V体育官网入口 - The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR-T cells

Dasatinib locks resting CD8⁺ and CD4⁺ CAR-T cells into a function-off state. (VSports手机版)

To evaluate the effect of dasatinib on CAR-T cell function, we first used a CD19-CAR with 4-1BB co-stimulation (CD19-CAR/4-1BB, fig. S1A) that conferred high rates of complete remissions in recent clinical trials (3). We enriched CAR-expressing T cells to ≥90 % purity (Fig. 1A) and performed co-culture assays with CD19-positive target cells (K562/CD19) in the presence or absence of dasatinib. With CD8+ CAR-T cells, complete blockade of target cell lysis was accomplished when dasatinib was used at a concentration of 100 nM (Fig. 1B, fig. S1B), which can be readily obtained in human serum (13). Treatment with 100 nM dasatinib also conferred complete inhibition of interferon γ (IFNγ) and interleukin 2 (IL-2) secretion (Fig. 1, C and D, fig. S1C) and CAR-T cell proliferation (Fig. 1E). The blockade was sustained for several hours after a single dasatinib administration at the time of assay set-up and accordingly, we did not observe induction of the activation markers CD25 and CD69 during the assay period of 24 hours (Fig VSports. 1F and fig. S1D). We observed partial inhibition of CAR-T cell cytolytic activity, cytokine secretion, and proliferation when dasatinib was used at concentrations ≤ 25 nM (Fig. 1, B-E).

CAR-T cell products in clinical application commonly contain CD8+ cytotoxic and CD4+ helper T cells. We confirmed that dasatinib was also effective with CD4+ CD19-CAR/4-1BB T cells (fig. S2) and found that 100 nM dasatinib completely abrogated the production of cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), IFNγ, and IL-2 after stimulation with target cells (fig V体育官网. S2D). Collectively, these data show that dasatinib can block the activation of resting CD8+ and CD4+ CAR-T cells after stimulation with target cells. The dose of dasatinib can be titrated to achieve partial and complete inhibition of CAR-T cell function.

Dasatinib is superior to dexamethasone in inhibiting CAR-T cell function.

We compared the inhibitory effect of dasatinib with that of dexamethasone, which is commonly used with the intention of exerting control over CAR-T cells (5). First, we analyzed the cytolytic activity of CD8⁺ CAR-T cells (CD19-CAR/4-1BB) in the presence of dexamethasone. When dexamethasone was added at assay set-up, there was no inhibitory effect (fig. S3A). However, after a 24-hour pre-treatment with dexamethasone, we observed partial inhibition of cytolytic activity (fig. S3A). Neither simultaneous treatment nor pre-treatment of CAR-T cells with dexamethasone resulted in reduced IFNγ secretion (fig. S3B), whereas IL-2 secretion was reduced in both settings (fig. S3B). These data correlated with reduced proliferation of CAR-T cells after exposure to dexamethasone compared to untreated CAR-T cells (fig. S3C). In contrast, treatment with dasatinib mediated complete inhibition of cytolytic activity, cytokine secretion and proliferation (fig. S3). Together, these data show that dasatinib is superior to dexamethasone in inhibiting CAR-T cell function. Dasatinib is able to confer immediate-onset and complete control over CAR-T cell function, whereas the inhibitory effect of dexamethasone is of delayed onset and incomplete, even when dexamethasone is used at very high doses of 100 μM.

Dasatinib prevents LCK phosphorylation and NFAT induction after CAR engagement. (VSports最新版本)

We sought to elucidate the molecular mechanism behind the inhibitory effect of dasatinib on CAR-T cell function. We performed western blots to determine the phosphorylation state of LCK (Y394), CAR-CD3ζ (Y142), and ZAP70 (Y319). In the presence of dasatinib, phosphorylation of all three proteins was inhibited (Fig. 2A and fig. S4). Quantitative analyses revealed that phosphorylation relative to non-dasatinib-treated CAR-T cells was reduced to 22 % (LCK), 13 % (CD3ζ), and 12 % (ZAP70) (Fig. 2B).

