Cell lines
Lenti-X 293 T cell line was purchased from Clontech (Cat#632180, California, United States) in August 2018, and the test report was provided by Clontech (California, United States). OVCAR-3 cell line was purchased from FuHeng Cell Center (Cat#FH0726, Shanghai, China) in February 2016. It was tested by STR Authenticated provided by FuHeng Cell Center, and the test report showed a complete match with the NIH: OVCAR-3 (ATCC HTB-161). Umbilical blood mononuclear cell (UBMC) was obtained from healthy donors in Beijing Obstetrics and Gynecology Hospital in March 2019. UBMC was tested for the positive rate of CD3 on the cell surface by flow cytometry after being activated. All cell lines were validated monthly to be mycoplasma free by PCR. Lenti-X 293 T cells were used to construct lentiviral expression vectors and were cultured in high glucose DMEM medium (Hyclone, Logan, United States) containing 5% fetal bovine serum (FBS, Hyclone, Logan, United States) and 1% penicillin-streptomycin solution (Hyclone, Logan, United States). OVCAR-3 cells were marked as OVCAR3-luc cells by luciferase, culturing in RPMI-1640 medium (Gibco, California, United States) supplemented with 10% FBS, 1% penicillin-streptomycin solution, and 0.1% insulin (Gibco, California, United States). After being activated by anti-CD3/CD28 magnetic beads (Novoprotein, Shanghai, China), UBMC was cultured in the GT-T551 H3 medium (TaKaRa, Osaka, Japan) containing 5%FBS and 40 IU/ml IL-2 (Novoprotein, Shanghai, China). All cells were cultured in an incubator (ESCO, Portland, United States) at 37 °C and 5% CO2. This study was approved by the Medical Ethics Committee, Beijing Obstetrics and Gynecology Hospital, Capital Medical University (2018-KY-026-01).
Construction of CAR molecule
After designing the sequences, primers and templates were synthesized by Sangon Biotech. According to the PCR principle, the single chain antibody fragments (scFv) of PD1, anti-MUC16 and PD1-antiMUC16 CAR were obtained. The main structures of PD1 and anti-MUC16 are PD-1etco and 4H11-VH-(Gly4Ser)3-4H11-VL. The primary structure of PD1-antiMUC16 is tandem of PD1 and anti-MUC16. The three scFv fragments were cloned into the pLVX-EFlα-IRES-mCherry plasmid (Clontech, California, United States) through EcoR I and Mlu I cloning sites and named PD1-antiMUC16 CAR, anti-MUC16 CAR, and PD1 CAR, respectively. The plasmid has been genetically engineered to be a second-generation CAR containing CD8a hinge region, CD8 transmembrane region, 4-1BB co-stimulation domain, and CD3ζ domains. The plasmids were amplified in bacterial solution, and the positive samples were selected by agarose gel electrophoresis and verified via sequencing analysis.
Lentivirus packaging
Set the experiment groups (pLV-PD1-anti-MUC16, pLV-anti-MUC16, and pLV-PD1) and Control group (control T). In each group, 13.7 μg plasmid was taken and mixed with packaging plasmid containing 3.43 μg pMD2.G, 3.43 μg pMDLg/pRRE, 3.43 μg pRSV-Rev (Addgene, Massachusetts, United States) to make DNA-mix. 7 × 106 cells Lenti-X 293 T were added into the DNA-mix and the same volume of polyethylenimine (PE1, Polyscience, Pennsylvania, United States), then cultured in the incubator. Fresh virus packaging medium containing Opti-MEM (Gibco, California, United States), 5% FBS, 1% L-glutamine (Gibco, California, United States), 1% sodium pyruvate (Gibco, California, United States), and 0.2% penicillin-streptomycin solution was supplemented after 6 h of culturing. After 24 h, we collected supernatant and obtained virus concentrate (210 μL), from which 6 μL was taken to infect 293 T cells again for virus titer detection. The rest of the virus concentrate was stored in a 4 °C refrigerator for preparation of CAR-T cells. After 48 h of infection, the infected cells were placed into 12-well plates (Corning, New York, United States). 5 μL Percp-cy5.5 antihuman PD-1 antibody (BD, New jersey, United States), and 10 μL FITC-Protein L antibody (ACRO, Delaware, United States) were added to each well, and incubated in the dark for 30 min. The flow cytometry (NovoCyte Advanteon, ACEA, Hangzhou, China) was used to detect the positive rate of PD1 and anti-MUC16 in Lenti-X 293 T cells. The viral titer was calculated according to the following formula:
