Background

T cells engineered with chimeric antigen receptors (CARs) represent a promising strategy for cancer treatment. However, many challenges remain in eradicating tumors [1, 2], including antigen escape [3, 4], immunosuppression in the tumor microenvironment [1, 5, 6] and lack of strictly tumor-specific targets [7]. To overcome these hurdles, incorporation of multiple strategies which can enhance antitumor efficacy and improve safety are required, and increasingly more combination strategies and synthetic biological approaches emerged. For instance, pre-clinical and clinical studies demonstrated that combining CAR-T with PD-1 blockade, either through targeted delivery of anti-PD-1 scFvc or genetic knock out of the PD-1 gene, shows enhanced tumor control compared with conventional CAR-T [8,9,10,11]. Synthetic regulatory circuits, like SynNotch/CAR, have been constructed to enable T cell activation to broaden the tool box for the design of more specific and safer T cell therapies for a wider range of tumor types [12].

Here, we present RB-340-1 as a combinatorial CAR/CRISPRi circuit that couples CRISPR interference (CRISPRi) to CAR signaling upstream and downstream to gene suppression of the endogenous Pd-1 gene to prevent CAR T cell exhaustion and enhance clinical outcomes. CRISPRi is a non-editing gene expression regulation strategy based on the nuclease de-activated CRISPR associated (dCas9) protein [13,14,15]. Coupling of dCas9 to a transcriptional repressor domain could silence expression of multiple endogenous genes [14]. RB-340-1 is engineered to express an anti-HER2 CAR single chain variable fragment (scFv) (4D5 clone) [16], with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a PD-1 promoter region-targeting short guide RNA (PD1sg). The other complex of RB-340-1 includes linker for activation of T cells (LAT) complexed to dCas9-Kruppel-associated box (Krab) via a TEV-cleavable linker. Upon antigen- encounter, the LAT-dCas9-Krab complex is cleaved by the TEV allowing nuclear translocation of dCas9-Krab to the transcription start site of the PD-1 gene. This conditional, non-gene editing and reversible suppression promotes resilience to checkpoint inhibition, and in vivo persistence and effectiveness against HER2-expressing oropharyngeal cancer xenografts. To make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels, which is named as RB-340-1F.

Currently, lentiviral transduction is a favorite method applied to deliver genes into T cells [17,18,19,20,21]. However, the efficiency of transduction of primary T cells with large payload lentiviral vectors is very low, especially when multiple lentiviral vectors are needed for transduction. To overcome this hurdle, we tried to utilize inhibitors of receptors involved in antiviral response. It is very well studied that TANK-binding kinase 1 (TBK1) and IƙB kinase ɛ (IKKɛ) regulate the production of type 1 interferons (IFNs), which trigger antiviral responses during viral infections [22, 23]. Therefore, blocking TBK1 activity should prevent the induction of interferons, reduce host response, and facilitate viral infection [24,25,26]. The compound BX795 has been reported as an inhibitor of the TBK1 and IKKɛ complex [27]. Moreover, a study in human natural killer (NK) cells found that BX795 boosted on average 3.8-fold the efficiency of lentiviral genetic modification [28]. Significant promotion of transduction was seen at 2 µM–10 µM concentration, without causing toxic effects on NK cells. Transduction efficiency only increased when BX795 was present during the transduction and did not increase when NK cells were pretreated and BX795 was absent during transduction, which means that the inhibition of antiviral response by BX795 is reversible. BX795 was also shown to promote lentiviral transductions in different cell lines. However, BX795 treatment has not been tested with human primary T cells yet. In this study, we use BX795 to promote RB-340-1F lentiviral transduction of human primary T cells and also detected the changes that BX795 treatment might bring to the transduced T cell product.

Methods

Cell lines

293T cells were purchased from ATCC (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS, Corning), and 1% Penicillin–Streptomycin (Invitrogen). The human head and neck squamous cell carcinoma line FaDu was obtained from the ATCC (Manassas, VA). FaDu/PD-L1 was generated by transducing lentiviral vector, pLenti6.3/V5TM-TOPOTM (Invitrogen), overexpressing PD-L1 (Human cDNA, NM_014143.4), followed by blasticidin selection. Fadu-PDL1 cells were maintained in the same medium as 293T cells.

