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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE310003 | GEO | 2026/02/05

REPOSITORIES: GEO

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Project description:A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive tumor microenvironment (TME), which is densely populated and supported by protumoral glioma-associated microglia and macrophages (GAMs). Targeting CD47, a don't-eat-me signal overexpressed by tumor cells, disrupts the CD47-SIRPalpha axis and induces GAM phagocytic function. However, antibody-mediated CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. To overcome these limitations, we combined local CAR T cell therapy with paracrine GAM modulation to effectively eliminate GBM. To this end, we engineered a new CAR T cell against epidermal growth factor receptor variant III (EGFRvIII) that constitutively secretes a signal regulatory protein gamma (SIRPgamma)-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in a dose-dependent manner in vitro and eradicated orthotopically xenografted EGFRvIII-mosaic GBM by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon tumor re-challenge. Combining anti-CD47 antibodies with anti-EGFRvIII CAR T cells failed to achieve a similar therapeutic effect, underscoring the importance of sustained paracrine GAM modulation. Multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry on GBM-xenografted brains showed that anti-EGFRvIII-SGRP CAR T cells accelerated GBM clearance, increased CD68+ cell trafficking to tumor scar sites and promoted GAM-mediated tumor cell uptake. In a peripheral lymphoma mouse xenograft model, anti-CD19-SGRP CAR T cells had superior efficacy to conventional anti-CD19 CAR T cells. Validation on human GBM explants revealed that anti-EGFRvIII-SGRP CAR T cells had a similar tumor-killing capacity to anti-EGFRvIII CAR monotherapy but showed a slight improvement in the maintenance of tumor-infiltrated CD14+ cells. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate immune TME. This results in the additive elimination of bystander EGFRvIII- tumor cells in a manner that overcomes the main mechanisms of CAR T cell therapy resistance, including tumor innate immune suppression and antigen escape.

Project description:Treatment efficacy with chimeric antigen receptor (CAR) T cell therapy in glioblastoma (GBM) is undermined by an immunosuppressive tumor microenvironment (TME). We previously showed that CAR T cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces anti-tumor activity against recurrent GBM and causes upregulation of programmed death-ligand 1 (PD-L1) in the TME. Here, we conducted a phase I trial to study the safety and tolerability of CART-EGFRvIII cells administered concomitantly with the PD-1 inhibitor pembrolizumab in patients with newly diagnosed, EGFRvIII+ GBM (n=7). Treatment was well tolerated in this small cohort without incidence of dose-limiting toxicity. However, no signal of efficacy was detected with a median progression-free survival of 5.2 months (90% CI, 2.9 – 6.0 months) and overall survival of 11.8 months (90 % CI, 9.2 – 14.2 months). We aimed to elucidate reasons for limited efficacy through correlative analyses. Using BBZ qPCR, we found circulating CAR T cells in 5 out of 7 patients at the time of repeat resection, but only in 1 patient in the tumor. However, shared T-cell receptors (TCRs) were found between the infusion product and the relapsed tumors, which could indicate an infiltration but lack of persistence of the CAR T cells. We further compared the tumor microenvironment of the tumors harvested before and after CAR+aPD1 administration using single cell RNA sequencing and observed comparable proportions of the major immune cell subsets. However, the myeloid and T cells infiltrating the tumors significantly evolved, with more exhausted, regulatory, and IFN-stimulated T cells at the relapse. At that time, the amount of IFN-stimulated T cells positively correlated with time from relapse to death. Together, these findings suggest that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicate a need to consider alternative immunotherapeutic strategies. ClinicalTrials.gov registration: NCT03726515.

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

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