Targeting Pediatric Neuroblastoma and Glioblastoma With The Combinatorial Therapy of IL-21 Secretion Oncolytic Virus and Anti-ROR1 CAR NK Cells

Author Type(s)

Faculty

Document Type

Abstract

Publication Date

2022

DOI

10.1002/pbc.29735

Journal Title

Pediatric Blood and Cancer

Department

Pediatrics

Abstract

Background: Children with recurrent and/or metastatic neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%) (1). Novel therapies are desperately needed for these poor risk patients. ROR1 is highly expressed on the majority of NB and GBM. Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (2). Oncolytic herpes simplex viruses (oHSVs) are a promising experimental therapy. C134 is a selective replication competent oHSV with enhanced viral gene expression (3). Objectives: To determine if C134-based human IL21 expression combined with anti-ROR1 CAR engineered exPBNK cells can efficiently target ROR1+ NB and GBM. Methods: ExPBNK cells were expanded with lethally irradiated K562-mbIL21cells as we previously described (1). ExPBNK cells were electroporated with anti-ROR1-CAR mRNA using maxcyte electroporator as we previously described (2). C021 was generated by modifying C134 to express human IL-21 gene. The supernatants of C134 and C021 (C134+hIL21) were generated as previously described (3). In vitro cytotoxicity of anti-ROR1 CAR NK against NB and GBM cell lines were examined at different E:T ratios. IFN-g, granzyme and perforin levels were evaluated by ELISA assays (1). In vivo anti-tumor effect was examined utilizing human NB tumor xenografted NSG mice (1,2). Results: C021 infected NB cells (MOI 0.025) generated hIL-21 in cell supernatants at 24 hours post infection (hpi) and peaked at 72hpi. Combining C134 with exPBNK cell therapy significantly enhanced the NK-mediated killing of NB and GBM cells (p < .05, p < .05). Combinations involving C021(C134+IL21) and anti-ROR1 CAR exPBNK cells had the greatest anti-tumor effect significantly enhancing NB and GBM cell death when compared to C134 + anti-ROR1 CAR exPBNK cell combinations (p < .05, p < .05). C021 addition to enhanced anti-ROR1 CAR exPBNK killing significantly increases IFN-g (p < .05), granzyme B (p < .05) and perforin (p < .05) secretion and significantly upregulated the NK activating marker CD25 (p < .05). Our in vivo animal study showed that C021 and anti-ROR1 CAR exPBNK cell combination reduced tumor burden in human NB xenografted NSG mice compared to untreated group (p < .05) and anti-ROR1 CAR exPBNK treated group (P = 0.056). Conclusion: Our data demonstrated the anti-tumor efficacy of the combination of oHSVs C021 with anti-ROR1 CAR exPBNK cells targeting NB and GBM cells In vitro and in vivo.

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