A TIP CAR efficiently associates within cells and dissociates upon addition of small molecule
TIP is a 16 amino acid peptide mimic of tetracycline which binds to the tetracycline repressor protein B (TetRB) with lower affinity than tetracycline18,19. We hypothesized that a split CAR incorporating TIP/TetRB could allow tetracycline-inducible dissociation of the signaling component from the antigen recognition component [Fig. 1a]. As an initial exploration, we generated an anti-CD19 CAR ectodomain incorporating the FMC63 single chain variable fragment (scFv)20 with a TetRB endodomain, co-expressed with a separate cytoplasmic TIP-eGFP fusion [Fig. 1b, and Supplementary Fig. S1(a)]. As controls, a similar split construct was generated lacking TIP as well as a “monolithic” CAR with an eGFP endodomain. Microscopy revealed that eGFP localized to the cell membrane in both the monolithic CAR and the split CAR, but not in the split CAR which lacked TIP. In the presence of 100 nM minocycline, the TIP-eGFP was displaced from the membrane and eGFP signal was dispersed throughout the cytoplasm [Fig. 1c]. These data suggested that the TIP/TetRB system could be used for inducible translocation of signaling domains from the cell membrane.
A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. (a) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. (b) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. (c) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. (d) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of SupT1 cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). (f) IFN-γ or (g) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).
TetRB/TIP CAR can be controlled by minocycline but has impaired maximal function
To functionally test the split TetRB CAR (‘TetCAR’) approach, the eGFP domains were replaced with 41BB-CD3ζ [Fig. 1d and Supplementary Fig. S1(b)]. Transduced PBMCs were co-cultured for 24 h with SupT1 cells expressing CD19-eGFP in the presence or absence of minocycline. Both standard “monolithic” CAR and the 41BB-CD3ζ TetCAR T cells efficiently lysed SupT1-CD19-GFP targets [Fig. 1e and Supplementary Fig. S1(c) and (d)]. However, whilst addition of minocycline had no effect on the control CAR, cytotoxicity was impaired after addition of minocycline to the TetCAR. There was no significant difference in cytotoxicity between the inhibited TetCAR and the TetCAR control that lacked the TIP sequence, suggesting 100 nM minocycline was sufficient to fully dissociate the TetCAR signaling domains. Secretion of IFN-γ and IL-2 was also quantified after co-culture [Fig. 1f,g]. In line with the cytotoxicity data, addition of minocycline had no effect on cytokine secretion by the control CAR, but significantly reduced IFN-γ secretion by the TetCAR. In the absence of Minocycline however, TetCAR secreted considerably less IFN-γ than the control monolithic CAR and IL-2 secretion was undetectable. These data show that addition of minocycline could inhibit the activation of the TetCAR; however, activity of the uninhibited TetCAR was less than that of a monolithic CAR.
Fab format and TetRB-attached CD28 co-stimulation improve TetCAR activity
To enhance maximal performance of TetCAR, several structural modifications were explored. We first tested incorporatation of the CD28 endodomain alongside different transmembrane domains or different linkers between the transmembrane domain and TetRB [Supplementary Fig. S2(a)]. These modifications had no improvement on TetCAR function [Supplementary Fig. S2(b)]. From the previous experiment, we noted that surface expression of TetCAR, as determined by recombinant CD19 binding, was lower than that of monolithic CAR [Supplementary Fig. S2(c) and (d)]. To increase the stability of TetCAR, further variants were constructed in a Fab-CAR format21 (Fab-TetCAR, outlined in Fig. 2a and Supplementary Fig. S2(e)), with either 41BB-CD3ζ or CD28-CD3ζ endodomains. Surface expression was increased in both Fab-TetCARs [Fig. 2b,c]. No differences in cytotoxicity of any variant were noted [Fig. 2d]. Inhibition of cytotoxicity was observed upon addition of minocycline in all variants, but this was only significant in the Fab-Tet-28z. Whilst Fab-TetCAR induced IFN-γ and IL-2 secretion were still lower in both variants compared with the control monolithic CAR, the Fab-TetCAR variants secreted higher levels of IFN-γ and IL-2 than their scFv-counterparts [Fig. 2e,f]. Despite the higher baseline levels of cytokine secretion in the Fab-TetCARs, addition of minocycline was still able to potently suppress cytokine secretion in both endodomain variants.