Lentiviral vectors production and titration

Second-generation, self-inactivating lentiviral vectors were produced with 293T cell line. 3 × 107 293T cells are seeded in T175 cell culture flasks. After 24 h, 70%–80% confluent 293T cells were transfected with 108 µl TransIT-LT1 (Mirus) mixed with 32 µg lentiviral vector plasmid, 16 µg psPAX2, and 8 µg pMD2.G packaging plasmid. ViralBoost Reagent (ALSTEM) was added to facilitate virus production. Viral supernatant was harvested 72 h post transfection. After clarification, the supernatant was concentrated by centrifugation at 10,000g through a 10% sucrose cushion for 4 h at 4 °C. The pelleted vectors were resuspended in RPMI 1640 Media (Gibco), aliquoted, and stored at − 80 °C for later use.

Lentiviral vector was titrated on HT1080 cells by serial dilution in DMEM supplemented with 10% FBS. 72 h post infection, the ratio of GFP or mCherry positive cells was detected by flow cytometry. Titer-transducing units/mL (TU/mL) was calculated using the following formula: Titer (TU/mL) = (N × P)/(V × D), where N = number cells per well, P = percent fluorescent positive cells, V = volume (μL) virus per well, D = fold dilution.

Human primary T cell isolation

Healthy donor leukopaks were purchased from PPA Research Group (pparesearch.com) and Miltenyi Biotec. Fresh peripheral blood mononuclear cells (PBMCs) were isolated by low-density centrifugation on Lymphoprep (Stem Cell Technology) according to the manufacturer’s instructions. CD3+ T cells were purified by negative selection using the Pan T Cell Isolation Kit Dynabeads Untouched Human T Cells (Invitrogen). In some experiments, T cells were enriched by CD4/CD8 positive selection using the StraightFrom® Leukopak® CD4/CD8 MicroBead kit, human (Miltenyi Biotec) and MultiMACS™ Cell24 Separator Plus (Miltenyi Biotec) according to the manufacturer’s instructions.

Lentiviral transduction of T cells

On day 0, cryopreserved Pan T cells or CD4+/CD8+ T cells were thawed and activated in 24-well plates pre-coated with anti-human CD3 antibody (Clone OKT3, 1 µg/ml, BioLegend) and anti-human CD28 antibody (Clone CD28.2, 1 µg/ml, BioLegend). T cell culture medium was composed of RPMI 1640 (Gibco), 10% Human Serum AB (GeminiBio), 2 mM GlutaMAX (Gibco), 50 µM 2-Mercaptoethanol (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 10 ng/ml recombinant human IL-7 (Gibco) and 10 ng/ml IL-15 (Gibco). On day 1, LdCK-GFP lentiviral vector and transduction enhancer TransPlus (Alstem) was added to T cell culture (total volume: 0.5 ml/well) with or without BX795 (final concentration 4 µM). Six hours after transduction, 1 ml fresh medium was supplemented to each well to support cell growth for overnight. On day 2, 1 ml supernatant was removed from each well. CAR-TEV-mCherry lentiviral vector and transduction enhancer TransPlus (Alstem) was added to T cell culture (total volume: 0.5 ml/well) with or without BX795 (final concentration 4 µM). Six hours after transduction, 1 ml fresh medium was supplemented to each well. On day 5, medium was changed, lentiviral vectors and all supplementary were washed out. Meanwhile, T cell stimulation was withdrawn via transferring cells into new plates. On day 6, GFP + and mCherry + double-transduced T cells were enriched through cell sorting (Sony SH800 Cell Sorter). The enriched cells were expanded until day 14 or day 21.

T cell killing assay

The cytotoxic capacities of the final products were evaluated in vitro against Fadu/PD-L1 tumor cells. In short, a total of 8 × 104 Fadu/PD-L1 target cells in 400 µL medium (DMEM supplemented with 10% FBS and 1% Penicillin–Streptomycin) was placed in triplicates into 48-well plates and incubated overnight for attachment. The following day, 400 µL of effector cells were added to obtain effector-to-target (E:T) ratios at 1:5 or 1:20. The amount of effector cells was calculated as CAR positive cells. 24 h after co-culture, supernatants were harvested to measure the secretion of IL-2, TNFα, and IFN-ɣ by following the directions of ELISA kits (DuoSet ELISA Development System, R&D Systems). Cells were collected for flow cytometry assay in the following 3 days.