We hypothesized that inhibition of CAR signaling by dasatinib would prevent induction of NFAT, a key transcription factor in activated CAR-T cells (10). Therefore, we co-transduced CAR-T cells with a reporter gene encoding a short-lived green fluorescent protein (GFP) mutant variant under control of an NFAT-inducible promotor (NFAT/GFP; fig. S5). Subsequently, we challenged CD8⁺ and CD4⁺ CAR-T cells with CD19⁺ Raji lymphoma cells and found that induction of the GFP reporter signal was indeed significantly reduced after dasatinib treatment [Fig. 2C; P=0.0495 (CD8⁺) and P=0.0006 (CD4⁺)].

These data suggested that dasatinib may be broadly effective with CAR constructs that contain a CD3ζ signaling module. Accordingly, we confirmed that dasatinib was also able to inhibit the effector functions of CD8⁺ and CD4⁺ T cells expressing a CD19-CAR with CD28 co-stimulation (CD19-CAR/CD28) (fig. S6 and fig. S7), as well as receptor tyrosine kinase like orphan receptor 1 (ROR1)-specific (fig. S8) and signaling lymphocytic activation molecule family member 7 (SLAMF7)-specific CARs (fig. S9). We found that the inhibitory effect of dasatinib was equally potent with CAR constructs that contained a CD28_CD3ζ vs. 4-1BB_CD3ζ signaling domain and identified 40 nM of dasatinib as the benchmark dose at which complete inhibition of CAR-T cell function was accomplished with each CAR construct (figs. S1, S2, S6-9). Together, these data show that dasatinib reduces the phosphorylation of LCK, CD3ζ, and ZAP70, resulting in limited induction of NFAT. This mechanism of action suggests that dasatinib is broadly applicable as a pharmacologic function-off switch for CAR constructs that use this signaling machinery.

The dasatinib-induced function-off state in CAR-T cells is not compromised by TCR stimulation.

The potent inhibitory effect of dasatinib on CD3ζ phosphorylation suggested that the function-off state in CAR-T cells could be sustained even when in trans stimulation was provided through the endogenous TCR. We generated polyclonal CD8⁺ CAR-T cell lines (CD19-CAR/4-1BB) that recognized the cytomegalovirus (CMV) pp65/HLA-A2 NLVPMVATV (NLV) epitope through their endogenous TCR. We confirmed co-expression of CD19-CAR and CMV-specific TCR by flow cytometry (fig. S10A). Then, we stimulated T cells with K562 cells that expressed HLA-A2 either alone (pulsed with pp65 NLV peptide) or in combination with CD19 and assessed function over time. In both cases, dasatinib completely blocked T cell activation and effector function, even when CD19-CAR and CMV-specific TCR were engaged simultaneously (fig. S10, B-E). Together, these data show that the dasatinib-induced function-off in CAR-T cells is not compromised by stimulation through the endogenous TCR.

Dasatinib pauses activated CAR-T cells in a function-off state.

We were interested in determining whether dasatinib was able to inhibit CAR-T cells that are already in an activated state, meaning in the process of executing their effector functions. To assess this, we activated CAR-T cells with antigen-positive target cells to turn them on, and then added dasatinib to turn them off. CD8⁺ CD19-CAR/4-1BB T cells rapidly started lysing target cells in the function-on phase. However, as soon as dasatinib was added to the culture (1 hour after assay set-up), target cell lysis stagnated, indicating that CAR-T cell function was switched off (Fig. 3A). Similarly, addition of dasatinib to activated CD8⁺ CAR-T cells interfered with subsequent cytokine secretion and proliferation (Fig. 3, B and C). Consistent with the mechanism of action revealed above, the inhibitory effect of dasatinib was less strong when CAR-T cells had progressed beyond a certain point of activation. If added later than 1 hour after turning CAR-T cells on, the inhibitory effect of dasatinib on subsequent proliferation was low (Fig. 3C). We reasoned that activated CAR-T cells that were not inhibited by dasatinib during a primary encounter with target cells would eventually be captured upon a sequential encounter with target cells. Accordingly, we performed a sequential stimulation study where Raji lymphoma cells were added every 24 hours to CD8⁺ and CD4⁺ CD19-CAR T cells that co-expressed the NFAT/GFP reporter gene. The data show that dasatinib was able to block T cell activation upon secondary and tertiary stimulation with target cells (fig. S11). In summary, these data demonstrate that dasatinib can pause activated CAR-T cells and induces a function-off state that is stable even when CAR-T cells encounter target cells sequentially.