Optimization of TetCAR surface expression and signaling. (a) Schematic overview of TetCAR constructs containing 41BB-ζ or CD28-ζ endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. (b) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. (c) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis. P values = FMC63-BBz versus Fab-Tet-BBz (***, 0.0003) or Fab-Tet-28z (***, 0.0002), FMC63-Tet-BBz versus Fab-Tet-BBz (ns, 0.089), FMC63-Tet-28z versus Fab-Tet-28z (*, 0.045). (d) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) control, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Šidák’s multiple comparisons within each group ± minocycline. P values for each construct + /- minocycline were: FMC63-Tet-BBz (**, 0.0023), FMC63-Tet-28z (***, 0.0003) and Fab-Tet-28z (*, 0.0279). (e) IFN-γ and (f) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the TetCAR groups at 0 nM minocycline (with Tukey’s multiple comparisons) or within these groups ± minocycline (with Šidák’s multiple comparisons). P values were; between FMC63-Tet-BBz and Fab-Tet-BBz (**, 0.0097, IFN-γ) and between FMC63-Tet-28z and Fab-Tet-28z (****, < 0.0001, IFN-γ and **, 0.0017, IL-2). P values between the TetCAR constructs ± minocycline were: FMC63-Tet-BBz (***, 0.0007, IFN-γ), FMC63-Tet-28z (*, 0.0409, IFN-γ), Fab-Tet-BBz (****, < 0.0001, IFN-γ and *, 0.0152, IL-2) and Fab-Tet-28z (****, < 0.0001 both IFN-γ and IL-2).
Addition of a membrane-proximal non-dissociating co-stimulatory domain further enhances cytokine secretion
Whilst maximal TetCAR cytokine release was improved by the Fab format, a deficit still remained. We reasoned that a TIP-tethered co-stimulatory domain signals ineffectively and we next introduced co-stimulatory domains between the transmembrane domain and TetRB, with and without additional TIP-tethered co-stimulation (outlined in Fig. 3a). As before, despite similar transduction efficiencies, CAR surface expression was significantly reduced in the Fab-TetCAR variants compared to the monolithic CAR, although differences between the Fab-TetCARs were not significant [Fig. 3b,c]. These variants were tested as before. All TetCAR variants induced similar cytotoxicity, although minocycline induced inhibition of cytotoxicity was less pronounced and was only significant with BB-Fab-Tet-z [Fig. 3d]. However, in contrast to 41BB, introduction of membrane proximal CD28 co-stimulatory domains restored cytokine secretion to that of the control monolithic CAR [Fig. 3e,f]. Notably, minocycline completely inhibited IFN-γ and IL-2 secretion from all Fab-TetCAR T cells, including the variant with CD28 and 41BB endodomains (28BB-Fab-Tet-z). This experiment was also performed using NALM6 cells as targets. The findings were similar to SupT1-CD19, although here cytotoxicity was significantly inhibited in all Fab-TetCARs after addition of minocycline [Fig. 3g]. However, cytokine release in response to NALM6, even by 28BB-Fab-Tet-z CAR, was reduced compared to the monolithic CAR [Fig. 3h,i], likely due to lower CD19 expression in NALM6 cells [Supplementary Fig. S2f].
