Lentiviral vector construction
Human codon-optimized S. pyogenes dCas9 was fused at the C-terminus with KRAB (Krüppel associated box) domain of Kox1 [12]. LAT-TCS-dCas9-KRAB was assembled by fusing LAT (Human cDNA, NM_001014987.2) with dCas9-KRAB-GFP (or dCas9-KRAB-Q8) and cloned into a modified pHR-SFFV lentiviral vector [34]; SFFV promoter was replaced with EF1α promoter, GZMB promoter or NFATRE promoter to get pHR-EF1αp, pHR-GZMBp and pHR-NFATREp lentiviral vectors (Fig. 1). As described previously [15], TCS sequence (ENLYFQ) was inserted in between LAT and dCas9 and was flanked by GS linker. Two nuclear export signals (NES, LALKLAGLDI and LQLPPLERLTL) flanked LAT to ensure the cytoplasmic localization of the chimera protein.
The HER2-specific scFv 4D5 sequence derives from the humanized mAb 4D5 Herceptin (trastuzumab) [17]. 4D5 scFv was amplified by PCR, based on the HER2 CAR vector purchased from Promab (PM-CAR-1024), linked to CD28 transmembrane and CD28 and CD3ζ intracellular signaling domains, and sub-cloned into pHR-EF1αp lentiviral vector. The tobacco etch virus (TEV) protease [15] was PCR amplified and cloned into the C-terminus of HER2 CAR. For HER2 CAR detection and enrichment, P2A-mCherry or P2A-tNGFR (truncated NGFR) was fused C-terminal to TEV. PD-1 sgRNA (PD-1sg) driven by mouse U6 promoter (mU6) were cloned into HER2 CAR-TEV vector upstream of the EF1α promoter for the ease of engineering the ChaCha system into human primary T cells [15].
For multiplexing experiments, PD-1sg and TIM-3 sgRNA (TIM-3sg) in tandem driven by mU6 and human U6 (hU6) promoters, respectively, were cloned into pHR-EF1αp lentiviral vector expressing HER2 CAR-TEV.
PD-1 sgRNA design and screening
A PD-1 sgRNA library was designed based on the predicted transcription factor binding sites and nucleosome distribution among the region spanning + / − 1 KB of the TSS of the targeted PDCD1 endogenous gene. The screening of PD-1 sgRNA was performed by transfection of 96 different PD-1 sgRNA candidates into dCas9-KRAB expressing Jurkat cells via Neon electroporation. The top 8 candidates with the most observed knockdown and fewest computationally predicted off-targets were cloned into the pLenti6 lentiviral vector for further validation in primary T cells. The top candidate PD-1 sgRNA #45 (GCTCCGCCTGAGCAGTGGAGA) was selected and cloned into pHR-EF1αp lentiviral vector expressing HER2 CAR-TEV as described above.
Lentiviral vector production
Second-generation, self-inactivating lentiviral supernatant was produced in the 293 T packaging cell line. In brief, 70% confluent 293 T T225 flask were co-transfected with 32 µg pHR-EF1αp lentiviral vector plasmid, and 16 µg psPAX2 (Gag/Pol) and 8 µg pMD2.G (VSVG envelope) packaging plasmid DNA using 108 µl TransIT-LT1 (Mirus) and 57 µl ViralBoost Reagent (ALSTEM). The 72-h viral supernatants were harvested, filtered through 0.45 µm PVDF membrane filter unit, and layered on top of 10% sucrose solution, and followed by high-speed centrifugation at 10,000×g for 4 h at 4 °C. Concentrated lentiviral stocks were frozen at -80 °C for future use.
