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. 2014 Aug 15;20(16):4262-73.
doi: 10.1158/1078-0432.CCR-13-2627. Epub 2014 Jun 11.

Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors

Edmund K Moon  1 , Liang-Chuan Wang  2 , Douglas V Dolfi  3 , Caleph B Wilson  4 , Raghuveer Ranganathan  2 , Jing Sun  2 , Veena Kapoor  2 , John Scholler  4 , Ellen Puré  5 , Michael C Milone  4 , Carl H June  4 , James L Riley  3 , E John Wherry  3 , Steven M Albelda  2
Affiliations

Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors

Edmund K Moon et al. Clin Cancer Res. .

Abstract

Purpose: Immunotherapy using vaccines or adoptively transferred tumor-infiltrating lymphocytes (TIL) is limited by T-cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine whether a similar tumor-induced inhibition occurred with genetically modified cytotoxic T cells expressing chimeric antigen receptors (CAR) targeting tumor-associated antigens.

Experimental design: Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4-1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin-expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways.

Results: CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction seemed to be multifactorial and was associated with upregulation of intrinsic T-cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD1, LAG3, TIM3, and 2B4).

Conclusions: Advanced-generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell-based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD1 pathway antagonism may augment human CAR T-cell function.

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Conflict of interest statement

The authors have no potential conflicts of interest to disclose.

