Inhibition Of IL-10 In The Tumor Microenvironment Potentiates Mesothelin-chimeric Antigen Receptor Nk-92MI-mediated Killing Of Pancreatic Cancer Cells
*Ramesh B Batchu1, *Oksana V Gruzdyn1, *Pavam S Tavva2, *Rajesh Dachepalli2, *Bala K Kolli1, Gamal Mostafa1, Donald W Weaver3, Scott A Gruber1
1John D. Dingell VA Medical Center, Detroit, MI;2Med Manor Organics Pvt. Ltd., Hyderabad, India3Wayne State University School of Medicine, Detroit, MI
OBJECTIVE(S): IL-10-mediated immunosuppression in the tumor microenvironment (TME) is an important impediment to developing effective immunotherapy for pancreatic cancer (PC). Although chimeric antigen receptor (CAR) T-cells kill PC cells in vitro, their activation in the clinical setting is often compromised by the TME. Previously, we demonstrated a significant reversal of TME-mediated inhibition of mesothelin (MSLN)-CAR T-cell activity against PC cells either by depleting IL-10 in autologous T-cells or by using the MSLN-NK-92MI CAR T-cell line which secretes IL-2, a cytokine supporting T-cell activation. In this study, we combined both of these approaches to further overcome TME-mediated inhibition of the ability of CAR T-cells to kill PC cells.
METHODS: Tumor-conditioned medium (TCM) simulating the TME was obtained from 48 h serum-free cultures of target BxPC-3 human PC cells with or without IL-10 depletion. MSLN-CAR vectors were electroporated into NK-92MI cells. IFN-γ and granzyme B secretion were measured by an ELISA kit and cytotoxicity by MTT assay.
RESULTS: When compared with mock electroporation, ELISA co-culture assays of MSLN-NK-92MI CAR T-cells with BxPC-3 target cells demonstrated robust secretion of IFN-γ (Fig.1A) and granzyme B (Fig.1B), both of which are crucial for the induction of cell death. We observed significant TCM-mediated inhibition of IFN-γ (p<0.01) and granzyme B (p<0.01) secretion in the same co-cultures, which was partially restored when TCM was IL-10 depleted (p<0.05 for both cytokines). Assays of BxPC-3 with MSLN-NK-92MI CAR T-cells demonstrated cytotoxicities of 60% (p<0.01) and 85% (p<0.01) at E:T ratios of 10:1 and 20:1, respectively, when compared with control (Fig. 1C). Addition of TCM to the co-cultures reduced these cytotoxicities to 10% (p<0.01) and 18% (p<0.05), respectively. However, IL-10 depletion from TCM blunted this reduction by increasing these cytotoxicities back to 30% (p<0.05) and 42% (p<0.05), respectively.
CONCLUSIONS: The ability of IL-2-secreting MSLN-NK-92MI CAR T-cells to kill PC cells may be further enhanced by depleting IL-10 from the local tumor microenvironment, potentially allowing for more effective clinical translation.
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