Removal of dasatinib rapidly releases CAR-T cells from their function-off state.

Next, we were curious whether the function-off state could be reversed and CAR-T cells be shifted back into a function-on state through removal of dasatinib. We co-incubated CD19-CAR/4-1BB T cells with target cells in the presence of dasatinib (function-off) and did not observe cytolytic activity, whereas in parallel cultures without dasatinib, target cell lysis started immediately (Fig. 4A). After 2 hours, dasatinib was removed from the co-cultures through a thorough medium change. The data show that after dasatinib removal, CAR-T cells rapidly went into a function-on state and commenced target cell lysis. Within 2 hours of dasatinib removal, CAR-T cells had achieved half of the specific lysis, and within 7 hours had achieved the same extent of specific lysis as untreated CAR-T cells.

We were interested in determining how long the function-off state could be maintained in CAR-T cells. We found that complete blockade of CAR-T cell function was sustained after 1 day and also after 7 days of continuous exposure to dasatinib, with no signs of the inhibitory effect wearing off (Fig. 4, B-E). Even after a 7-day exposure to dasatinib, removal of the drug reignited CAR-T cell function, as evidenced by specific high-level cytolytic activity, IFNγ, and IL-2 secretion and productive proliferation, which occurred with similar kinetics as in CAR-T cells that had never been exposed to dasatinib (Fig. 4, B-E). We determined the percentage of live, early apoptotic, and late apoptotic T cells at several time points during and after the 7-day exposure to dasatinib and did not observe a negative effect of dasatinib treatment on CAR-T cell viability (Fig. 4F). In summary, these data show that dasatinib induces a function-off state in CAR-T cells that can be sustained for several days without affecting CAR-T cell viability and rapidly reversed into a function-on state after removal of the drug.

VSports最新版本 - Dasatinib pauses activated CAR-T cells in a function-off state in vivo.

To obtain proof of concept for dasatinib as a pharmacologic on/off switch for CAR-T cells in vivo, we used a lymphoma xenograft model in immunodeficient NOD.Cg-Prkdc^scid IL-2rg^tm1Wjl/SzJ (NSG) mice. Mice were inoculated with FFLUC/GFP-expressing Raji lymphoma cells on day -7 and received CD19-CAR/4-1BB modified or control T cells on day 0. In this set of experiments, we administered dasatinib between day 3 and day 5 after T cell transfer to create a ‘function-on-off-on’ sequence (Fig. 5A). In the first phase (day 0 to day 3), CD19-CAR T cells commenced exerting their anti-lymphoma activity and were strongly activated, as demonstrated by bioluminescence imaging (BLI) (Fig. 5, B-D) and serum cytokine analysis (function-on) (Fig. 5E). In the second phase after CAR-T cell transfer (day 3 to day 5), we administered dasatinib to a subgroup of mice. Dasatinib rapidly induced a function-off state and halted anti-lymphoma reactivity, as evidenced by increasing BLI signal (Fig. 5C) and decreasing IFNγ in serum from each of the mice in this subgroup (fig. S12). In contrast, the BLI signal did not increase and IFNγ remained stable during this phase in mice that had received CD19-CAR T cells but no dasatinib. In the third phase (after day 5), administration of dasatinib was discontinued to allow CAR-T cells to revert back into their function-on state. Indeed, CAR-T cells rapidly resumed their anti-lymphoma function as revealed by decreasing BLI signal (Fig. 5C) and increasing serum IFNγ in each of the mice (fig. S12). Similar data were obtained in analogous experiments with T cells expressing a CD19-CAR with CD28 co-stimulation (Fig. 5F).