Flow cytometry

Cells were washed once with FACS buffer composed of 1x DPBS without Ca2+ and Mg2+ (Invitrogen) and 2% heat-inactivated FBS(Corning), then incubated with appropriate amounts of antibody at 4 °C for 30 min, protected from light. The labeled cells were then washed and re-suspended in FACS buffer. For cell-mediated cytotoxicity assay, CountBright™ absolute counting beads (Invitrogen) were added and mixed with cells prior to data acquisition. Data acquisition was performed on CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed through Kaluza software (Beckman Coulter). LdCK-GFP and CAR-TEV-mCherry expression was determined by fluorescent labels. In some experiment, LdCK and MSLN-CAR-TEV without fluorescent labels were used, their expression was assessed through detecting their tags with antibodies Q8-PE (clone QBEND/10, ThermoFisher) and NGFR-FITC (clone ME20.4, BioLegend). The antibodies used for T cell phenoty** were CD3-APC (clone SK7), hCD45-AF700 (clone HI30), CD4-PerCP (clone SK3), CD8-BV510 (clone SK1), CD27-PE-Cy7 (clone M-T271), CD28-BV605 (clone CD28.2), and CD279/PD1-BV421 (clone EH12.2H7) from BioLegend. The antibodies used for cell-mediated cytotoxicity assay were CD274/PDL1-PE-Cy7 (clone MIH1, Invitrogen), CD340/HER2-BV605 (clone 24D2, BD Biosciences), CD3-APC (clone SK7), hCD45-AF700 (clone HI30), CD4-PerCP (clone SK3), CD8-BV510 (clone SK1), and CD279/PD1-BV421 (clone EH12.2H7) from BioLegend. Live/dead discrimination was determined using LIVE/DEAD® Fixable Near-IR Dead Cell Stain (Invitrogen).

Statistical analysis

For preparation of graphs and statistical analysis, Microsoft Excel and GraphPad Prism8 were used. P < 0.05 was considered statistically significant. Significance of findings was defined as: ns = not significant; *, P < 0.05; **, P < 0.01.

Results

Establishment of human primary T cell transduction procedure for RB-340-1F

RB-340-1 is a combinatorial CAR/CRISPRi circuit delivered into T cells by two lentiviral constructs, LdCK and CAR-TEV (Fig. 1a). LdCK encodes linker for activation of T cells (LAT) complexed to dCas9-Krab via a TEV-cleavable sequence (TCS). CAR-TEV encodes an anti-HER2 (4D5) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a short guide RNA (PD1sg) targeting the transcription start site (TSS) of the PD-1 gene. The integrated provirus sequence between two LTRs is around 10 kb for LdCK, and is around 8 kb for CAR-TEV. In this study, green fluorescent protein and m-cherry were used as reporters in LdCK and CAR-TEV, respectively. To distinguish the platform using fluorescent labels from RB-340-1 without fluorescent labels, we name it as RB-340-1F.

Fig. 1
figure 1

RB-340-1F two-vector platform and T cell transduction workflow. a Schematic representation of the first lentiviral vector LdCK-GFP and the second lentiviral vector CAR-TEV-mCherry. b The percentages of cells expressing the activation markers CD69, CD25, and cell surface protein LDLR. T cells were stimulated in anti-CD3/anti-CD28 pre-coated plate (CD3/CD28 activation). T cells cultured in non-coated plate were used as control (no activation). Results are the averages of two donors. c T cell transduction workflow and the time window to add BX795. The final concentration of BX795 in cell culture is 4 µM

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Human primary T cells need to be activated before transduction [19, 29,30,31]. To identify the proper timing for transduction, we documented the status of activation of primary T cells by testing the level of expression of canonical activation markers following stimulation with anti-CD3 plus anti-CD28 (Fig. 1b). CD69 is an early T cell activation marker [32] and was detectable within hours of stimulation. CD25 is the alpha chain of the IL-2 receptor [33] and became up-regulated 18 h after stimulation. Low-density lipoprotein-receptor (LDLR), the major entry port of VSV-G pseudo-typed lentiviral vectors in human cells [34] was also up-regulated 18 h after stimulation. Thus, we selected 18 h as the earliest time point to expose lentivirus to T cells. Lentiviral transduction was tried at different time windows from 18 h to 72 h. Several other factors that affect lentiviral transduction, including virus multiplicity of infection (MOI) and application of transduction enhancers like Transplus, were also investigated. Based on these preliminary tests, we generated an optimized transduction protocol for our two-vector and large payload RB-340-1F platform (Fig. 1c).

