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Abstract 2753: Interleukin-10 and transforming growth factor-beta do not suppress tumor killing activity of PBMC-derived SUPLEXA cells


 Introduction: SUPLEXA cells are peripheral blood mononuclear cells (PBMCs) being developed by Alloplex Biotherapeutics as an autologous adoptive therapy for cancer. The novel nature of SUPLEXA cells is that they are developed from peripheral blood mononuclear cells (PBMCs) that have been activated and expanded by engineered leukocyte stimulator cell lines (ENLIST cells), which are melanoma cell lines that express immunomodulatory proteins to program the tumor killing phenotype of SUPLEXA cells. We find that the SUPLEXA cell manufacturing process reproducibly results in a mixture of NK cells, CD8 T cells, and γδ T cells with broad tumor cell killing activity. In this study, we evaluated the effects of two major immunosuppressive cytokines found in the tumor microenvironment (IL-10 and TGF-β) on the tumor cytolytic function and cytokine release by SUPLEXA cells.
 Results: SUPLEXA cells were prepared by a 14-day process of PBMC induction by ENLIST cells for 5 days followed by 9 days expansion in cytokines. SUPLEXA cell mediated tumor cytolytic assays using red fluorescent protein (RFP) expressing M14 melanoma cells as targets were performed in the absence or presence of IL-10 or TGF-β (1.25 - 10 ng/ml). Cytokine levels in 48-hour culture supernatants from cytolytic reactions and SUPLEXA or M14 target cells alone were measured using a 36-plex Luminex assay. Without cytokine addition, SUPLEXA mediated cytolysis was 47.3% at 8:1 and 30.9% at 4:1 effector to target (E:T) ratios. Significant increases in SUPLEXA cell tumoricidal activity was observed with addition of IL-10 (74.3% at 8:1 and 51.7% at 4:1 E:T ratios at 2.5 ng/ml IL-10 added, P &It 0.001 by two-way ANOVA). Adding TGF-β caused a reduction in cytolytic activity that was not significant (36.5% at 8:1 and 20.8% at 4:1 E:T ratios with 2.5 ng/ml TGF-β added). Supernatants from cytolysis assays showed that SUPLEXA cells produced IFNγ, MIP-1α, RANTES, IL-8, Gro-α, MIG, and IP-10 in response to tumor cells. IL-10 significantly augmented IFNγ, RANTES, and MIP-1α production by SUPLEXA cells in a dose-dependent fashion. In contrast, TGF-β reduced IFNγ, MIP-1α, Gro-α, RANTES, and IP-10 production, but increased IL-8 production by SUPLEXA cells.
 Conclusions: An in vitro approach was used as a surrogate for what might occur in patients with solid tumors to test whether potent immune suppressive cytokines that are present in the tumor microenvironment might inhibit SUPLEXA cell function. Our findings indicate that IL-10 and TGF-β do not significantly reduce the tumor killing activity of SUPLEXA cells. Instead, we discovered that IL-10 significantly enhances SUPLEXA cell function and cytokine production, which suggests that SUPLEXA cells may acquire heightened tumoricidal activity and promote immune surveillance by augmenting cytokine and chemokine levels within the tumor microenvironment when given therapeutically to cancer patients.
 Citation Format: Frank Borriello, Joshua W. Keegan, James A. Lederer. Interleukin-10 and transforming growth factor-beta do not suppress tumor killing activity of PBMC-derived SUPLEXA cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2753.