We analyzed peripheral blood at several time points throughout the experiment, and on day 7, found a lower frequency of CAR-T cells in the subgroup of mice that had received dasatinib, suggesting their proliferation had been effectively controlled during the function-off phase (Fig. 5G). However, with subsequent follow-up, we found a potent anti-lymphoma effect in each of the mice that had gone through the ‘function-on-off-on’ sequence, demonstrating that the inhibitory effect of dasatinib was indeed reversible and that the therapeutic effect of CD19-CAR T cells had not been compromised by this intervention (Fig. 5C).

Collectively, these data demonstrate that dasatinib can be used as a pharmacologic control switch to steer CAR-T cells in vivo. The data show that administration of dasatinib for a short-term interval does not compromise the anti-lymphoma efficacy of CD19-CAR T cells in this in vivo model and confirm the favorable pharmacodynamic attributes of dasatinib, including rapid onset and complete reversion of the inhibitory effect that we established in our in vitro experiments.

Dasatinib can be used as an emergency drug to prevent cytokine release syndrome.

We sought to determine whether dasatinib is suitable as an emergency drug to prevent fatal CRS in a murine model we recently reported (7). In this model, C.B.Igh-1b/GbmsTac-Prkdc^scidLyst^bg N7 (SCID/beige) mice are engrafted with Raji lymphoma cells and then treated with CD19-CAR/CD28 T cells, which causes rapid-onset CRS with a high rate of mortality within the first 48 hours (Fig. 6A). In this model, we created a function-on-off-on sequence, and due to the aggressive nature of CRS, commenced administering dasatinib at 3 hours after CAR-T cell transfer for a 30-hour period. We measured human serum cytokines at 8 hours after CAR-T cell transfer and found significantly lower concentrations of IFNγ (P<0.0001), IL-2 (P=0.0002), GM-CSF (P<0.0001), and tumor necrosis factor α (TNFα) (P=0.0006) in each of the mice that had received dasatinib compared to mice that had not (Fig. 6B). We repeated this analysis at 16 hours after CAR-T cell transfer and detected no increase in serum cytokines, demonstrating that cytokine production from CAR-T cells was under complete control (Fig. 6B). We also found lower concentrations of murine IL-6 that is produced by endogenous innate immune cells as part of CRS pathophysiology (Fig. 6C). During acute CRS in the first 48 hours after CAR-T cell transfer, we observed a high rate of mortality, with only 25% survival in the group of mice that had not received dasatinib (Fig. 6D). By contrast, in the dasatinib treatment group, 70% of mice were alive at the 48-hour interim analysis. With subsequent follow-up until 168 hours, survival rates remained stable in both groups (Fig. 6D).

Collectively, these data show that a short treatment course with dasatinib can prevent fatal CRS after CAR-T cell transfer in mice. These data highlight the potential of dasatinib as a pharmacologic on/off switch for CAR-T cells to steer their function and to reduce toxicity in clinical settings.

"V体育官网" Study Design

In vitro experiments and experiments in NSG mice were done three times with T cells from different healthy donors. Experiments in SCID/beige mice were done once. In mouse experiments, the group size was determined by biostatistical calculation using GPOWER based on expected effect size, which is the mean difference in standard deviation (SD) units detectable with 80 % power at a 2-sided 0.05 level of significance with 2-sample t-test, and the SD fraction, which is SD as a fraction of biologically meaningful difference. Mice were randomly allocated to treatment groups before tumor injection (NSG model) or before T cell infusion (SCID/beige model). Data analysis was based on objectively measurable data; the investigators were not blinded to group allocation during data collection and analysis.

Human subjects

Blood samples were obtained from healthy donors who provided written informed consent to participate in research protocols approved by the Institutional Review Board of the University of Würzburg [Universitätsklinikum Würzburg (UKW)], or were purchased from the New York Blood Center (IRB-exempted) and handled following all required ethical and safety procedures set forth by the Memorial Sloan Kettering Cancer Center (MSKCC). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over Ficoll-Hypaque (Sigma).