Primary T cell isolation and CAR T cell production
Healthy donor leukopak were purchased from www.pparesearch.com. Fresh peripheral blood mononuclear cells (PBMCs) were isolated by low-density centrifugation on Lymphoprep (Stem Cell Technology) according to the manufacturer’s instructions. Pan T cells were isolated using Dynabeads Untouched Human T Cells (Invitrogen). To generate CAR-T cells, at day 0 cryopreserved Pan T cells were thawed and activated in 24-well plate coated with human anti-CD3 (OKT3, 1 µg/ml, Biolegend) and human anti-CD28 (αCD28, 1 µg/ml, BD Pharmingen™) antibodies in T cell culture medium (RPMI supplemented with 10% human serum, 2 mM GlutaMAX, 50 µM 2-Mercaptoethanol (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). Recombinant human IL-7 and IL-15 (Gibco) were both provided at 10 ng/ml. T cells were transduced with lentiviral vectors carrying LAT-dCas9KRAB-GFP or LAT-dCas9KRAB-Q8 at day 1 and HER2 CAR-TEV-mChr/PD-1sg or HER2 CAR-TEV-tNGFR/PD-1sg at day 2 after activation. At day 5, T cells activation was removed via transferring transduced T cells into a tissue-culture-treated 24-well plate. At day 6, double-transduced T cells were enriched through cell sorting (Sony Cell Sorter) for GFP and mCherry or Q8 and tNGFR double positive T cells. After cell sorting, T cells were maintained at 0.5 × 106 − 1 × 106 cells per ml in T cell culture medium with IL-7 and IL-15 (10 ng/ml). CAR-T cells were used for in vitro assays or implanted into mice at day 14 or 21 of manufacturing run.
Cell lines
The human head and neck squamous cell carcinoma line FaDu was obtained from the ATCC (Manassas, VA). FaDu-PD-L1 (named for simplicity FaDu hereafter) was generated by transducing lentiviral vector, pLenti6.3/V5™-TOPO™ (Invitrogen), overexpressing PD-L1 (Human cDNA, NM_014143.4), followed by blasticidin selection. Cells were cultured in complete media (DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco). All cells were routinely tested for potential mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza).
Quantification of HER2.CAR and dCas9 vector copies in transduced T cells
Engineered cells were pelleted, washed with PBS and DNA was extracted using DNeasy blood and tissue kit (Qiagen). PCR amplification was performed using primers for lentiviral backbone (RRE sequence), as well as specific primers for CAR and LdCK. RPP30 amplification was used as cell number control. Droplets were generated using the QX200 droplet generator (Bio-Rad) according to the manufacturer protocol. Amplification in droplets was measured using QX200 Droplet reader (Bio-Rad) and analyzed using Quantasoft software.
Flow cytometry
Human HER2 and PD-L1 expression on tumor cells was detected using human HER2-PE-CY7 (clone 24D2, Biolegend) and PD-L1-APC (clone MIH1, eBioscience). HER2 4D5 CAR was detected using the anti-trastuzumab idiotype Alexa Fluor 647-conjugated antibody (clone 2661E, R&D system). HER2 4D5 CAR was also detected using human recombinant HER2 protein conjugated with Alex Fluor 647. Human T cells surface phenotype and transduction efficiency were assessed using the following antibodies: NGFR-FITC (clone ME20.4, Biolegend), Q8 (clone QBEND/10, ThermoFisher), CD45-AF700 or CD45-BV605 (clone HI30, Biolegend), CD3-APC (clone SK7, Biolegend), CD4-PerCP (clone SK3, Biolegend), CD4-BB700 (clone SK3, BD Bioscience), CD8-BV510 (clone SK1, Biolegend), CD27-PE-CY7 (clone M-T271, Biolegend), CD28-BV605 (clone CD28.2, Biolegend. Expression of T cell inhibitory receptors was analyzed using PD-1-BV421 or PD-1-BV605 (clone EH12.2H7, Biolegend), TIM-3-BV605 or TIM-3-PE-CY-7 (clone F38-2E2, Biolegend), CD39 (clone A1, Biolegend), LAG-3-BV711 (clone 11C3C65, Biolegend). Live/dead discrimination was determined using LIVE/DEAD fixable Near-IR dead cell stain kit (ThermoFisher). Flow cytometry results were analyzed using Kaluza software (Beckman Coulter).
