BackgroundGlioblastoma (GBM) is a deadly brain malignancy with a dismal prognosis. While immunotherapy holds great promise for GBM treatment, most have failed due to a suppressive tumor microenvironment (TME). Antigen heterogeneity and adenosine signaling are two immunosuppressive mechanisms in GBM. The CD73-adenosine axis plays a multifaceted role in GBM pathogenesis and drives the dysfunction of NK cells in GBM TME.1,3 Our NKG2D-chimeric antigen receptor (CAR)-natural killer (NK) cells have shown anti-tumor activity when combined with CD73 blockade in vivo.2 To further extend the potency of these cells against GBM and address antigen heterogeneity in GBM, we combined the local blockade of CD73 with multi-antigen-targeting engineered NK cells. In order to improve treatment assessment, PET/MR imaging was employed to enable detailed, non-invasive assessment of tumor progression. Imaging assessment of adoptively-transferred CAR- NK cells was also developed to determine the fate of NK cell delivery to the tumor site over time.MethodsWe generated multifunctional engineered NK (E-NK) cells that express an anti-CD73 scFv, which is cleavable by GBM-associated proteases, an NKG2D-CAR, as well as a GD2 CAR, which can actively target the GD2 antigen overexpressed on GBM (Figure 1A). For E-NK cell radiolabeling, zirconium-89 (89Zr, ½ life = 78 Hr) radiotracer was attached covalently to the E-NK cell surface via conjugation with DFO-Bz-NCS in a range of doses from 50–600 µCi.ResultsAn optimal balance between labeling efficiency and cell viability was attained at 120 µCi 89Zr resulting in 39% labeling efficiency and 46% cell viability over for 48 hours. After labeling, the NK cells maintained their in vitro killing activity against GBM cells (figure 1B). The 89Zr labeled E-NK cells were administered intravenously in mice containing intracranial GBM10 tumors at week 5 post-implant. PET imaging was performed at 1 and 2 days later and gamma imaging ex vivo at 4 days. Free 89Zr was visible diffusely throughout the body with low levels in the brain. The majority of 89Zr labeled E-NK cell groups localized to the lungs with detectable activity elsewhere in various organs (figure 1C and 1D).Abstract 138 Figure 1PET imaging and gamma counting of the engineered NK cellsFigure 1 (A) Multifunctional, responsive CAR constructs; (B) In vitro killing activity against GBM43 cells after co-incubation with 89Zr labeled NK cells at an E:T ratio of 10 for 4 h with LDH assay (N=3); (C) & (D) In vivo PET imaging and ex vivo gamma counting with 89Zr at week 5 in 10 mice during 4 days, GBM intracranial implantation to NSG male mouse, 89Zr, 89Zr + NK cell, or 89Zr + E NK cell (7 × 106 cells with 500 µCi) was administered through intravenous injection, Qimage was used for the PET/MRI co-registration and analysisConclusionsWe generated multifunctional E-NK cells which showed the improved killing of GBM cells using novel targeting approaches, including the blockade of CD73-mediated adenosinergic signaling. We also optimized E-NK cell radiolabeling with 89Zr for GB10 therapy in vitro and in vivo fate mapping against a xenograft of patient-derived GBM.AcknowledgementsWe gratefully acknowledge the Walther Oncology Embedding Program, Indiana University Simon Cancer Center, and In Vivo Therapeutics Core.ReferencesWang J, Matosevic S. NT5E/CD73 as correlative factor of patient survival and natural killer cell infiltration in glioblastoma. J Clin Med 2019;8(10):1526.Wang J, Lupo KB, Chambers AM, Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunother Cancer 2018;6(1):136.Yan A, Joachims ML, Thompson LF, Miller AD, Canoll PD, Bynoe MS. CD73 promotes glioblastoma pathogenesis and enhances its chemoresistance via A2B adenosine receptor signaling. J Neurosci 2019;39(22):4387.Flink J, Muzi M, Peck M, Krohn K. Multimodality brain tumor imaging: mr imaging, PET, and PET/MR imaging. J Nucl 2015;5(10):1554–1561.
TIM-3 Expression Is Downregulated on Human NK Cells in Response to Cancer Targets in Synergy with Activation