VSports app下载 - Experiments in NSG mice

The UKW Institutional Animal Care and Use Committee approved all experiments in NSG mice. NSG mice (female, 6 to 8 weeks old) were purchased from Charles River. Mice were inoculated with 1×10⁶ FFLUC/GFP-expressing Raji cells via i.v. injections (tail vein). Native Raji cells were obtained from the German collection of microorganisms and cellcultures (DSMZ) and transduced with FFLUC/GFP encoding lentivirus. Tumor burden and distribution were analyzed by serial BLI on an IVIS Lumina imager (Perkin Elmer): mice received 0.3 mg/g luciferin i.p. and images were acquired 10 minutes after luciferin injection in small binning mode at an acquisition time of 1 second to 1 minute to obtain unsaturated images. Data were analyzed using LivingImage Software (Caliper), and the average radiance was analyzed in regions of interest that encompassed the entire body of each individual mouse. Mice were treated with 5×10⁶ CAR-modified or control untransduced T cells (CD4:CD8 ratio = 1:1) i.v. after 7 days of tumor engraftment. Dasatinib was administered i.p. at a dose of 10 mg/kg. The presence of human cytokines in serum was measured using multiplex cytokine analysis (Thermo Fisher Scientific).

Experiments in SCID/beige mice

The MSKCC Institutional Animal Care and Use Committee approved all experiments in SCID/beige mice. Experiments were done as previously reported (7). In short, primary human T cells were purified from buffy coats by negative bead selection (Pan T cell Isolation Kit, Miltenyi) and activated with CD3/CD28 CTS Dynabeads (Thermo Fisher Scientific) at a ratio of one bead per cell. T cells were cultured in XVIVO15 (Lonza) supplemented with 5% human serum (Gemini), 10 mM HEPES (Invitrogen), 2 mM GlutaMAX (Invitrogen), 1x MEM vitamin solution (Invitrogen), and 1 mM sodium pyruvate (Invitrogen). Additionally, the medium was supplemented with 50 IU/ml penicillin-streptomycin (Invitrogen) and 60 IU/ml recombinant IL-2 (Novartis). SCID/beige mice (Taconic, female, 6 to 8 weeks old) were inoculated with 3 x 10⁶ FFLUC/GFP-expressing Raji tumor cells [American Type Culture Collection (ATCC)] via i.p. injection, and tumors were left to grow for 20 days. Tumor burden was evaluated by BLI using IVIS Imaging System (Perkin Elmer) and LivingImage Software (Caliper) 2 days before CAR-T cell transfer. Mice were treated with 3 × 10⁷ CD19-CAR/CD28_ζ (7) CAR-modified T cells via p. injection. Dasatinib was administered i.p. in 5 doses (every 6±1 hour) at 10 mg/kg starting 3 hours after CAR-T cell injection. Blood was collected from mice by tail clipping. Serum cytokines were measured by cytometric bead array (BD).

Statistical analysis

Data from in vitro experiments were analyzed and plotted with Prism (GraphPad). Shown is the mean value + SD unless indicated otherwise. Specific lysis was analyzed using two-way ANOVA with Sidak’s multiple comparison test. Cytokine secretion and proliferation was analyzed using ordinary one-way ANOVA with Holm-Sidak’s multiple comparison test or Kruskal-Wallis test with Dunn’s multiple comparison test as indicated in the figure legends. Data on quantitative western blots were analyzed by Kruskal-Wallis test with Dunn’s multiple comparison test. In NFAT/GFP reporter experiments, GFP expression was analyzed by one-way ANOVA with Holm-Sidak’s multiple comparison test. In NSG mouse experiments, data sets were analyzed by Mann-Whitney test (tumor progression/regression) and two-way ANOVA with Sidak’s multiple comparison test (cytokine secretion, flow cytometry). In SCID/beige experiments, data sets were analyzed by unpaired t-test (cytokines) and Mantel-Cox test (survival). Original data are included in data file S1.