Figures

Fig 1
Fig 1. CAR T cells slow tumor growth but do not cause regression
(A) 5x106 EMMESO tumor cells were injected into the flanks of NSG mice. After they grew to ~200 mm3 in size, 20x106 mesoCAR T cells were injected via tail vein and tumors were measured for 39 days post injection. T cells were able to slow growth by day 18 but were unable to eradicate flank tumors. (B) 5x106 EMMESO tumor cells were injected into the flanks of NSG mice. After they grew to ~150 mm3 in size, 20x106 FAPCAR T cells were injected via tail vein and tumors were measured for 41 days post injection. T cells were able to slow growth by day 11 but were unable to eradicate flank tumors (* p<0.05).
Fig 2
Fig 2. CAR T cells infiltrate, survive, proliferate, and retain transgene expression in tumors
39 days after tail vein T cell injection, flank tumors were harvested and digested and the quantity of human CD3+ cells were assessed. The percentage (A) and absolute number (B) of intra-tumoral CAR T cells was much higher than non-transduced T cells. (9.7% (8.2x106) CAR T cells vs. 0.16% (2.5x105) NT T cells (p<0.05)) The percentage (C) and absolute number (D) of intra-splenic CAR T cells was much lower than non-transduced T cells (<0.05% (3.89x102) CAR T cells vs. 0.25% (2.82x104) NT T cells (p<0.05)). The percentage (E) and absolute number (F) of intra-tumoral CAR T cells was also assessed at earlier time points and demonstrated a marked increase over time. (0.07% (2.16x104) in the first week after T cell injection to 9.7% (7.98x106) in the 6th week after T cell injection. (G) When myc-tagged mesoCAR TILs were isolated from flank tumors 40 days after injection, they retained surface expression of myc-tagged mesoCAR after infiltration into tumors. Percentage of Myc-tag expressing T cells after infiltration was actually higher than at the time of injection (42.7% vs. 13.6%)
Fig 3
Fig 3. CAR T cells undergo tumor-induced hypofunction of cytolytic and cytokine secretion ability
(A) Cytotoxicity: Flank mesoCAR TILs from Day 39 demonstrated hypofunctional killing of EMMESOffluc compared to cryo mesoCAR T cell controls (98.7% vs. 12.3%, p<0.001). TILs/T cells were cocultured with firefly luciferase expressing EMMESO tumors at 20:1 E:T ratio for 18 hours. Measurement of luciferase activity of remaining tumor cells was used to calculate % killing. B. IFNγ secretion: Flank mesoCAR TILs demonstrate hypofunctional secretion of IFNγ in response to EMMESOffluc tumor compared to cryo mesoCAR T cell controls (17,387pg/ml vs. 1689pg/ml, p<0.001). Coculture was performed at a ratio of 20:1 (effector to target) for 18 hours in 37°C, 5%CO2. EMPffluc targets were also tested to confirm antigen-specific tumor killing ability of both the cryo mesoCAR T cells and the mesoCAR TILs. (C) Isolated spleen infiltrated T cells demonstrated much less loss of tumor killing function as compared to TILs (A) and (D) much less loss of tumor induced IFNγ secretion as compared to TILs (B). (E) Flank FAPCAR TILs from Day 28 demonstrated hypofunctional killing of 3T3mFAP-ffluc compared to cryo FAPCAR T cell controls (24% vs. 0%, p<0.01). TILs/T cells were cocultured with firefly luciferase expressing 3T3p and 3T3mFAP tumors at 10:1 E:T ratio for 18 hours. Measurement of luciferase activity of remaining tumor cells was used to calculate % killing. (F) IFNγ secretion: Flank FAPCAR TILs demonstrate hypofunction secretion of IFNγ in response to 3T3mFAP-ffluc tumor compared to cryo FAPCAR T cell controls (2653.7pg/ml vs. 60.85pg/ml, p<0.01).
Fig 4
Fig 4. CAR TIL hypofunction progresses rapidly after injection and may be due to defects in proximal signaling
(A) Upon exposure to either albumin-labeled beads (negative control) or mesothelin-labeled beads, the mesoCAR TILs were found to be hypofunctional in their ability to produce IFNγ in response to the antigen-coated beads. (B) Upon exposure to 0.1ug/ml PMA and 2ug/ml ionomycin, the mesoCAR TILs demonstrated preserved ability to produce IFNγ as measured by intracellular cytokine staining by flow cytometry. (C) Upon exposure to either albumin labeled beads (negative control), mesothelin labeled beads, or CD3/CD28 beads (positive control) for 15 minutes, mesoCAR TILs were found to be hypofunctional in their ability to produce signal via phosphorylation of ERK in response to antigen or CD3/28 stimuli. (D) When flank tumors were harvested at early (day 5), mid (day 17), and late (day 39) timepoints, isolated EMMESO TILs were cocultured with firefly luciferase expressing EMMESO tumor at 20:1 E:T ratio. After 18 hours, % killing was calculated after measuring luciferase activity of the remaining tumor cells. We demonstrated that EMMESO TILs undergo rapidly increasing tumor-induced hypofunction after IV injection .
Fig 5
Fig 5. CAR TIL cytolytic and cytokine secreting functions recover after rest away from tumor. DGK and pSHP1 expression is increased in hypofunctional CAR TILs
Human TILs isolated from flank EMMESO tumors at Day 39 and “rested” away from tumor for 24 hours demonstrated significant recovery in their (A) EMMESOffluc tumor killing ability and (B) their ability to secrete IFNγ in response to EMMESOffluc tumor. (C) Compared to freshly harvested EMMESO TILs, resting away from tumor led to recovery of antigen-specific signaling function of EMMESO TILs as measured by detection of phosphorylated ERK. (D) DGKa and DGKz and pSHP1 significantly elevated in TILs freshly isolated from EMMESO. Expression of both isoforms of DGK as well as pSHP1 decreased dramatically after over night rest of TILs.
Fig 6
Fig 6. PDL1 blockade and DGK inhibition are able to restore CAR TIL function
10ug/ml of anti-PDL1 antibody was added to the co-culture killing assay and was able to restore (A) mesoCAR TIL killing of EMMESOffluc (B) and tumor-induced mesoCAR TIL secretion of IFNg after fresh isolation from flank tumor. (C) 1uM of type I and II DGK inhibitor was added to the co-culture killing assay and was able to restore mesoCAR TIL killing of EMMESOffluc with (D) minimal increase in tumor-induced mesoCAR TIL secretion of IFNg after fresh isolation from flank tumor. 25ug/ml of SSG was added to the co-culture killing assay and was able to restore (E) mesoCAR TIL killing of EMMESOffluc (F) and tumor-induced mesoCAR TIL secretion of IFNg after fresh isolation from flank tumor.
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