Plasmid construction and production of recombinant lentiviral vectors
The tandem-CAR construct is a second-generation vector consisting of the following components in-frame from the 5′ end to the 3′ end: the CD8 signal peptide sequence, anti-BCMA-scFv (C11D5.3)[18], anti-CD19 scFv (FMC63AA 1-267, GenBank ID: HM852952.1), the hinge and transmembrane regions of the CD8α molecule, the cytoplasmic domain of CD28, and the CD3 zeta signaling domain. The sequence was synthesized by Tsingke Biological Technology (Shanghai, China) and cloned into the pUT plasmid backbone (Unicar-Therapy Biomedicine Technology Co., Ltd., Shanghai, China). The newly-constructed lentiviral vector is referred to as tan-CAR. We also prepared the scFv domain CAR vectors CD19-CAR and BCMA-CAR with the CD8 signal peptide sequence anti-CD19 scFv (FMC63AA 1-267, GenBank ID: HM852952.1) or anti-BCMA-scFv (C11D5.3), the hinge and transmembrane regions of the CD8α molecule and cytoplasmic domain of CD28 and the CD3 zeta signaling domain. Lentiviruses were generated from these constructs via transient transfection of HEK293T cells.
Cell lines
All the cell lines were purchased from the American Tissue Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Medical Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA). K562 cells were stably transduced with the lentiviral constructs encoding CD19 or BCMA and luciferase. Following transduction, single luciferase-positive cells were selected for clonal expansion. K562-CD19-luc and K562-BCMA-luc stable cell lines were generated by this method. Myeloma cell line 8226 were transduced with the lentiviral constructs encoding CD19 to obtain the tumor cells expressing both CD19 and BCMA.
Preparation of CAR T cells
Healthy donor-derived peripheral blood mononuclear cells were isolated from blood by gradient centrifugation using Lymphoprep™ (Oriental Hua Hui, Beijing, China) followed by CD3+T-cell enrichment by positive selection using a magnetic bead separation method (Miltenyi Biotec, Bergisch Gladbach, Germany). CD3+T cells were cultured and activated in vitro using anti-CD3/CD28 monoclonal antibodies (Miltenyi Biotec) in a 5% CO2 atmosphere at 37 °C for 18–24 h. The activated T cells were then transduced with lentivirus (CD19-CAR, BCMA-CAR and tandem CAR) for 48 h. We also tansduced the BCMA CAR (D1) followed by CD19-CAR (D2) to obtain the T cells expressed two scFv by transduced two lentivirus. After transduction, the CAR T cells were cultured and expanded in a 5% CO2 atmosphere at 37 °C for 14 days in AIM-V medium (Gibco, Grand Island, NY, USA), supplemented with 100 IU/mL recombinant human interleukin-2 (IL-2; Peprotech, Rocky Hill, NJ, USA), 5 ng/ml recombinant human IL-7 (Peprotech), 5 ng/mL recombinant human IL-15 (Peprotech) and 10% autologous plasma[19].
Flow cytometry
For the flow cytometry assays, the cells were harvested and washed twice with 1 mL of phosphate-buffered saline (PBS) containing 2% FBS (Gibco). To determine transduction efficiency and the CD4/CD8 ratio, the CAR T cells were labeled with the recombinant protein L-FITC (ACRO Biosystems, Beijing, China), anti-CD4-PE-Cy7 (eBioscience, San Diego, CA), and antipCD8-APC (eBioscience) for 45 min at 4 °C in the dark. For detection of the CD19 CAR-expressing T cells, the CAR T cells were incubated with human CD19 protein-FITC (ACRO) for 45 min at 4°C in the dark. For detection of the BCMA-CAR T cells, the CAR T cells were labeled with human BCMA protein-FITC (ACRO) for 45 min at 4°C in the dark. The cells were washed twice before analysis by Attune NxT flow cytometer (Thermo Fisher, Waltham, USA).
T-cell activation assay
T-cell activation was evaluated by measuring CD69 expression on tan-CAR T cells in response to 24-h co-culture with target cells. Un-transduced (NC) T cells were used as negative controls, and the T cells transduced with CD19-CAR or BCMA-CAR served as positive controls. After co-culture, the cells were harvested and washed twice with 1 mL of PBS containing 2% FBS and then labeled with CD69-PE (BiolegendCA, USA), CD3-FITC (Biolegend), and protein L-FITC (ACRO) for 20 min at room temperature in the dark. CD69 expression in CAR T cells as detected by flow cytometry was used as a marker of CAR T-cell activation.
Quantitation of T-cell proliferation
Cell proliferation assays were performed using a Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) assay kit (Abcam, Cambridge, UK) following the manufacturer’s instructions. In brief, the CAR T cells were labeled with 2.5 µM CFSE and then co-cultured with Raji cells which treated with mitomycin before to stop the division, at a stimulator to responder ratio of 2:1 (106 CAR T cells/mL) for 5 days in 96-well plates in 200 µL serum-free AIM-V (Gibco) medium per well. Flow cytometry was performed using an Attune NxT flow cytometer (Thermo Fisher) to detect changes in CFSE intensity. FlowJo V10 software (TreeStar, San Carlos, CA, USA) was used for data analysis.
