, Glioblastoma, Cancer stem cell, NKG2D on September 27, 2021 by guest. Protected copyright. Background 5-year survival rate of glioblastoma patients is still less than Glioblastoma is the most aggressive cancer of the central 10%, due to the biological complexity of brain tumors [1]. nervous system. Over the past few decades, although some The blood-brain barrier, a highly selective semipermeable progress has been made in traditional treatment options, border that separates the circulating blood from the brain consisting of surgery, radiotherapy, and chemotherapy, the and extracellular fluid in the central nervous system, pre- vents most cancer therapeutics from entering the brain par- enchyma [2]. Only three new treatments, with minor * Correspondence: [email protected]; [email protected] † improvement in prognosis, have been approved by the Dong Yang, Bin Sun and Hongjiu Dai are co-first authors. 2Nanjing Kaedi Biotech Co. Ltd, 18 Zhilan Road, Nanjing 211100, Jiangsu, FDA for glioblastoma since 2005, including temozolomide, China bevacizumab, and tumor-treating fields [3]. Therefore, there 1 Key Laboratory of Animal Models and Human Disease Mechanisms of is an urgent need to develop new treatment strategies for Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of glioblastoma. Sciences, 32 East Jiaochang Road, Kunming 650223, Yunnan, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yang et al. Journal for ImmunoTherapy of Cancer (2019) 7:171 Page 2 of 13 J Immunother Cancer: first published as 10.1186/s40425-019-0642-9 on 9 July 2019. Downloaded from Recently, immunotherapy using engineered T cells NKG2DL dependent manner [20]. NKG2D ligand expressing chimeric antigen receptors (CAR) showed re- (NKG2DL) overexpression was reported in glioblastoma markable curative effects in multiple tumor types, espe- stem cells (GSCs), suggesting that NKG2D-expressing cially, in the case of hematologic malignancies [4]. CARs CAR-T cells may also have an effect on GSCs [21]. are artificial fusion proteins that include an extracellular In this study, we constructed CAR-T cells expressing antigen-binding domain, a transmembrane domain, and the extracellular domain of human NKG2D, as well as intracellular T-cell signaling domains (such as CD3ζ the 4-1BB and CD3ζ signaling domains, and investigated alone or in combination with one or more costimulatory the efficacy of these NKG2D-expressing CAR-T cells components) [5]. It’s reported that T cells can cross the against human glioblastoma cells and CSCs. blood-brain barrier and infiltrate the brain by diffusion [6]. Thus, CAR-T cell-based immunotherapy for glio- Methods blastomas could overcome the difficulty of drug delivery. Cell culture So far, CAR-T cells against IL-13Ra2, EGFRVIII, and U-251 MG, T98G, U-87 MG and HTB185 cell lines were HER2 have been tested in glioblastoma clinical treat- purchased from China Infrastructure of Cell Line Re- ment and have showed promising results [7–9]. More- sources (Kunming, China). The cells were tested via short over, CAR-T cell immunotherapy targeting EphA2, tandem repeat (STR) profiling in October 2018. The cells MUC1, EGFR, PD-L1, and PDPN are currently undergo- were cultured in DMEM containing 10% fetal bovine ing clinical studies in glioblastoma, but the results have serum (Millipore, USA), 100 U/ml penicillin, and 100 mg/ not yet been made public [10–12]. These reports indi- ml streptomycin (Life Technologies, USA). The GSC-3# cate that CAR-T cell therapy could be a new treatment cell line, isolated from primary glioblastoma tissues, was for glioblastoma patients. However, there are still some described previously [22]. U-251 MG and U-87 MG sus- limitations that prevent the application of such therap- pended cell spheres, named U251-CSC and U87-CSC, re- ies, such as the heterogeneity of glioblastoma and im- spectively, were established using serum-free neural stem munosuppression of tumor microenvironment. Thus, it cell medium composed of DMEM/F12, EGF (20 ng/ml, is still necessary to develop new CARs for the treatment Life Technologies, USA), bFGF (20 ng/ml, Life Technolo- of glioblastoma. Natural killer group 2 member D gies, USA), and B27 (1x, Life Technologies, USA) [23, 24]. (NKG2D) ligands, including two MHC class I-related All cell lines were cultured in a humidified incubator with chains and six UL16-binding proteins, were reported to 5% CO2 at 37 °C. be expressed at low levels in healthy human tissues and to be upregulated in most cancers [13]. NKG2D- Vector design expressing CARs were demonstrated to be effective The CD19 targeting domain and the extracellular do- http://jitc.bmj.com/ against most cancer types, such as multiple myeloma, main of human NKG2D was synthesized (Idobio, China) ovarian carcinoma, and lymphoma [14]. CAR-T cells ex- and cloned into a CAR-encoding lentivirus backbone pressing the full length murine NKG2D were also re- and named CD19-BBZ CAR and NKG2D-BBZ CAR, re- ported to remarkably prolong the survival of mice spectively, containing a CD8 hinge spacer and trans- bearing intracranial xenograft of mouse glioma cells membrane domain, 4-1BB, and CD3ζ endo-domains. [15]. However, the exact role of NKG2D-expressing The CARs were cloned into the LentiGuide-Puro plas- on September 27, 2021 by guest. Protected copyright. CARs in human glioblastoma remains to be elucidated. mid (Addgene, USA) and expressed under the control of The existence of cancer stem cells (CSCs) in glioblast- an EF1a promoter. oma is considered one of the major causes of the conven- tional therapy failure and cancer recurrence [16, 17]. Lentiviral package Previous studies have shown that the targeted killing of The lentiviral plasmids were co-transfected into CSCs could effectively inhibit glioblastoma tumorigenesis HEK293T cells with the packaging plasmids psPAX2 and and prolong the survival of glioma-bearing mice [18]. pCMV-VSVG (Addgene, USA) at a ratio of 10:8:5 as Therefore, development of specific and effective thera- described before [25]. peutic approaches to eliminate CSCs is urgently required. Due to the strong resistance to radiotherapy and chemo- CAR-T cell preparation therapy, immunotherapy shows more promising potential Blood collected from three healthy donors was used to to eradicate CSCs. Many surface markers of CSCs have isolated T cells respectively using the RosetteSep™ Hu- been used as target antigens for immunotherapy in cancer man T Cell Enrichment Cocktail (STEMCELL, Canada) treatment, such as CD133, CD90, ALDH, and EpCAM, and the T cells were cultured in RPMI 1640 medium and revealed encouraging anticancer activity [19]. Previ- (Life Technologies, USA) containing 10% FBS (Life ous studies have proved that natural killer cells could Technologies, USA) with 200 U/ml IL-2 (PeproTech, efficiently target osteosarcoma stem cells in an NKG2D- USA). T cells were activated by CD3/CD28 Dynabeads Yang et al. Journal for ImmunoTherapy of Cancer (2019) 7:171 Page 3 of 13 J Immunother Cancer: first published as 10.1186/s40425-019-0642-9 on 9 July 2019.