Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function.

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved in vivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both in vitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies.

Poxvirus-Based Active Immunotherapy with PD-1 and LAG-3 Dual Immune Checkpoint Inhibition Overcomes Compensatory Immune Regulation, Yielding Complete Tumor Regression in Mice.

Poxvirus-based active immunotherapies mediate anti-tumor efficacy by triggering broad and durable Th1 dominated T cell responses against the tumor. While monotherapy significantly delays tumor growth, it often does not lead to complete tumor regression. It was hypothesized that the induced robust infiltration of IFNγ-producing T cells into the tumor could provoke an adaptive immune evasive response by the tumor through the upregulation of PD-L1 expression. In therapeutic CT26-HER-2 tumor models, MVA-BN-HER2 poxvirus immunotherapy resulted in significant tumor growth delay accompanied by a robust, tumor-infiltrating T cell response that was characterized by low to mid-levels of PD-1 expression on T cells. As hypothesized, this response was countered by significantly increased PD-L1 expression on the tumor and, unexpectedly, also on infiltrating T cells. Synergistic benefit of anti-tumor therapy was observed when MVA-BN-HER2 immunotherapy was combined with PD-1 immune checkpoint blockade. Interestingly, PD-1 blockade stimulated a second immune checkpoint molecule, LAG-3, to be expressed on T cells. Combining MVA-BN-HER2 immunotherapy with dual PD-1 plus LAG-3 blockade resulted in comprehensive tumor regression in all mice treated with the triple combination therapy. Subsequent rejection of tumors lacking the HER-2 antigen by treatment-responsive mice without further therapy six months after the original challenge demonstrated long lasting memory and suggested that effective T cell immunity to novel, non-targeted tumor antigens (antigen spread) had occurred. These data support the clinical investigation of this triple therapy regimen, especially in cancer patients harboring PD-L1neg/low tumors unlikely to benefit from immune checkpoint blockade alone.