Reconfiguration of endodomain positions enhances TetCAR function. (a) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41BB or CD28 endodomains, with a TIP-CD3ζ or TIP-41BB-CD3ζ domains. (b) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. (c) Median fluorescent intensity of CAR expression on surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 5 donors from 2 experiments. Statistical analysis was through one-way ANOVA between the groups, p values were; between FMC63-BBz and each Fab-TetCAR (****, < 0.0001). Differences between Fab-TetCARs alone were analyzed by one-way ANOVA but were not significant. (d) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; FMC-Tet-BBz (*, 0.0119). (e) IFN-γ and (f) IL-2 release after 24 h of co-culture with SupT1-CD19 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = 28-Tet-z (*, 0.0484), 28BB-Fab-Tet-z and 28BB-Fab-Tet-BBz (****, < 0.0001). P values for IL-2 = 28-Tet-z (*, 0.0150), 28BB-Fab-Tet-z (****, < 0.0001), 28-Fab-Tet-BBz (**, 0.0029) and 28BB-Fab-Tet-BBz (***, 0.0007). (g) Killing of NALM6 after 48 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; BB-Fab-Tet-z (**, 0.0083), BB-Fab-Tet-BBz (*, 0.0189) and 28-Fab-Tet-z, 28BB-Fab-Tet-z, 28-Fab-Tet-BBz and 28BB-Fab-Tet-BBz (****, < 0.0001). (h) IFN-γ and I) IL-2 release after 48 h of co-culture with NALM6 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = BB-Fab-Tet-z (*, 0.0433), 28-Fab-Tet-z (*, 0.0387), 28BB-Fab-Tet-z (****, < 0.0001) and 28BB-Fab-Tet-BBz (**, 0.0020). P values for IL-2 = 28BB-Fab-Tet-z (*, 0.0450).
Tunable control of optimized TetCAR activity through dose-dependent, minocycline inhibition
The two most promising constructs (28-Fab-Tet-z and 28BB-Fab-Tet-z) were taken forward for more detailed characterization. To evaluate CAR inhibition over a range of minocycline concentrations, CAR T cells were cocultured at a 1:4 E:T ratio with SupT1-CD19-GFP with fourfold increasing doses of minocycline from 0.02 to 1600 nM. Here, cytotoxicity was compared relative to an inert TetCAR that could bind to CD19 but lacked any signaling capacity [constructs outlined in Supplementary Fig. S3(a) and (b)]. The inhibition of cytotoxicity to SupT1-CD19-GFP [Fig. 4a] increased with minocycline concentration up to 100 nM, at which point a plateau was reached. 28BB-Fab-Tet-z was more potently inhibited by minocycline, reaching 99% (± 31% SD) of live targets relative to the inert TetCAR, compared to 68% (± 44% SD) with 28-Tet-z. The IC50 was 4.5 nM for 28-Tet-z and 2.3 nM for 28BB-Tet-z. Both Fab-TetCARs tested also showed a similar dose-dependent reduction in both IFN-γ and IL-2 with increasing concentrations of minocycline, fully inhibiting cytokine secretion at concentrations > 6.25 nM [Fig. 4b,c]. The IC50 for 28-Fab-Tet-z and 28BB-Fab-Tet-z were 0.21 nM and 0.24 nM for IFN-γ and 0.34 nM and 0.44 nM for IL-2 secretion. In addition to IFN-γ and IL-2, secretion of a number of other effector cytokines by 28BB-Fab-Tet-z showed a similar dose-responsive decrease in response to minocycline [Supplementary Fig. S3(c)]. Lastly, inhibition of cytokine secretion by 28BB-Fab-Tet-z was also tested after addition of tetracycline and tigecycline, a glycylcycline derivative of tetracycline. Both small molecules inhibited IFN-γ and IL-2 secretion, however this required higher concentrations than minocycline (> 100 nM) and had no effect at lower doses [Supplementary Fig. S3(d) and (e)].
TetCAR activity can be fine-tuned in vitro with a dose-dependent response to minocycline. (a) Killing of SupT1-CD19-GFP after 24 h of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600 nM were added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, ± SD. n = 4 donors from 1 experiment. (b) IFN-γ and (c) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at various minocycline doses. Data shows mean ± SD, n = 4 donors from 1 experiment. (d) IL-2 secretion from FMC63-BBz or 28BB-Fab-Tet-z CARs 1–5 h after co-culture with SupT1-CD19-GFP at a 2:1 E:T ratio. 100 nM minocycline was added to separate wells every hour. Data shows the mean (± SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. Color coded bars indicate the number of hours that the co-cultures were exposed to minocycline for. n = 4 donors from 2 independent experiments. (e) Cytotoxicity or (f) IL-2 secretion by 28BB-Fab-Tet-z CARs after coculture with SupT1-CD19 at a 1:1 E:T ratio. Inhibition by minocycline was removed by washing cells with complete media at 48, 24 and 2 h before addition of SupT1-CD19 targets. Wash steps are indicated by “[W]”. Data shows mean (± SD) % of live targets relative to NT T cells (e) or mean (± SD) IL-2 secretion (f) after 24 h. n = 3 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with multiple comparisons between the 28BB-Fab-Tet-z CAR under different conditions. P values for cytotoxicity (e) were: 48 h wash versus no wash (*, 0.0118), 24 h wash versus no wash (**, 0.0050) and no drug versus no wash (**, 0.0044). P values for IL-2 secretion (f) were: 48 h wash versus no wash (**, 0.0015), no drug versus no wash (**, 0.0044), no drug versus 2 h wash (*, 0.0103) and 48 h wash versus 2 h wash (**, 0.0033).
