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target, oﬀ-tumor’ side eﬀects must be considered. UPAR expression in cancer and cancer-associated Apicidin improves its eﬃcacy for CARs; however, its rare expression in normal cells such as some macrophages, some endothelial cells and respiratory epithelial cells is a challenge. The spatio-temporal restriction of uPAR expression may be a natural coping strategy; however, embellishing or arming CARs using strategies such as switchable CAR-T (toxicity controlled by antigen dosage) should be investigated further .
Based on clinical evidence and cell line characteristics about uPAR, we generated a novel anti-uPAR CAR consisting of an antigen re-cognition domain via not scFv but a natural ligand amino-terminal fragment (ATF), a part of the A chain of uPA to mimic the natural binding between uPA and uPAR to construct the third generation CAR (ATF-CAR) T cells and then investigated ATF-CAR T cells against ovarian cancer cells in vitro studies.
2. Materials and methods
2.1. Clinical serum samples
45 patients with malignant ovarian tumor and 42 patients with benign ovarian tumor as control were enrolled to test uPA and soluble uPAR levels in serum. Patients with renal and hepatic function im-pairment, coronary insuﬃciency or active infections, subjects using anticoagulantsor antiaggregant drugs and malignant tumor in other systems were excluded. No patient had undergone chemotherapy or radiotherapy before surgery. The Ethical Committee at the second hospital of Jilin University approved this study and informed consent was received from every patient. Patients with malignant ovarian cancer were staged according to the Federation International of Gynecology and Obstetrics (FIGO). Blood samples were collected on the day before surgery in tubes containing EDTA as an anticoagulant and serum separator. Grouping and statistics depended on biopsy patholo-gical reports after surgery. Obtained serum supernatants were tested for uPA and uPAR levels by enzyme-linked immunosorbent assay (ELISA).
Human ovarian cancer cells ES-2 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human ovarian cancer cells SKOV3, HO8910, A2780, C13 K, RH30 (human rhabdo-myosarcoma cells) and 293 T (human embryonic kidney fibroblast cells) were purchased from the Chinese Academy of Sciences Shanghai Institute for Biological Sciences-Cell Resource Center. Human ovarian cells were maintained in RMPI-1640 (Hyclone, Logan, UT, USA) sup-plemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (10,000 units penicillin and 10 mg streptomycin/ml in 0.9% sodium chloride, Sigma-Aldrich, Ayrshire, UK). RH30 and 293 T cells were maintained in complete medium composed by DMEM high glucose with 10% heat-inactivated FBS, and 1% penicillin-streptomycin. All of the cells were maintained under standard conditions (37 °C and 5% CO2 in a humi-dified incubator) and split 1:3 every three days.
2.3. Design of CAR and construction of plasmids
The lentiviral vector encoding anti-uPAR ATF-CAR was constructed based on the third generation CAR consisting of two more co-stimu-lating domains. ATF is the amino-terminal fragment of uPA, which contains 157 amino acids and includes the uPAR-binding region but completely lacks the catalytic domain of uPA. We used ATF to replace scFv for usual CAR design with less immunogenicity and shorter length. The lentiviral vector was purchased from Genechem (Shanghai, China).The ATF-CAR vector consisted of the following components in frame from 5′ end to 3′ end: ATF, the hinge and transmembrane regions of the CD8a molecule, the cytoplasmic domains of CD28 and 4-1BB Biomedicine & Pharmacotherapy 117 (2019) 109173
(CD137), CD3ζ signaling domain, and 2A-eGFP. The corresponding DNA sequence of the ATF-CD8a-CD28-CD137-CD3ζ was codon-opti-mized for its optimal expression in human cells using the online codon optimization tool and was synthesized by Integrated DNA Technologies. The control lentivirus was from the wild type lentiviral vector mainly containing 2A-eGFP.
2.4. Lentivirus production and concentration
The complete CAR sequence was cloned into the BamHI restriction site of the lentiviral, yielding ATF-CA8a-CD28-CD137-CD3ζ-2A-eGFP. The 2A-eGFP presented as a control vector. Lentiviral particles con-taining the ATF-CAR or control vector were produced by transfection of 75% confluent 293 T cells with the aforementioned vectors, together with the packaging constructs psPAX2, pMD2G using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, USA). 72 h-post-transfection, the conditioned medium was harvested and filtered through a 0.45 μM filter unit (Millipore, USA). High-titer lentiviral particles were con-centrated 30-fold by ultracentrifugation (Beckman, USA) for 2 h at the speed of 280,000 rpm. The pallet was re-suspended with Dulbecco’s phosphate-buﬀ ;ered saline (PBS) and the virus titer was tested by transfecting 293 T cells more than 108/ml stored at −80 °C.