$$ \mathrm{Viral}\ \mathrm{Titer}\left(\mathrm{TU}/\mathrm{ml}\right)=\frac{\left(\mathrm{Number}\ \mathrm{of}\ \mathrm{Infected}\ \mathrm{Cells}\times \mathrm{Positive}\ \mathrm{Rate}\right)}{\mathrm{Viral}\ \mathrm{Volume}\left(\mathrm{ul}\right)}\times 1000 $$
(1)
T cell transduction
Set the experiment groups (pLV-PD1-anti-MUC16, pLV-anti-MUC16, and pLV-PD1) and Control group (control T). After thawing, 2.5 × 106 UBMC were added into phosphate buffer saline (PBS, Hyclone, Logan, United States) with a volume of 10 times, which was then centrifuged at 1000 rpm for 15 min (BT-320C, Baiyang, Beijing, China). Then an appropriate T cell complete medium and 25 μL magnetic beads were added. The mixture was cultured in the incubator for 48 h. Lentiviral supernatants were collected, of which 200 μL per well was added into the 12-well plate coated with 200 μg NovoNectin (Novoprotein, Shanghai, China) overnight. Meanwhile, control T well was added with 200 μL GT-T551 H3 medium. All wells were added with 800 μL UBMC (2.5 × 105cells/well) and cultured in an incubator. After culturing for 6 h, 2 mL of T cells complete growth medium (GT-T551 H3 + 5%FBS + 40 IU/ml IL-2) was added and T cells were re-infected for the following day. After the second infection for 96 h, 1 × 106 cells was taken out from each well, washed twice with PBS, removed of the magnetic beads, and stained with 5 μL Percp-cy5.5 antihuman PD-1 antibody and 10 μL FITC-Protein L antibody for 30 min in the dark. Then it is followed by washing with PBS twice and detection for the positive rate of CAR structure by the flow cytometry. Finally, when CAR-T cells were successfully prepared, the positive ratio of the other two CAR-T cells was adjusted according to the lowest positive ratio of three CAR molecules by increasing the number of control T cells.
Preparation of target cells
After thawing, OVCAR3-luc cells were cultured in RPML1640 medium containing 20% FBS, 1% glutamine, 1% sodium pyruvate, 1% penicillin and streptomycin,0.01 mg/mL insulin. It was seeded in T25 bottle with a density of 5 × 105/4 cm2 in an incubator (37 °C, 5% CO2). Both MUC16 plasmid and PDL1 plasmid (Juventas, Tianjin, China) were mixed with the PEI-DNA mix to complete lentiviral packaging by PEI transfection, and were harvested for MUC16 lentiviral expression vector, PDL1 lentiviral expression vector, and MUC16-PDL1 lentiviral expression vector. The lentiviral concentrate was harvested at 24 h and 48 h, followed by infecting OVCAR3-luc cells. Flow cytometry measured the positive rate of MUC16 and PDL1 on the OVCAR3-luc cell surface after 96 h of re-infection.
Flow Cytometry
One million CAR-T cells were stained with 5 μL Percp-cy5.5 antihuman PD-1 antibody and 10 μL FITC-Protein L antibody for an antigen-antibody binding reaction. After 30 min of dark incubation, dissociative antibodies uncombined with antigens were washed by PBS. CAR molecule expression was measured for fluorescence intensity by flow cytometry. This method was used to detect viral titer and transfection rate of T cell. The cytotoxicity of CAR-T cells was evaluated via detecting the luciferase expression of tumor cells after stained with 70 μL Steady-Glo. Simultaneously, the transfection efficiency of tumor cells was evaluated via detecting green fluorescent protein (GFP).
Enzyme-linked Immunosorbent assay (ELISA)
The human IFN-gamma ELISA kit, IL-2 ELISA kit, and TNF-alpha ELISA kit (all kits, Dakewe, Beijing, China) were used to measure the concentrations of IFN-γ and IL-2, TNF-α, respectively. According to the instructions of the ELISA kit, three samples were processed, and the standard curve was prepared. Then the fluorescence value was measured by the enzyme-labeled instrument (TECAN, Mannedorf, Switzerland). The cytokines quantity was then calculated.