In this protocol, T cells were stimulated in anti-CD3 and anti-CD28 pre-coated plates in T cell culture medium supplemented with recombinant human IL-7 and IL-15. LdCK vector was added to the T cell culture on day 1 (18–24 h) (MOI = 10), CAR-TEV vector was added on day 2 (MOI = 20). Lentiviral vectors were washed out on day 5. Meanwhile, T cell stimulation was withdrawn via transferring cells to non-coated plates. To enrich double transduced cells, GFP and mCherry double positive cells were sorted on day 6. These double positive cells were cultured and expanded until day 14 or day 21. With this protocol, we achieved around 4%–9% double transduced cells on day 6, depending on the donors (data not shown).

RB-340-1F lentiviral transduction of human primary T cells is promoted by BX795 treatment

To promote RB-340-1F T cell transduction efficiency, we tested different strategies, including BX795 treatment. BX795 was added to T cells along with lentiviral vectors and the other transduction enhancer Transplus. The final concentration of BX795 in cell culture is 4 µM. Six hours after transduction, BX795 concentration is diluted to 1.3 µM due to the supplement of more fresh medium to cells. For RB-340-1F, BX795 was added twice along with two lentiviral vectors on day 1 and day 2 (see “Methods”, Fig. 1c). On day 5, BX795 was washed out. No more BX795 treatment was needed after cell transduction.

We tested BX795 treatment with T cells from four donors. The dot plots in Fig. 2a display transgene expression assessed on day 6 by flow cytometry from one representative donor. Figure 2a shows that BX795 treatment promoted both LdCK-GFP and CAR-TEV-mCherry transduction rate dramatically. In the four donors, without BX795 treatment, LdCK-GFP transduction rate is around 12.4%–18.1%, CAR-TEV-mCherry transduction rate is around 17.3%–27.5%. With BX795 treatment, LdCK-GFP transduction rate was increased to 24.0%–31.2%, CAR-TEV-mCherry was increased to 28.9%–43.7%. The higher transduction rate by the second vector CAR-TEV-mCherry might be caused by higher MOI and shorter transgene sequence of the second vector. Collected and averaged data from four donors are shown in Fig. 2b. The total transduced T cells, including GFP +/mCherry-, GFP +/mCherry + , and GFP-/mCherry + cells, increased from 31.5% to 47.78%. We observed a 1.45-fold increase for GFP single positive population (GFP +/mCherry-), 1.28-fold increase for mCherry single positive population (GFP-/mCherry +), and 2.28-fold increase for the double positive population (GFP +/mCherry +), which represent the complete RB-340-1F product to be enriched for further expansion. Not only was there a significant increase in the percentage of transduced T cells, but median fluorescence intensity (MFI) of transduced cells was also statistically significantly increased by BX795 treatment (Fig. 2c, d). Fourteen days after cell sorting, GFP and mCherry double positive cells were analyzed by PCR for vector copy number (VCN). We found that VCN was increased by 1.38-fold with BX795 treatment, which indicates that the higher transgene expression was caused by more gene integration.

Fig. 2
figure 2

Effect of BX795 treatment on RB-340-1F lentiviral vectors transduction efficiency. a Transgene expression was assessed by flow cytometry on day 6. b T cell transduction rate with the absence of BX795 (BX795-) or presence of BX795 (BX795+). GFP+/mCherry-: GFP single positive population. GFP+/mCherry + : GFP and mCherry double positive population. GFP-/mCherry+ : mCherry single positive population. c MFI of mCherry in CAR-TEV-mCherry transduced cells. d MFI of FITC in LdCK-GFP transduced cells. All results are the averages of four donors, except for figure A, which is flow cytometry plot from a representative donor. *p < 0.05; **p < 0.01

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We sustainably saw transduction promotion with BX795 treatment in different experiments. The effect of BX795 was also observed when we worked with the other Refuge CAR-T platforms. These results clearly demonstrate that BX795 treatment enhances RB-340-1F and the other large payload lentiviral transduction of human primary T cells.