HER2 Micro-bead preparation and HER2 beads stimulation
The ectodomain of recombinant human HER2 (Thr 23–Thr 652, Acro Biosystems) was chemically biotinylated by mixing 30 μg of HER2 protein with 20 molar excess of EZ-Link NHS-PEG4-Biotin (Thermo Scientific) in sodium bicarbonate buffer (pH 8.3). After incubation for 2 h at 4 °C on a rotator, the biotinylated HER2 was purified using Zeba desalting columns (7 kDa MWCO). For the conjugation of biotinylated HER2 onto the microbeads, 3 mg of Dynabeads of 2.8 μm in diameter coated with streptavidin (M-280 Streptavidin, ThermoFisher) were first washed twice and re-suspended in 1 ml of PBS. 3 or 15 μg of biotinylated HER2 protein was added into each microtube containing the bead suspensions, for low or high densities, respectively. Each microtube was quickly vortexed to homogenize the solution. After an overnight incubation at 4 °C on a rotator, the microbeads were washed once in PBS and further incubated with PBS supplemented with 2% (w/v) of biotinylated BSA for 1 h at 4 °C on a rotator. Finally, the microbeads were washed twice and re-suspended in PBS with 0.02% NaN3 for storage. The surface density of HER2 on microbeads was evaluated by flow cytometry using mouse anti-human HER2 antibody (clone 191924, R&D Systems) and QiFiKit (Agilent). The surface density values (ABC/μm2) were measured to be 1487 and 3628, for HER2 Low and High beads, respectively.
For HER2 beads stimulation experiments, approximately 80,000 CAR-T cells were cultured in 96-well flat bottom plates and HER2 low or high beads were added at a 1:1 beads to HER2 CAR-T cells ratio. Three days after beads stimulation, CAR-T cells were harvested and expression of HER2 CAR (tNGFR) and LdCK (Q8) were analyzed by flow cytometry.
Co-culture experiments and cytokine production
For regular co-culture experiments, approximately 80,000 FaDu tumor cells were seeded in 48-well flat bottom plates and CAR-T cells were added 6 h later at indicated effector:target (E:T) ratios and cultured for 6 days. Triplicate wells were plated for each condition. Culture supernatants were collected and analyzed for IFN-γ, IL-2 and TNF-α by ELISA (Biolegend). Residual tumor cells, CAR-T cell proliferation and surface expression of PD-1 were analyzed by flow cytometry.
For repetitive tumor stimulation experiments, CAR-T cells were co-cultured with FaDu tumor cells at Day 0 (20,000 CAR-T cells; 80,000 FaDu cells; E:T = 1:4) and re-stimulated with 1,00,000 FaDu cells at day 4, 8 and 12. Non-stimulated CAR-T cells were maintained in culture in the presence of 100 U ml−1 IL-2. At indicated time points (day 4, 8, 12 and 16), co-culture supernatants were analyzed for IFN-γ, IL-2 and TNF-α by ELISA (Biolegend). Stimulated and non-stimulated CAR-T cells and surface expression of PD-1, TIM-3, LAG-3 and CD39 were analyzed by flow cytometry.
Animal experiments
After counting, 0.5 × 106 or 1 × 106 FaDu tumor cells were re-suspended in 100 µl of PBS plus matrigel (Corning) and subcutaneously injected into the right flank of 6-to-8-week-old female immune deficient NSG mice (JAX laboratory). When average tumor size reached close to 100 mm3, mice were randomized into different groups (see Additional files 3, 4, 5: Figures S3–5 for details), and relevant mice received anti-PD-L1 (atezolizumab, 10 mg/kg) intravenously. At the following day, for the model of intratumoral administration of CAR-T cells, a total of 0.3 × 106, 0.25 × 106 or 0.1 × 106 of HER2 CAR-T cells, as specified in the figure legends, were injected intratumorally in a volume of 20 µl; for the model of intravenous administration of CAR-T cells, 1 × 106 or 3 × 106 of HER2 CAR-T cells were intravenously injected in a volume of 100 µl. In both models, mice in the atezolizumab group were continuously treated with atezolizumab (5 mg/kg) twice every week. Tumor dimensions were measured biweekly with digital calipers, and tumor volumes were calculated using the formula V = ½ (length × width2). Mice were humanely euthanized according to IACUC protocol and tumor were resected immediately after euthanasia for further analysis.
Isolation of tumor-infiltrating CAR-T cells
Solid tumor tissue was collected, rinsed with PBS, and mechanically dissociated using the gentleMACS dissociator (Miltenyi), and single-cell suspensions were stained with the described antibodies and analyzed by flow cytometry.
Statistical analysis
Statistical analyses for significant differences between groups were conducted using unpaired two-tailed t tests using GraphPad Prism8. Survival curves were compared using the log-rank Mantel-Cox test. A p < 0.05 was considered statistically significant. Significance of findings was defined as: ns not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001.