Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

Lentiviral vector construction and production

The lentiviral vector was designed as previously described [13, 41]. LdCV vector contains the following: human codon-optimized S. pyogenes dCas9 which was fused at the C-terminus with VP64-p65-Rta (VPR). LAT-TCS-dCas9-VPR was assembled by fusing LAT (Human cDNA, NM_001014987.2) with dCas9-VPR-Q8 and cloned into a modified pHR-SFFV lentiviral vector (PMID: 26830878). SFFV promoter was replaced with EF1α promoter. 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 second vector harbors the HER2-specific CAR linked to the tobacco etch virus (TEV) protease (PMID: 29263378), which was designed as previously described (PMID: 34743703). For HER2 CAR detection and enrichment, P2A-tNGFR (truncated NGFR) was fused C-terminal to TEV. IL-12A sgRNA (IL12Asg) and IL-12B sgRNA (IL12Bsg) were controlled by human U6 promoter (hU6) and H1 promoter, respectively. Guide RNAs 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 (PMID: 29263378). For multiplexing experiments, IL12Asg and IL12Bsg in tandem with described promoters, were cloned into pHREF1αp lentiviral vector expressing HER2 CAR-TEV.

Second-generation, self-inactivating lentiviral supernatant was produced in the HEK293 T packaging cell line as previously described (PMID: 34743703). The 72-h viral supernatants were harvested, filtered through 0.45 μm PVDF membrane filter unit, layered on top of 10% sucrose solution, and followed by high-speed centrifugation at 10,000g for 16 h at 4 °C. The concentrated lentiviral pellet was resuspended on RPMI media and frozen at − 80 °C for future use.

Lentiviral titration was performed in 96 well flat bottom treated plate. HT1080 cells were adjusted at a density of 500,000 cells/ml and 4 ml of cell mix were combined with 16 μl of Transplus™ (Alstem). The mix was aliquoted in 100 μl/well and incubated at 37 °C for one hour. Crude and concentrated virus were thawed on ice. Four serial dilutions were performed, mixing 15 μl of virus and 135 μl of cell media. 100 μl of each dilution were then transferred onto seeded cells and incubated 3 days at 37 °C. Cells were then washed with PBS, trypsinized and incubated with 100 μl of antibody mixture NGFR-FITC (clone ME20.4, Biolegend 1:100) and Q8-PE (clone QBEND/10, ThermoFisher 4:100) for 30 min at 4 °C. Cells were washed twice in FACS buffer and resuspended in 200 μl FACS buffer. Samples were run in cytoFlex (Beckman) and FITC or PE positive cells were screened. For titer calculation selected wells (for dilution factor) presented 1–20% fluorescent positive cells and titer was calculated using the following formula:

$$\mathrm\,(\mathrm/\mathrm) = (50.000 \times \mathrm) / (100 \times \mathrm).$$

IL-12 sgRNA design and screening

An arrayed screen for IL-12 sgRNAs was performed. IL-12A and IL-12B sgRNA libraries were designed as previously described [13]. The screening of IL-12 was performed by transfection of 26 sgRNA for p35 and 31 sgRNA for p40 into dCas9-VPR expressing Jurkat cells. The top 5 candidates for IL-12A and IL-12B (highest knockdown and fewest computationally predicted off-targets) were further tested in primary T cells. The selected top candidates were cloned into pHR-EF1αp lentiviral vector expressing HER2 CAR-TEV as described above.

Primary T cell isolation and CAR T cell production

Primary T cell isolation and CAR-T cells production were performed as previously described [13]. At day 1 after activation, T cells were transduced with lentiviral vectors carrying LAT-dCas9VPR-Q8 (at MOI = 10), followed by a second transduction with HER2 CAR-TEV-tNGFR/IL-12sgRNAs (at MOI = 10) at day 2. At day 5, T cells activation was removed, and transduced T cells were cultured in a 24-well plate. At day 6, double-transduced cells were enriched for double positive, through cell sorting (Sony Cell Sorter) for Q8 and tNGFR. After cell sorting, T cells were cultured up to day 14 in T cell culture media with IL-15 and IL-7 (10 ng/ml). CAR-T cells were used for in vitro assays or implanted into mice at day 14 of manufacturing run.

Cell lines

Human embryonic kidney 293 cells (HEK293T), human fibrosarcoma (HT1080) and cells from hypopharyngeal tumor of a squamous cell carcinoma patient (FaDu) were obtained from ATCC (Manassas, VA). FaDu-PD-L1 (named FaDu-PDL1) was generated as previously reported [13].

All cells were routinely tested using the MycoAlert Mycoplasma Detection Kit (Lonza).

Quantification of HER2.CAR and dCas9 vector copies in transduced T cells

Genomic DNA was extracted from engineered cells using DNeasy blood and tissue kit (Qiagen). PCR amplification was performed using primers for RRE sequence in the lentiviral backbone, for CAR and LdCV. RPP30 gene was used as reference 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.

Co-culture experiments and cytokine production

Supernatant from co-cultured cells was harvested and analyzed for human IFN-γ, IL-2 and TNF-α by ELISA (Biolegend) and human IL-12 by Quantikine HS ELISA (R&D system).

HER2 micro‑bead preparation and HER2 beads stimulation

Beads were prepared as previously reported [13, 26].

Approximately 80,000 CAR-T cells were cultured in 96-well flat bottom plates and HER2 beads were added at a 1:1 bead to HER2 CAR-T cells ratio, in TBM media. Three days after beads stimulation, CAR-T cells were harvested and analyzed.

Animal experiments

After counting, 0.5 × 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 file 1: Fig. S2 for details), and relevant mice received anti-PD-L1 (atezolizumab, 10 mg/kg) intravenously. At the following day, a total of 0.3 × 106 of HER2 CAR-T cells, as specified in the figure legends, were injected intratumorally in a volume of 20 µl. 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 Miltenyi gentleMACS dissociator. The cell suspension was filtered using a MACS SmartStrainer, the strainer was washed with 10 ml of RPMI media and samples were centrifuged 500g for 5 min. Pellet were resuspended in 1.5 ml of CS10 freezing media and 300 ul were dispensed in each cryovial. Single-cell suspensions were subsequently thawed and stained using the described antibodies and analyzed by flow cytometry.

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). 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 or BV786 (clone M-T271, Biolegend), CD28-BV605 or BV711 (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). Mouse cells were assessed using the following antibodies: Flow cytometry results were analyzed using Kaluza software (Beckman Coulter).

Statistical analysis

Unpaired two-tailed t-test was used to analyze significant differences between groups. Statistical analyses were performed using GraphPad Prism8. Survival curve were compared using the log-rank Mantel-Cox test.

Significance of findings was defined as: ns not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001.

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