Cytotoxicity assays
Cytotoxicity was determined via quantitation of lactate dehydrogenase activity in the supernatants of effector and target cell co-cultures using the Cytotoxicity Detection Kit (Promega, Madison, WI, USA) following the manufacturer’s protocol. All the transduced CAR T cells (effector, E) were co-cultured with cells of the target cells that overexpressed CD19, BCMA, or both antigens (target, T) at E:T ratios of 5:1, 2.5:1, and 1:1, respectively. Target and effector cells were seeded in 96-well plates in a total volume of 100 µL of serum-free RPMI 1640 media (Gibco) and incubated at 37 °C for 6 h. After co-culture, 50 μL of cell-free supernatant from each well was transferred to a new 96-well plates and mixed with equal volume of lactic acid dehydrogenase substrate mixture for 20 min at room temperature in the dark. The absorbance was recorded at 492 nm using a full wavelength reader Multiskan GO (Thermo Scientific). Tumor (target cell) lysis was calculated with the following formula: % lysis = (experimental LDH release − spontaneous LDH release) / (maximum LDH release − spontaneous LDH release) × 100.
Detection of CD107a
To evaluate CD107a expression on the cell surface as an indirect marker of degranulation, 106 CAR T cells were co-cultured with target cells at a 5:1 ratio in 96-well plates with a total of 200 µL of AIM-V (Gibco) medium per well for 6 h. The Golgi inhibitor monensin (Invitrogen, Carlsbad, CA, USA) was added before the incubation. Cocktails (Invitrogen) were added to the positive control group before co-culture. After a 6-h incubation, cells were labeled with anti-CD107a-APC, anti-CD3-FITC, and anti-CD8-PE. All the antibodies were purchased from Becton, Dickinson and Company Co., Ltd (Franklin Lakes, NJ, USA). Cells were collected, washed twice with PBS, and flow cytometry analysis was performed on an Attune NxT flow cytometer (Thermo Fisher). The results were analyzed by FlowJo V10 software (TreeStar).
Analysis of cytokine release
Cytokine release was evaluated using a Th1/Th2 Cytometric Bead Array (CBA) Kit II (BD Bioscience) according to the manufacturer’s instructions. Briefly, CAR-transduced T cells were co-cultured with the various K562 cell transfectants at an E:T ratio of 5:1 in a 96-well plate with a total volume of 200 μL of RPMI 1640 medium (Gibco). After 24 h in co-culture, cell-free supernatants were harvested and the levels of various cytokines were evaluated. The capture microspheres for seven specific cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-a, and IL-17A) were first mixed and then incubated with the sample and fluorescent antibody for 3 h. The mixture was washed and cytokine concentrations were determined by flow cytometry (Thermo Fisher). The concentration of each cytokine was calculated from standard curves.
Mouse xenograft model
Mouse experiments were performed with the approval of the Institutional Animal Care and Use Committee of East China Normal University. Four-to-six-week-old male NOD/scid/γc−/− (NSG) mice were purchased from Biocytogen Co., Ltd (Beijing, China). Xenograft models were established via injection of K562-CD19-luc and/or K562-BCMA-luc cells. A total of 7 × 106 mixed tumor cells at a ratio of 1:1 in 200 μL PBS were injected into mice via the tail vein on day 0. The mice were then randomly divided into four groups that received either (a) 107 CD19-CAR T cells (n = 3), (b) 107 BCMA-CAR T cells (n = 3), (c) 107 tan-CAR T cells (n = 3) or (d) 107 un-transduced T cells (n = 3, negative control) via the intravenous route on both days 8 and 10. The mice injected with K562-CD19-luc or K562-BCMA-luc were treated with CD19-CAR T or BCMA-CAR T cells, respectively. Tumor progression was monitored by bioluminescent imaging every four days beginning on day 7. The mice were sacrificed when moribund or upon the development of hind-limb paralysis. For in vivo imaging, the mice were injected intraperitoneally with 150 mg/kg D-luciferin (Yeasen, Shanghai, China) and imaged under isoflurane anesthesia using the Xenogen-IVIS system (LI-COR Biosciences, Lincoln, NE, USA). Fluorescence was quantified using Living Image software (IVIS Lumina Series, PerkinElmer, Waltham, MA, USA).
Statistical analysis
Statistical analyses were carried out using GraphPad Prism 8.0. Biological replicates of in vitro (n = 3) and in vivo data (n = 3) are presented as the mean ± SD. Statistical analysis was performed to assess differences between individual treatment groups and the un-transduced control group using one-way ANOVA. Statistically significant findings were defined as *p < 0.05.