Cytotoxicity assay and cytokine release assay
Target cells: OVCAR3-luc cells, OVCAR3-PDL1-luc cells, OVCAR3-MUC16-luc cells, and OVCAR3-MUC16-PDL1-luc cells; Effector cells: PD1-antiMUC16 CAR-T cells, antiMUC16 CAR-T cells, PD-1 CAR-T cells, control T cells (negative group). Target cells was adjusted for a density of 2 × 105cells/mL by GT-T551 H3, and seeded into the black flat 96-well U-bottomed plate (50 µL/well). Effector cells were adjusted for a density of 3.2 × 106cells/mL by GT-T551 H3, and seeded into the same 96-well plate (50 μL/well) at effector to target(E/T) ratios of 1:1, 4:1, 8:1 and 16:1, respectively. Simultaneously, target cells (50 μL/well) were cultured alone and with 50 μL GT-T551 H3 medium. The plate was put in the incubator at 37 °C for 4 h. Afterwards, all cells were stained with 70 μL Steady-Glo (Promega, Wisconsin, United States) per well for 20 min in the dark. The fluorescence value was detected by flow cytometry and the killing rate of various CAR-T cells on tumor cells was calculated according to the following formula:
$$ \mathrm{Killing}\ \mathrm{Rate}\%=\frac{\left(\mathrm{Fluorescencemax}\ \mathrm{of}\ \mathrm{target}\ \mathrm{cells}-\mathrm{Fluorescence}\ \mathrm{of}\ \mathrm{experimental}\ \mathrm{well}\right)}{\mathrm{Fluorescencemax}\ \mathrm{of}\ \mathrm{target}\ \mathrm{cells}}\times 100\% $$
(2)
The CAR-T cells were co-cultured with target cells, at 1:1 ratio(1 × 104 T cells and 1 × 104 target cells) in a V-bottomed 96-well plate for 48 h in the incubator, followed by harvesting supernatant through centrifugation (2500 rpm, 5 min), and detection for the release of IFN-γ, IL-2, TNF-α, respectively by ELISA kit.
Xenograft mice models for in vivo treatment
All animal studies were approved by the Medical Ethics Committee, Beijing Obstetrics and Gynecology Hospital, Capital Medical University (2018-KY-026-01). In-house bred NPG mice (NOD.Cg-PrkdcscidIl2rgtm1Vst/Vst) were obtained from Beijing Vitalstar Biotechnology Co., Ltd. Twenty healthy NPG mice (females, 35–41 days old, 18-21 g in weight) were raised in specific pathogen-free (SPF) conditions and fed with autoclaved food and water. For the xenograft models, NPG mice were intraperitoneally injected with 5 × 105 OVCAR3-MUC16-GFP-PDL1-luc cells and 50 μL Matrigel (Corning, New York, United States). After 48 h, mice were intraperitoneally injected with 100 μL D-Luciferin and Postassium Salt (Sciencelight, Shanghai, China) for 6 min before being put in an isoflurane-oxygen mixture gas anesthetic box containing 3% isoflurane (RWD, Shenzhen, China) for 2 min. The tumor burden was then measured by IVIS Spectrum and analyzed by Living Image, version 4.3, software (Perkin Elmer). The study subjects were distributed randomly into four groups (n = 5 for each group) on day 0, and each group was intraperitoneally injected with CAR-T cells (1 × 106cells per mouse). Imaging was performed on days 7, 14, 21 and 28 to monitor the tumor changes. Mice were euthanized by carbon dioxide asphyxiation when tumour volume exceeded 2000 mm3, when the weight loss over 20%, or when they lost their ability to eat autonomously. The mice were placed in a transparent box that released pure carbon dioxide after conformed to the above criteria. When mice were observed to faint, carbon dioxide continued to be released for 2 min for euthanization. The survival time of each mouse was recorded and the survival curve by GraphPad Prism 8.3.0 software drawn.
Statistical analysis
Statistical analysis was performed using SPSS 23.0 software. Data are shown as mean ± 1 standard deviation (SD). For the in vitro killing assay, the significance of different groups was determined using nonparametric tests. For the in vivo assay, Student’s t-test was used to distinguish the difference between groups. The value of P < 0.05 was considered significant. The mice survival curve was drawn using GraphPad Prism 8.3.0 software.