Minocycline induces rapid and reversible inhibition of TetCAR signaling
To further examine the kinetics of minocycline-induced inhibition of TetCAR effector function, IL-2 secretion was assessed 1–5 h after co-culture with SupT1-CD19 targets. Each hour, 100 nM of minocycline was added to relevant wells [Fig. 4d]. As expected, the control CAR was unaffected by minocycline addition and induced detectable IL-2 secretion after 3 h. This was mirrored by 28BB-Fab-Tet-z in the absence of minocycline, however addition of minocycline at different time points was able to inhibit further cytokine secretion within 2–3 h. To ensure that the inhibition of TetCAR was reversible and that effector function could be restored upon removal of minocycline, 28BB-Fab-Tet-z cells were incubated overnight with 100 nM minocycline, then washed with media 48, 24 or 2 h before activation with SupT1-CD19 targets. Removal of minocycline 48 h before activation restored full TetCAR activity relative to a non-inhibited control, as measured by cytotoxicity and IL-2 secretion [Fig. 4e,f]. Washing at shorter timepoints (24 or 2 h) before activation only partially restored effector function.
28-Fab-Tet-z and 28BB-Fab-Tet-z CAR T cells are comparable with a gold-standard monolithic CAR
To ensure that 28-Fab-Tet-z and 28BB-Fab-Tet-z CAR T cells had no deficit in cell killing, we evaluated cytotoxicity at decreasing effector to target ratios, ranging from 1:1 to 1:32, to “stress” cytolytic function. There was no difference in cytotoxicity to SupT1-CD19-GFP or NALM6 at any E:T ratios in comparison to control CAR [Fig. 5a]. CAR T cell proliferation was also determined by co-culture with mitomycin C-treated SupT1-CD19-eGFP, NALM6, Raji cells or Raji CD19 knock-out cells [Fig. 5b]. In the absence of minocycline, both TetCARs proliferated to SupT1-CD19 similarly as the control CAR. However, in line with our observations for cytokine secretion, proliferation of the TetCARs was lower in response to NALM6 targets. The proliferative response of the TetCARs to Raji cells also appeared slightly lower than the control CAR, however this decrease was not significant. As expected, in the absence of CD19 on the Raji-CD19KO targets, none of the CARs proliferated above the NT T-cells. A single dose of 400 nM minocycline on day 0 was sufficient to significantly reduce TetCAR proliferation in response to SupT1-CD19 and Raji cells. A similar trend was observed with NALM6, however this was not significant due to poor response seen in the absence of minocycline in this setting.
Effector function of CD28-containing TetCAR matches 41BBζ control CAR. (a) Killing of SupT1-CD19-GFP after 24 h or NALM6 after 48 h of co-culture with CAR-T cells at 1:1–1:32 E:T ratio. Data shows mean ± SD, n = 4 donors from 2 independent experiments. (b) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1:2 E:T ratio for 7 days. To relevant wells, 400 nM of minocycline was added on day 0. Graphs show mean (± SD) number of RQR8+ T cells (filled bars) or total CD3+ T cells (white bars) for each target. n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n = 3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group ± minocycline (with Šidák’s multiple comparisons). P values for SupT1-CD19 were 28-Fab-Tet-z (**, 0.0028) and 28BB-Fab-Tet-z (**, 0.0050). P values for Raji were 28-Fab-Tet-z (*, 0.0320) and 28BB-Fab-Tet-z (*, 0.0304). (c) Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows geometric mean (± SD) in CD3+ T cells. n = 4 donors, from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for Lag3 expression were 28-Fab-Tet-z (**, 0.0078) and 28BB-Fab-Tet-z (*, 0.0207). (d) Percentage of naïve (CD62L+, CD45RA+), Tcm (central memory; CD62L+, CD45RA–), Tem (effector memory; CD62L–, CD45RA–) or Temra (terminally differentiated effector memory; CD62L–, CD45RA+) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows mean (± SD) in CD3+ T cells. n = 4 donors, from 2 independent experiments.
