Lentiviral vectors construction
The extracellular and transmembrane portions of dPD1z derived from PD1 receptor (Uniprot Entry Q15116, amino acids (aa) 1-191), and CARPD-L1z contained a high-affinity anti-PD-L1 scFv that derived from Atezolizumab. The cytoplasmic domains of dPD1z and CARPD-L1z both contain 4-1BB (Uniprot Entry Q07011, aa 214–255), TLR2 (Uniprot Entry O060603, aa 636–784) and the CD3ζ (Uniprot Entry P20963, aa 52–164). The scFvs of CARMSLNz and CAR19z derived from SS1 and FMC63 respectively, in tandem with CD28 (Uniprot Entry P10747, aa 180–220), TLR2 and CD3ζ. GL vector contained the firefly luciferase reporter gene (GenBank ABA41653.1, aa 1-550) and eGFP reporter genes (GenBank YP_009062989.1, aa 1-239) linked through 2A peptide. The gene of MSLN (GenBank NP_001170826.1, aa 1-622) was overexpressed in H460GL generated H460-MSLNGL. The uPD-L1 contained the full-length gene of PD-L1 (GenBank NP_001254635.1, aa 1-176) and labeled with a truncated CD19. DNA sequences were synthesized by Genscript (Nanjing) Co., Ltd. (Nanjing, China) and cloned into the second-generation lentiviral vector pWPXLd.
Lentivirus particles were produced in HEK-293T cells via PEI MAX 40K (Polyscience, 24765-1) transfection. HEK-293T cells were co-transfected with the pWPXLd-based lentiviral plasmid and two packaging plasmids, psPAX2 and pMD2.G. Lentivirus-containing supernatants were harvested at 48 and 72 hours post-transduction and filtered through a 0.45-µm filter.
CAR T Cells Manufacture
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Lymphoprep (StemCell Technologies, 07851). T cells were negatively selected from PBMCs using a Pan T cell isolation kit (MiltenyiBiotec, 130-096-535) and activated using microbeads coated with anti-human CD2, anti-human CD3 and anti-human CD28 antibodies (MiltenyiBiotec, 130-091-441) for 2 days in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. On day 2 post-activation, T cells were transduced with lentiviral supernatants in the presence of 8 µg/ml polybrene (Sigma-Aldrich, TR-1003-G). Twelve hours post-transduction, T cells were cultured in fresh medium containing IL-2 (300 IU/ml), subsequently, fresh medium was added every 2–3 days to maintain cell density within the range of 0.5–1 × 106/ml. Healthy PBMC donors provided informed consent for the use of their samples for research purposes, and all procedures were approved by the Research Ethics Board of the Guangzhou Institutes of Biomedicine and Health (GIBH).
Cells And Culture Conditions
HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, C11995500BT). H460 (human large cell lung cancer), MKN-28 (human gastric carcinoma), SMMC-7721 (human hepatoma carcinoma), HeLa (human cervical cancer) and NALM6 (CD19+ acute lymphoblastic leukemia) cell lines were obtained from ATCC and maintained in RPMI-1640 (Gibco, C11975500BT). GL-expressing cell lines were generated through transduction of the parental cell line with a lentiviral supernatant containing GL and were sorted for GFP expression on a FACS Aria TM cell sorter (BD Biosciences). DMEM and RPMI-1640 medium were supplemented with 10% heat-inactivated FBS (Vigonob, XC6936T) and 1% penicillin/streptomycin. All cells were cultured at 37 °C in an atmosphere of 5% carbon dioxide.
Flow cytometry was performed on a BD LSRFortessa cytometer, and data were analyzed using FlowJo software. The antibodies used, including anti-human CD3-PE-cyanine 7 (clone: UCHT1), anti-human CD4-APC (clone: GK1.5), anti-human CD4-APCcy7 (clone: GK1.5), anti-human CD8-PE (clone: 53 − 6.7), anti-human CD8-Pecpcy5.5 (clone: 53 − 6.7), anti-human CD25-PE (clone: PC61.5), anti-human CD69-APC (clone: H1.2F3), anti-human CD19-APC (clone: 1D3) and anti-human PD-L1-APC (clone: M1H1) were purchased from eBioscience. Anti-human Mesothelin-PE (clone: sc-33672) was purchased from Santa Cruz Biotechnology. All FACS staining was performed on ice for 30 minutes, and cells were then washed with PBS containing 1% FBS before cell cytometry. PB, spleen and tumor samples from xenograft mice were treated with a red blood cell lysis buffer (Biolegend), and the cells were stained with the corresponding antibodies.
The target cells H460GL, MKN-28GL, SMMC-7721GL, HeLaGL and H460-MSLNGL (10^4 cell/well) were incubated with CAR T or negative control T cells at the indicated ratios in triplicate wells of U-bottomed 96-well plates. Target cell viability was monitored 24 h later by adding 100 µl/well of the substrate D-Luciferin (potassium salt) (YEASEN, 40901ES03) at 150 µg/ml. Background luminescence was negligible (< 1% of the signal from the wells with only target cells). The viability percentage (%) was equal to the experimental signal/maximal signal × 100, and the lysis percentage was equal to the 100–viability percentage.
Cytokine Release Assays
Enzyme-linked immunosorbent assay (ELISA) kits for IL-2, IFN-γ, TNF-α and GM-CSF were purchased from eBioscience, and all ELISAs were performed according to the manufacturer’s protocols. T cells were co-cultured with target cells at a 1:1 E/T ratio for 24 h, then the culture supernatants were collected and analyzed by ELISA kits.
Quantitative Real-time PCR
mRNA was extracted from cells with TRIzol reagent (Thermo Fisher, 15596018) and reverse-transcribed into cDNA using a PrimeScript™ RT reagent kit (Takara, RR047A). All reactions were performed with TransStart Tip Green qPCR SuperMix (TransGene, AQ142-11) on a Bio-Rad CFX96 real-time PCR machine using the following primers: human PD-L1 forward, 5’–CCTACTGGCATTTGCTGAACGCAT-3’, and human PD-L1 reverse, 5’-ACCATAGCTGATCATGCAGCGGTA-3’.
Xenograft Models And In Vivo Assessment
Animal experiments were performed in the Laboratory Animal Center of the GIBH, and all animal procedures were approved by the Animal Welfare Committee of GIBH. All protocols were approved by the relevant institutional animal care and use committee (IACUC). All mice were maintained in specific pathogen-free (SPF)-grade cages and provided autoclaved food and water. To develop the lung cancer cell line xenograft models, 5 × 105 H460GL cells in 200 µl of PBS were injected subcutaneously into the right flanks of NOD-SCID-IL2Rg-/-(NSI) mice aged 6–8 weeks. Ten days after tumor cell transplantation, 5 × 106 CAR T cells were injected through the tail vein of mice. Tumors were measured at indicated days with a caliper to determine the subcutaneous growth rate.
To develop the first-generation PDXs, surgical tumor samples, including lung cancer, gastric carcinoma and hepatoma carcinoma were transplanted subcutaneously into 3 to 6 NSI mice. Tumors that reached an approximate size of 1000 mm3 were removed and passed into secondary recipients for expansion for further cancer research. Tumors were cut into 2 × 2 × 2 mm3pieces and transplanted into the right flanks of NSI mice. Then, 10, 15, 20 or 24 days after tumor transplantation, mice were infused with CAR T cell or control T cells. In total, 5 × 106 CAR T cells were injected one time into each mouse. Tumors were measured with a caliper, and tumor volume was calculated using the following equation: (length × width × width)/2.