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Background Glioblastoma (GBM) is the most common malignant brain tumor, accounting for 51% of all malignant central nervous system tumors, 1 with a dismal median survival of 12-15 months with current therapies. New therapeutics are urgently needed. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is upregulated in GBM and play a key role in maintenance and tumorigenicity of glioma stem-like cells (GSCs),2 a subpopulation associated with GBM recurrence and resistance. Our group has successfully ex vivo expanded functional peripheral blood NK cells (exPBNK) and electroporated with anti-ROR1 CAR mRNA.3 Oncolytic herpes simplex viruses C134, a neurovirulent deleting γ134.5 and expressing the human cytomegalovirus IRS1, is a promising experimental therapy.4 We previously demonstrated enhanced cytotoxicity against neuroblastoma using HSV expressing human IL-21(HSV C021) in combination with ROR1.CAR.5 NKTR-255 is an investigational IL-15Rα-dependent, polymer-conjugated IL-15 agonist that promotes NK cell expansion. Here, we investigate the anti-tumor effects of anti-ROR1 CAR NK with and without HSV C021 and/or NKTR-255 against GBM.Methods NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells and anti-ROR1 CAR mRNA was electroporated to exPBNKs as we previously described. 3 NKTR-255(40ng/ml) and HSV C021(MOI=0.1) were added for in vitro cytotoxicity assays. IFN- γ, granzyme B, perforin and IL-21 levels were examined by ELISA.5 ROR1 positive M059K were used as target cells.Results The GBM cell line M059K showed high ROR1 expression ( figure 1A). The ROR1.CAR NK demonstrated significantly enhanced cytotoxicity against GBM cells and elevated IFN-γ secretion compared to unmodified NK cells (p<0.0001) (figure 1B). HSV with IL-21 expression alone induced 28.07±6.49% tumor cell death and significantly enhanced the in vitro cytotoxicity of both mock NK cells or ROR1.CAR NK against GBM at 48 hours (p < 0.05). Secreted IL-21 was detected in the supernatant of C021-infected GBM cells at an MOI of 0.1,0.01 or 0.001, with levels peaking at 48 hours post-infection and lasting for 7 days. When combined with NKTR-255, HSV C021 further increased cytotoxicity of mock NK cells from 0.29 ± 1.93% to 77 ± 0.66%, and of ROR1.CAR NK cells from 40.04 ± 9.76% to 90.62 ± 0.7% (p < 0.0001) (figure 1B). Increased IFNγ secretion was also confirmed by ELISA assay (p < 0.05) (figure 1C).Conclusions The combination of HSV C021 with NKTR-255 significantly enhanced ROR1.CAR cytotoxicity against GBM in vitro, accompanied by elevated IFN γ secretion. In vivo evaluation using humanized NSG models is currently underway.References Ostrom QT, Price M, Neff C, Cioffi G, Waite KA, Kruchko C, Barnholtz-Sloan JS. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the USA in 2016-2020. Neuro Oncol. 2023 Oct 4;25(12 Suppl 2):iv1-iv99.Zhu H, Cheng L, Liu D, Ma X, Chen Z, Fan H, Li R, Zhang Y, Mi H, Li J, Zhang S, Yu X, Shu K. ROR1 facilitates glioblastoma growth via stabilizing GRB2 to promote c-Fos expression in glioma stem cells. Neuro Oncol. 2025 Mar 7;27(3):695-710.Chu Y, Nayyar G, Tian M, Lee DA, Ozkaynak MF, Ayala-Cuesta J, Klose K, Foley K, Mendelowitz AS, Luo W, Liao Y, Ayello J, Behbehani GK, Riddell S, Cripe T, Cairo MS. Efficiently targeting neuroblastoma with the combination of anti-ROR1 CAR NK cells and N-803 in vitro and in vivo in NB xenografts. Mol Ther Oncol. 2024 May 24;32(2):200820.Cassady KA, Bauer DF, Roth J, Chambers MR, Shoeb T, Coleman J, Prichard M, Gillespie GY, Markert JM. Pre-clinical Assessment of C134, a Chimeric Oncolytic Herpes Simplex Virus, in Mice and Non-human Primates. Mol Ther Oncolytics. 2017 Mar 1;5:1-10.Chu Y, Tian M, Saini U, Ayala-Cuesta J, Klose K, Mendelowitz AS, Foley K, Ozkaynak MF, Luo W, Cripe TP, Lee DA, Cassady KA, Cairo MS. Combinatorial immunotherapy with anti-ROR1 CAR NK cells and an IL-21 secreting oncolytic virus against neuroblastoma. Mol Ther Oncol. 2024 Dec 21;33(1):200927.Abstract 650 Figure 1A) Expression of ROR1 in GBM cell line. In vitro cytotoxicity B) and IFN-gamma release C) of unmodified exPBNK or ROR1.CAR NK, with/without NKTR-255 and/or HSV C021, against GBM cells at 10:1 ratio