Developing a multimodal therapy for glioblastoma using oncolytic virus delivering CD19 and EGFRvIII antigens and bi-specific CARs

Glioblastoma (GBM) is the most malignant primary brain tumor with no cure. While chimeric antigen receptor (CAR)-T cell therapy has exhibited considerable success for hematopoietic malignancies, CAR-T therapy for GBM is limited by heterogeneous tumor antigen expression, antigen loss after treatment with CAR-T targeting single antigens, and an immunosuppressive tumor microenvironment (TME). To address these challenges, we developed a multimodal immunotherapy platform for effective GBM treatment by combining bispecific CAR-T (BiCAR-T) cells with oncolytic virus carrying dual antigens and membrane-bound cytokines. Specifically, we developed a dual-antigen-encoding oncolytic virus (OVDual) that delivers truncated CD19 (CD19t) and EGFRvIII (EGFRvIIIt) antigens to GBM cells and BiCAR-T cells that are equipped with both CD19 CAR and EGFRvIII CAR. We demonstrated that OVDual effectively delivers CD19t and EGFRvIIIt to GBM cells and that BiCAR-T cells exhibited superior tumor-killing efficacy when combined with OVDual. To improve immune cell expansion/persistence and cytotoxicity, we engineered an OV expressing membrane-bound IL-15 and IL-21 (OVmIL15/21) and showed that OVmIL15/21 enhanced the expansion and killing activity of BiCAR-T and BiCAR-NK cells against GBM cells in vitro and boosted the anti-tumor effect in vivo. The combination of OVDual with BiCAR-T or BiCAR-NK cells allowed us to address the main challenges associated with inefficient CAR-T therapy for GBM, including heterogeneous antigen expression, antigen escape, and the immunologically 'cold' TME, and cytokine-armed OV further boosted the expansion/persistence and cytotoxicity of CAR immune cells. The multimodal platform developed in this study offers promising potential for effective GBM therapeutic development. Overall design: GBM cells were infected with an oncolytic virus encoding truncated CD19 and EGFRvIII antigens at a multiplicity of infection (MOI) of 1 for 4 hours, together with an oncolytic virus encoding membrane-bound IL-15 and membrane-bound IL-21 (mIL15/mIL21) or a control virus encoding GFP. After infection,Bi-specific CAR-T cells were added at a 1:1 effector-to-target (E:T) ratio. Following 48 hours of co-culture, cells were collected for RNA extraction using TRIzol reagent. The purified RNA samples were subsequently submitted to the vendor for bulk RNA sequencing (RNA-seq) analysis.