Many cancers (unlike normal cells) are deficient in argininosuccinate synthetase-1 (ASS1), a critical enzyme required for arginine synthesis from citrulline, which makes them dependent on importing extracellular arginine. Pegylated arginine deiminase (ADI-PEG 20) depletes the external supply of arginine by degrading it to citrulline thereby affecting growth and viability of cancer cells while sparing normal tissues which express ASS1. ADI-PEG 20 is being investigated in clinical studies in arginine auxotrophic cancers and is well-tolerated. Arginine is used in a number of metabolic pathways including those affecting tumor growth and immune cell regulation. The anti-tumor properties of ADI-PEG 20 have been extensively investigated, however, its impact on immune cells is largely unknown. Therefore, we have investigated potential implications of ADI-PEG 20 treatment for tumor immune environment. We studied how ADI-PEG 20 affects T cell subsets in healthy donor human peripheral blood mononuclear cells (PBMCs) under resting and activation conditions and expression of immunosuppressive programmed death-ligand 1 (PD-L1) on cancer cell lines. PBMCs were treated with ADI-PEG 20 under resting and activation conditions and were characterized by immune cell phenotyping using flow cytometry. For PBMC stimulation we used anti-CD3/CD28 beads or phytohemagglutinin (PHA). During stimulation (and not under resting conditions) ADI-PEG 20 markedly boosted CD69 expression on T cells (both CD4+ and CD8+) while moderating their exhaustion (CTLA-4 and PD-1 levels remained low, similar to that at a resting state). Moreover, ADI-PEG 20 reduced accumulation of regulatory T cells (Treg) induced by anti-CD3/CD28 beads. The effect of ADI-PEG 20 on PD-L1 expression in ASS1-low cancer cell lines was analyzed by flow cytometry and RT-qPCR. ADI-PEG 20 treatment induced PD-L1 upregulation on some cancer cell lines in vitro, the magnitude of the upregulation was cell line dependent and in most it was much lower than that induced by 200 ng/mL IFNγ. Based on our findings that ADI-PEG 20 downmodulates Treg cells we have hypothesized that ADI-PEG 20 may improve immunogenicity of non-immunogenic tumors. To test this hypothesis we used poorly immunogenic syngeneic mouse melanoma B16-F10 model. IHC analysis of the tumor sections revealed that five out of six ADI-PEG 20 treated animals had a large number of T cells in their tumors; only one ADI-PEG 20 treated mouse had very little tumor T cell infiltrate, similar to the non-treated controls. This demonstrates that ADI-PEG 20 can improve tumor immunogenicity. ADI-PEG 20 also inhibits growth of the B16-F10 tumor in vitro and in vivo. In summary, we discovered that ADI-PEG 20 increases healthy human donor effector T cell activation and blocks their exhaustion while reducing accumulation of Tregs in vitro. While ADI-PEG 20 induced PD-L1 expression in some cancer cell lines in vitro this increase was relatively small in most studied cell lines and is unlikely to significantly contribute to tumor immune evasion. Importantly, ADI-PEG 20 treatment resulted in a marked increase in tumor infiltrating T cells in a poorly immunogenic B16-F10 melanoma model. Our data suggests that in addition to the direct anti-tumor effects ADI-PEG 20 can help boost tumor immune surveillance and could be a good primer for an additional anti-tumor immune therapy.

Citation Format: Elena Brin, Katherine Wu, Hsin-Tze Lu, Yudou He, Zhaoming Dai, Wei He. Arginine deiminase can boost anti-tumor immune surveillance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A31.