328 Promoting NK survival and function within the tumor microenvironment (2022)

<h3>Background</h3> The efficacy of CAR-NK cellular therapies in solid tumors is limited by insufficient survival and expansion within the suppressive tumor microenvironment (TME). To augment CAR-NK survival and anti-tumor function, CAR-NK cells are typically administered with exogenous cytokines or other signal modifiers that converge on signal transducer and activator of transcription-5 (STAT5) activation, an essential signaling node for NK survival and function.<sup>1</sup> However, systemic administration of these cytokines or signal modifiers is associated with toxicity,<sup>2</sup> unintended bolstering of TME components,<sup>3</sup> and CAR-NK rejection.<sup>4</sup> Thus, novel strategies to enhance CAR-NK function within the TME are needed. To promote CAR-NK survival and function in the TME while bypassing the need for exogenous signal dependency, we genetically modified GD2-specific CAR-NK cells to express a constitutively active IL-7 receptor complex, termed C7R (C7R.GD2-NK), that intrinsically confers persistent STAT5 activity within NK cells. <h3>Methods</h3> To mimic challenges of the TME, we created an <i>in vitro</i> TME model by preconditioning CD14<sup>+</sup> monocytes with CHLA255 neuroblastoma cells for 72 hours, converting them to an inhibitory M2-TAM phenotype expressing suppressive cytokines and ligands. 4 days after the addition of the C7R.GD2-NK cells to this immunosuppressive milieu, we assessed NK survival, expansion, cytokine secretion, and anti-tumor function. <h3>Results</h3> Resting C7R.GD2-NK cells exhibited 3.4-fold (± 0.7; n=4 donors, p&lt;0.005) higher phosphorylated STAT5 (pSTAT5) compared to resting GD2-NK cells. C7R.GD2-NK cells alone in long-term culture expanded for 14 days without exogenous cytokine support but not indefinitely. In short-term co-cultures with GD2<sup>+</sup> CHLA255 cells, C7R.GD2-NK demonstrated similar IFNγ levels as GD2-NK stimulated with exogenous IL-2 or IL-15 (mean% GD2-NK IFNy<sup>+</sup> of 26.1, 21.2, and 22.4, respectively). In the <i>in vitro</i> TME model, C7R.GD2-NK cells expanded less than IL-15- or IL-2-stimulated GD2-NK cells (mean fold expansions of 2.8-, 4.5-, and 4.3-fold, respectively) but demonstrated similar anti-tumor activity. Furthermore, cell culture supernatants from C7R.GD2-NK treated conditions exhibited similar levels of NK effector cytokines IFNγ, TNF-α, and GM-CSF to exogenous cytokine-stimulated GD2-NK cells. Ongoing experiments in an <i>in vivo</i> TME xenograft model will assess the benefit of C7R in CAR-NK activity. <h3>Conclusions</h3> C7R drives STAT5 activity in GD2 CAR-NK cells within TME conditions without the need for exogenous cytokines. The NK-intrinsic STAT5 signal is sufficient to promote NK survival, expansion, and function within the TME, potentially without the side effects of exogenous cytokines or cytokine modulators. Thus, C7R may be a viable alternative therapeutic strategy to augment CAR-NK efficacy in solid tumors. <h3>References</h3> Gotthardt D, Sexl V. STATs in NK cells: The good, the bad, and the ugly. <i>Front Immunol</i>. 2017; <b>7</b>:694 Dutcher JP, Schwartzentruber DJ, Kaufman HL, Agarwala SS, Tarhini AA, Lowder JN, Atkins MB. High dose interleukin-2 (Aldesleukin) – expert consensus on best management practices-2014. <i>J Immunother Cancer</i>. 2014; <b>2</b>:26 Nelson B. IL-2, Regulatory T Cells, and Tolerance. J Immunol. 2004; <b>172</b>:3983-3988 Berrien-Elliot M, <i>et al</i>. Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy. <i>Blood</i>. 2022; <b>139</b>:1177-1183