Isolation, ex vivo expansion followed by reinfusion of activated autologous lymphocytes is a successfully utilized and promising clinical treatment for both solid and liquid tumors. Although it has been routine for most clinical and experimental laboratories to rely on interleukin-2 (IL-2) to support T cell expansion this cytokine can result in substantial expansion and induction of regulatory T cells (Tregs), AICD, exhaustion and terminal differentiation. IL-2 signals when the three subunits of IL-2 receptor, namely α, β and γ form a heterotrimeric protein. We have recently described a novel re-targeted form of IL-2 which utilizes NKG2D rather than IL-2Rα to form the high affinity receptor complex for IL-2 signaling. This cytokine (OIL-2), targets signaling solely to NKG2D-expressing cells but its role in expanding CD8+ T cells for adoptive T cell therapy is unknown

In direct comparison to IL2, OIL-2 resulted in significantly improved expansion of CD8+ T cells (70-fold in vs 345-fold increase in 3 weeks; p<0.002), reduced cell death/apoptosis (% cell viability of 32.73±3.01 vs 73.9±11.28; p<0.017), improved memory formation (%Tcm of 9.867±7.28 in IL-2 vs 25.5±2.78 in OIL-2; p<0.039) and increased effector functions (%IFN-γ+ cells were 43.5±3.15 vs 71.4±1.4; p<0.09). To evaluate the signaling pattern we inhibited PI3K using wortmannin but saw no decrease in the efficiency of OIL-2 (%IFN-γ + cells reduced non-significantly from 70.66±3.154 to 62.1±1.531). We next evaluated phosphorylation of AKT and noted an increase in AktSer473 phosphorylation (MFI of 457±16.58 in IL2 vs 667±75.52 in OIL-2; p<0.003). However, the levels of pSTAT5 after activation was lower in OIL-2 cultured T cells (MFI of 869.2±123.6 vs 574.4±41.42; p<0.001). Taken together our data indicate a pattern of mTOR2 activation by OIL-2 vs mTOR1 by wild-type IL-2. We also found a decrease in %Tregs and FOXO1, and p53 levels in OIL-2 cultured T cells. To fully evaluate our cytokine, we expanded anti-gp100 TCR transgenic CD8+ T cells in either wild-type IL2 or OIL-2 and demonstrated an improvement in the control of B16 melanoma (151.73±40.01vs 62.59±22.49 mm3 on day 13 of growth). We also showed that in-vitro lysis of B16 tumor cells were enhanced by OIL-2 cultured T cells (% B16 lysis of 19.5±7.62 vs 49±7.32; p<0.001).

In conclusion we demonstrate that our first-in class cytokine is more effective and offers a superior method for ex vivo T cell expansion. OIL-2 mediated CD8+ T cell stimulation occur via an altered signaling complex which causes T cell metabolic re-programming. As a result, OIL-2 expanded T cells persist and offers better tumor clearance and provide superior protection against malignancy over T cells expanded through alternative protocols.

Citation Format: Anirban Banerjee, Yizhan Guo, Sarah Hein, Alexander S. Krupnick. A novel first-in-class common-gamma chain cytokine facilitates expansion of CD8+T cells and offers a superior cellular source for adoptive T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 939.