Optimizing Chimeric Antigen Receptor (CAR)-T Cell Therapy Against Glioblastoma Through Tandem CARs Against IL13Rɑ2 and EGFRvIII

Abstract

Adoptive cell therapy with chimeric antigen receptor (CAR)-T cells represents a significant advancement in personalized cancer treatment. While CAR-T cell therapy has demonstrated remarkable response rates in hematological malignancies, significant challenges remain in designing CAR-T cells against solid tumors. This is in part due to the heterogeneous expression of tumor antigens in solid tumors. Glioblastoma (GBM) is a solid tumor with a poor prognosis and a median length of survival of 15 months. Current treatments offer minimal benefit, and there is a dire need for more effective and less toxic therapeutics. Currently, efforts to develop CAR-T cell therapies against GBM are underway. Two antigens, in particular, EGFR variant III (EGFRvIII) and IL13Rα2 are attractive targets as they are both enriched on cancer cells, yet absent on healthy brain tissue. We are addressing antigen heterogeneity in GBM through the use of tandem CAR-T cells that target both IL13Rα2 and EGFRvIII. These dual-targeting CAR-T cells can increase potency by targeting two different antigens, while also retaining efficacy with the presence of one antigen. We designed 2nd generation CARs targeting IL-13Rα2 (CAR-IL13Rα2) and EGFRvIII (CAR-EGFRvIII) individually and concurrently (Tandem CAR). Our in vitro model utilized of the GBM cell lines U87 and U251, a GBM-derived cancer stem cell line that forms neurospheres, BT74, and K562, artificial antigen presenting cells, expressing either IL13Rα2+ or EGFRvIII+. In vitro cytotoxicity assays indicated that the Tandem CAR demonstrates superior killing capacities in mixed populations of IL13Rα2+ and EGFRvIII+ target cells at effector:target ratios of 10:1, 3:1, and 1:1, both for U87 and U251. The differential cytotoxicities of the Tandem CAR versus single antigen CARs were evident as early as several hours into the assay and continued up until 72 hours. There is also comparable or superior cytotoxicity of the Tandem CAR against the BT74 cell line, varying at different effector:target ratios. Activation assays, based on CD69 expression, and long-term proliferation assays indicate that each target antigen is sufficient to trigger Tandem CAR effector function at a level comparable to CAR-IL13Rα2 and CAR-EGFRvIII. In vivo data with subcutaneous implantation of K562 expressing either IL13Rα2 or EGFRvIII in the flanks of mice, as well as intracranial implantation of BT74, intracranial implantation of mixed U87 populations, and intracranial implantation of mixed U251 populations, demonstrate similar or superior anti-tumor activity of the Tandem CAR compared to the monospecific CARs. Altogether, our data demonstrate that targeting multiple GBM antigens through the Tandem CAR not only has comparable activity to single antigen CARs but may also be superior in terms of cytotoxicity and efficacy in heterogeneous tumor populations.