BX795 treatment changes CD4+ :CD8+ compositions in RB-340-1F double transduced T cells

To further dissect the effect of BX795 on transduced T cells, we performed T cell phenoty** analysis on day 6 before cell sorting. We found that BX795 treatment did not obviously change CD4+ :CD8+ compositions in the bulk population (the mixture of transduced and non-transduced cells) on day 6 (Fig. 3a). When gating on CD4+ cells or CD8 + cells, we saw transduction promotion in both populations. In the CD4+ population, the percentage of double transduced cells was increased from 8.22% to 17.56%, which is 2.2-fold change. In CD8 + population, the percentage of double transduced cells was increased from 1.65% to 5.94%, which is 3.7-fold (Fig. 3b). The higher fold change in CD8 + cells was consistently observed in all our experiments, and was also observed when BX795 was applied to the other Refuge CAR-T platforms (data not shown). These results indicate that BX795 might promote lentiviral transduction better in CD8+ cells, or transduced CD8+ cells grow faster with BX795 treatment. Since we did not see higher CD8+ ratio in bulk population on day 6 (Fig. 3a), we speculate that BX795 does not promote CD8 + cell growth. The mechanism underlying the different promotion extent between CD4+ cells and CD8+ cells remains not clear.

Fig. 3
figure 3

Effect of BX795 treatment on RB-340-1F transduction of CD4+ T cells and CD8+ T cells. a Percentage of CD4+ cells and percentage of CD8+ cells in the bulk population on day 6 before cell sorting. b Percentage of double transduced cells in CD4+ or CD8+ cells on day 6. BX795-: no BX795 treatment. BX795+ : with BX795 treatment. The above results are the average of four donors. c Percentage of CD4+ cells in double transduced cells on day 6 and day 13. d Percentage of CD8+ cells in double transduced cells on day 6 and day 13. BX795-: no BX795 treatment. BX795+ : with BX795 treatment. The above results are the average of three donors. *p < 0.05; **p < 0.01

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When gating on double transduced T cells, we saw higher CD8+ ratios in the BX795 treated group comparing to the group without BX795 treatment (Fig. 3d). The difference is enlarged along with T cell expansion after cell sorting. One week after cell sorting (day 13), the percentage of CD8+ cells in BX795 treated group became 23.90%, which is much higher than 13.84% of CD8+ ratio in the group without BX795 treatment (Fig. 3d). Meanwhile, the percentage of CD4 + cells in double transduced cells dropped from 85.39% to 75.28% (Fig. 3c). Although we are not sure if BX795 treatment affects lentiviral transduction of CD4+ cells and CD8+ cells in different extent or affects growth rate of transduced CD8+ cells, there is no doubt that BX795 treatment increased CD8+ ratio in RB-340-1F double transduced cells.

BX795 treatment does not dramatically affect transduced T cell expansion and designated CAR-T cell function

The previous study in NK cells reported that BX795 treatment did not cause immediate toxic effects on NK cells up to 10 µM [28]. In this study, we used an even lower concentration, 4 µM BX795, in all the experiments, and did not observe obvious cell death during or after BX795 treatment. The viability of BX795-treated cells was around 90% and did not show difference compared with non-treated cells. We recorded T cell growth rate at various time points (day 3, day 6, day 10), and found that cell expansion rate in the BX795 treated group is comparable to that in the non-BX795 treated group (Fig. 4a). Cell surface markers CD27 and CD28 are frequently used to define stages of T cell differentiation and are related with T cell activation and expansion [35,36,38]. It was subsequently found to specifically block the TBK1 and IKKɛ complex [27]. It is well known that TBK1 and IKKɛ regulate the production of type 1 IFNs during viral infection [22, 23]. Moreover, type 1 IFNs have been reported to influence the fate of CD4+ and CD8+ T cells during the initial phase of antigen recognition, and are also required for CD8+ T cell expansion and memory formation during lymphocytic choriomeningitis (LCMV) infection contributing to the formation of CD8+ T cell memory and effector function in response to vesicular stomatitis virus infection [39,40,41]. Although the effect of type 1 IFNs on T cells in the above situations may not be exactly the same as in conditions related to lentiviral transduction of human primary T cells, it should be recognized that blocking TBK1 and IKKɛ signaling via BX795 may alter T cell phenotype and function. Further characterization of signaling pathways involved in this response is needed to incorporate BX795 into T cell product manufacturing.

Conclusions

This study demonstrates that BX795 treatment promotes lentiviral transduction of combinatorial CAR/CRISPRi circuit RB-340-1F into human primary T cell, with the averages at 2.3-fold change. After BX795 treatment, the percentage of CD8+ cells in RB-340-1F double transduced T cells was increased, while cell growth and CAR-T cell function were not dramatically affected. This observation indicates that the use of BX795 is an effective and safe approach to promote RB-340-1F T cell production. Moreover, single vector transduction can also benefit from using of BX795 by overcoming the transduction efficiency plateau often observed in human primary T cells. This approach might have broader use for other T cell or other immune cell therapy products.