306 Selective immunometabolic reprogramming of NK cells to enhance their metabolic fitness against glioblastoma

Background Despite advancements in immunotherapy, the immunosuppressive tumor microenvironment (TME) continues to be a significant barrier in aggressive malignancies such as glioblastoma (GBM). 1 2 SLC1A5, a vital glutamine transporter, is lost by NK cells when glutamine is depleted in the TME, but tumor cells overexpress it and consume more glutamine.2–4 The activation, proliferation, and anti-tumor activity of NK cells are all hampered by this metabolic imbalance. We devised a dual strategy, genetically engineering NK cells to overexpress SLC1A5, boosting their glutamine uptake and resilience, and designing bispecific antibody to selectively block SLC1A5 on GBM43 cells, starving tumors and redirecting glutamine to NK cells.Methods After inhibiting glutamine uptake and metabolism against GBM43 cells, we assessed cytotoxicity and activation of NK cells. The expression of SLC1A5 was assessed in NK cells obtained from donors and patients. Glycolytic and mitochondrial activities were measured using ECAR and OCR metabolic profiling. Glutamate uptake and SLC1A5 expression were investigated in NK and GBM43 co-cultures. NK cells overexpressing human SLC1A5 were engineered using lentiviral transduction; click chemistry and flow cytometry were utilized to assess glutamine uptake, activation, and cytotoxicity. WB and binding experiments verified that bispecific antibody we generated targeted SLC1A5 on GBM cells. Selective glutamine uptake was evaluated in co-cultures of SLC1A5-NK cells and GBM43 treated with the bispecific antibody. The effectiveness and selectivity of this dual strategy are being assessed in vivo using orthotopic GBM xenografts.Results While cell survival remained unchanged, NK cell cytotoxicity against GBM43 cells was dramatically decreased when glutamine metabolism was disrupted. Significant changes in acidity and oxygen consumption were found via metabolic assays, demonstrating critical role glutamine plays in NK cell activity. GBM43 cells showed greater absorption and SLC1A5 expression, outcompeting NK cells for glutamine. NK cell viability was significantly reduced by SLC1A5 inhibition with V9302 ( figure 1). Both alone and in co-culture with GBM43, SLC1A5-overexpressing NK cells demonstrated higher glutamine uptake, improved activation, and increased cytotoxicity (figure 2). The bispecific antibody successfully shifted metabolic balance in favor of NK cell anti-tumor activity by selectively inhibiting glutamine uptake in GBM43 cells while promoting NK cell function.Conclusions By genetically modifying NK cells to increase glutamine uptake and using a bispecific antibody to specifically starve tumor cells while sparing NK cells, our two-pronged strategy directly tackles glutamine competition, metabolic basis of immunotherapy resistance. This approach is a promising step toward improving immunotherapy responses in GBM by restoring NK cell metabolic fitness in TME.Acknowledgements Authors gratefully acknowledge the funding from the Purdue Center for Cancer Research (NIH grant P30 CA023168) and the V Foundation for Cancer Research, Indiana Clinical and Translational Sciences Institute, funded in parts by award number UL1TR001108.References Ma G, Zhang Z, Li P, Zhang Z, Zeng M, Liang Z, Li D, Wang L, Chen Y, Liang Y, Niu H. Reprogramming of glutamine metabolism and its impact on immune response in the tumor microenvironment . Cell Communication and Signaling 2022;20(1):114. https://doi.org/10.1186/s12964-022-00909-0Nachef M, Ali AK, Almutairi SM, Lee S-H. Targeting SLC1A5 and SLC3A2/SLC7A5 as a potential strategy to strengthen anti-tumor immunity in the tumor microenvironment. Frontiers in Immunology 2021;12. https://doi.org/10.3389/fimmu.2021.624324Loftus RM, et al. Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice. Nat. Commun. Jun. 2018;9(1):2341. doi: 10.1038/s41467-018-04719-2Wang B, Pei J, Xu S, Liu J, Yu J. (2024). A glutamine tug-of-war between cancer and immune cells: Recent advances in unraveling the ongoing battle. Journal of Experimental & Clinical Cancer Research : CR, 43, 74. https://doi.org/10.1186/s13046-024-02994-0Ethics Approval The human blood was obtained by the protocol approved by Purdue University, IRB:2024-168 The animals were handled according to the protocol approved by Animal care and use facility: 1112000342Abstract 306 Figure 1(a) Effect of SLC1A5 blocking by V9302 on NK viability after 24, 48 and 72 hours, no significant difference between donor viability, one-way annova: ns; (b) SLC1A5 expression on NK cells in variable glutamine concentration, expression of SLC1A5 on GBM43 is significantly more than on pNKs, one-way ANNOVA; Donor 1 vs. GBM43, p=0.0022, Donor 2 vs GBM43 and Donor 3 vs GBM43, p<0.0001; (c ) SLC1A5 expression on NK and GBM43 cells in co-culture; (d) Glutamine uptake in nmol by pNK and GBM43 when cultured separately in variable glutamine concentration, glutamine uptake by pNK is significantly lower than by GBM43, p=0.0013; (e) Glutamine uptake by pNK and GBM43 when co-cultured in transwellAbstract 306 Figure 2(a) Plasmid lentiviral vector of transgene SLC1A5 and mCherry detection marker and CMV promoter; (b, c) Transduction efficiency in GBM43 cell line; (d) Transduction efficiency in pNK; (e) Transduction in pNK with BX795