Background Glioblastoma (GBM) is the most lethal form of primary brain tumor in adults, with a 95% five-year mortality rate. Current therapy consists of surgical resection, chemotherapy, and radiotherapy. However, there remains an urgent need for novel therapies as recent trials have failed to improve overall survival.¹ A new approach to target GBM is the administration of Chimeric Antigen Receptor (CAR) T cells to treat relapsed/refractory disease.² To date, multiple clinical trials employing CAR T cells targeting disparate antigens in the context of GBM have been completed; and analysis of data accrued from these trials identified antigen loss and the upregulation of T cell inhibitory pathways to be major limitations of CAR T cell antitumor efficacy.^3,4 Accordingly, we have identified B7-H3 as a potential target antigen for CAR T cell therapy of GBM, and the Programmed Death-1/Programmed Death Ligand-1 (PD-1/PD-L1) axis as an inhibitory pathway suitable for combinatorial immunotherapy with CAR T cell therapy of GBM.

Methods B7-H3 expression on U251, U87, and 4 primary GBM cells lines was evaluated by flow cytometry, while patient tumor samples were analyzed by immunofluorescence. B7-H3 targeting CAR T cells were engineered by transducing primary human T cells with a retroviral vector encoding either a CD28-based CAR, or a CD28-based CAR containing a PD-1 blocking scFv. In vitro luciferase-based killing assays, and Luminex-based quantification of cytokine release were used to characterize CAR function. NSG mice were engrafted with U251 GBM cell line and treated with a single peripheral infusion of CAR T cells. Tumors were harvested at various time points following CAR T cell treatment and characterized for T cell infiltrate, T cell phenotype, and expression of checkpoint pathways.

Results We demonstrate that B7-H3 is a suitable antigen for CAR T cell therapy of GBM, and that B7-H3-targeting CAR T cells are able to safely, and efficiently control disease progression in a disease-relevant xenograft model. We also show that GBM tumor cells upregulate PD-L1 in direct response to CAR T cell activity, and that CAR T cells upregulate PD-1 following activation through the CAR. We subsequently demonstrate that concomitant PD-1 blockade augments the antitumor capabilities of B7-H3-targeting CAR T cells through increased T cell engraftment and improved effector function to confer durable, long-term remissions in a disease-relevant preclinical model.

Conclusions These data demonstrate the concomitant PD-1 blockade is safe and efficient method for improving the antitumor capabilities of B7-H3 targeting CAR T cells in the context of GBM.

References

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2. Bagley SJ, Desai AS, Linette GP, June CH, O’Rourke, DM. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro Oncol. 2018; 20: 1429–1438.

3. Brown CE, Badie B, Barish ME, Weng L, Ostberg JR, Chang W-C, Naranjo A, Starr R, Wagner J, Wright C, Zhai Y, Bading JR, Ressler JA, Porgnow J, D’Apuzzo M, Forman SJ, Jensen MC. Bioactivity and safety of IL13R 2-redirected chimeric antigen receptor CD8+ T cells in patients with recurrent glioblastoma. Clin Cancer Res. 2015; 21: 4062–4072.

4. O’Rourke DM, Nasrallah MP, Desai A, Melenhorst JJ, Mansfield K, Morrissette JJD, Martinez-Lage M, Brem S, Maloney E, Shen A, Isaacs R, Mohan S, Plesa G, Lacey SF, Navenot J-M, Zheng Z, Levine BL, Okada H, June CH, Brogdon JL, Maus MV. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med. 2017; 9: eaaa0984.

Ethics Approval The human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #09-156.