CD28 co-stimulation does not increase exhaustion or terminal differentiation in Fab-TetCAR T cells
Although CD28-CD3ζ endodomains can drive potent CAR T cell activation, this can limit memory formation and skew populations towards short-lived effector cells22. To show that Tet-CARs containing a CD28 endodomain were functionally equivalent to control 41BB-CD3ζ monolithic CAR, expression of Lag3/Tim3 or CD62L/CD45RA was evaluated. After the 7-day co-culture with target cells [Fig. 5c,d and Supplementary Fig. S4(a) and (b)], both Lag3 and Tim3 expression on CD3+ T cells were similar in the TetCARs compared with the standard CAR. Likewise, the proportions of the memory populations were similar between the TetCARs and the control CAR. Taken together these data show that even during maximal activation, TetCAR constructs containing CD28 endodomains did not drive an enhanced expression of exhaustion markers or skew differentiation of activated T cells.
28BB-Tet-CAR can be functionally regulated by minocycline in vivo
Lastly, we evaluated the activation and inhibition of 28BB-Fab-Tet-z CARs in vivo. NSG mice were engrafted with NALM6 engineered to express firefly luciferase (NALM6-Fluc); 4 days later, different cohorts were treated with either 5 × 106 NT, FMC63-BBz, or 28BB-Fab-Tet-z CAR T cells, with or without minocycline. An additional cohort of 28BB-Fab-Tet-z treated mice were treated with minocycline 3 days after T cell transfer, during the peak of the initial anti-tumor response [Fig. 6a]. Minocycline was given at a dose of ~ 16 mg/kg (0.4 mg per mouse) i.p. every 1–2 days. There was a significant reduction in tumor burden with 28BB-Fab-Tet-z and FMC63-BBz versus NT T cells in the absence of minocycline [Fig. 6b,c]. However, there was a non-significant trend to shorter tumor control with 28BB-Fab-Tet-z, which resembled the reduced activation and proliferation of the TetCAR in response to NALM6 targets in vitro. Whilst the addition of minocycline on day 0 had no effect on the FMC63-BBz group, early inhibition of 28BB-Fab-Tet-z completely abrogated the tumor control seen in the absence of minocycline. Injection of minocycline after initial tumor control (on day 3) was also able to inhibit subsequent TetCAR activity. Overall, these data show that although 28BB-Fab-Tet-z CARs are less potent than the FMC63-BBz CAR, they nevertheless provide significant tumor control, which can be regulated by treatment with minocycline in a relevant tumor model in vivo. Furthermore, inhibition of TetCAR function can be initiated during and after CAR T cell activation in vivo, mirroring a clinically relevant application of this technology.
28BB-Fab-Tet-z function and inhibition in vivo. (a) Overview of in vivo experiment. NSG mice were injected i.v. with 0.5 × 106 NALM6-FLuc tumor cells. On day 0, mice were randomly assigned based on tumor burden to receive 5 × 106 non-transduced (NT), FMC63-BBz or 28BB-Fab-Tet-z CAR T cells. Groups were further divided with some to receive 0.4 mg minocycline i.p. every 1–2 days, starting either on day 0 or day 3. (b) Bioluminescence radiance (photons/s/cm2/sr) of NALM6-FLuc tumors in mice in select groups. (c) Geometric mean radiance (photons/s/cm2/sr) of NALM6-FLuc cells, in mice in all groups treated with NT, FMC63-BBz (± minocycline) or 28BB-Fab-Tet-z CAR T cells (± minocycline). n = 4 mice per group from 1 experiment. Table shows statistical analysis through one-way ANOVA with multiple comparisons between groups at each time point.
