AMPK agonism improves In vivo CAR T cell function across a range of platforms and clinical indications Free

The metabolic profile of adoptive cell therapies profoundly impacts their subsequent in vivo persistence and function. In these studies, we wished to determine the in vivo consequence of exposing chimeric antigen receptor (CAR) T cells to Compound 991, a direct AMPK agonist, during in vitro culture.

Using a NOD-scid IL2Rgamma^null (NSG) mouse model, we first injected 1e6 Nalm6 cells into NSG recipients, followed one week later by 3e6 CD28 co-stimulated CAR T cells (+/- 991 exposure in vitro). Mice receiving DMSO-treated CAR T cells (control cohort) experienced a delay in leukemia relapse, but eventually succumbed to lethal and overwhelming leukemia burden. In stark contrast, 54% of mice receiving CAR T cells pre-treated with Compound 991 remained leukemia-free throughout the experiment (6/11), with 73% of these mice surviving until the end of the experiment on day 70 (p<0.001).

We next asked whether AMPK activation would benefit CAR T cells containing a 4-1BB costimulatory domain. 991 treatment profoundly increased the in vitro respiratory capacity of 4-1BB CAR T cells, as measured on the Seahorse metabolic analyzer (p<0.0001), and markedly enhanced the cytotoxic capacity of these cells across a range of effector:target ratios in an IncuCyte killing assay (p<0.0001). In vivo, 991-treated 4-1BB CAR T cells vastly outperformed DMSO-treated CAR T cells in relation to their anti-leukemia efficacy, extending the median time of relapse by >4 weeks and improving median survival by more than 30 days (p<0.0001).

Next, we wished to determine whether 991-mediated benefits translated into CAR responses against solid tumors. One million luciferase+ Nalm6 cells (mixed 1:1 with Matrigel) were subcutaneously injected into NSG recipients, followed by injection three weeks later of 2e6 CD19-targeting CAR T cells (+/- 991 treatment). Tumor burden was measured via weekly IVIS imaging and survival measured to day 87. 5/9 mice receiving DMSO-treated CAR T cells experienced lymphoma relapse, compared to only 2/10 mice receiving 991-treated cells (p=0.04). Further, all five relapsing DMSO-treated CAR T cell mice succumbed to unrestrained tumor growth, while zero recipients of 991-treated CAR T cell died during the course of the experiment (p=0.0067). Importantly, flow cytometry revealed significant splenic infiltration of Nalm6 cells in relapsing DMSO-treated recipients at the time of euthanization, while metastatic activity was absent in the 991-treated group.

As the CAR T cell field is gradually moving away from prolonged stimulation and expansion, we next addressed the question of how AMPK agonist treatment could be incorporated into an abbreviated workflow. In proof-of-principle studies, we truncated our T cell transduction and agonist treatment to a 6-day time window instead of 11. Agonist treatment at the time of Dynabead stimulation was not well tolerated. However, treatment of primary human T cells with Compound 991 on days 2 and 4 of culture generated an enhanced respiratory capacity, including marked increases in maximal oxygen consumptions rates and spare respiratory capacity. Importantly, levels of reactive oxygen species did not increase in agonist treated cells, despite a noted increase in respiratory capacity, likely as a result of increased expression of anti-oxidants including Thioredoxin 1 in 991-treated cells (p=0.0264). Importantly, transduction efficiency on day 2, in the presence of 991, was equivalent between DMSO and 991-treated groups, achieving >85% CAR transduction, with only a transient decrease in cell recovery on day 4 (which corrected by day 6). Finally, we sought a mechanism for the increased leukemia/lymphoma control following Compound 991 pre-treatment. 991-treated CAR T cells, recovered 20 hours post-injection from the bone marrow of Nalm6-bearing recipients, upregulated multiple T cell activation markers including CD25, CD98, CD137 (4-1BB), and CD279 (PD-1) (all p<0.05).

From these data, we conclude that 991 treatment increases in vivo anti-tumor efficacy in both CD28- and 4-1BB co-stimulated CAR T cells, is compatible with cell therapies targeting solid tumor, is amenable to a truncated protocol of CAR T cell generation, and is at least partially dependent upon increased early activation of CAR T cells in the early moments of antigen encounter. Taken together, these data suggest and support multiple potential avenues for translation of this novel therapeutic approach to the clinic.