EXTH-50. Development of EGFR/IL13Ra2 bispecific CAR T cells to overcome heterogeneous glioblastoma

Abstract Tumor heterogeneity in glioblastoma (GBM) remains a great challenge for chimeric antigen receptor (CAR) T cell immunotherapy, as antigen-negative tumor populations can evade targeted destruction and drive recurrence. Epidermal growth factor receptor (EGFR) and interleukin-13 receptor alpha 2 (IL13Rα2) are promising targets in GBM. While murine mAb 806-derived CAR T cells effectively target overexpressed wild-type EGFR (EGFRwt) and the mutant EGFRvIII variant-expressing cells, repeated administration of this murine antibody-derived therapy risks immunogenicity. To address tumor escape and immunogenicity concerns in the setting of monovalent CAR T cell therapy, we have developed and optimized a bispecific CAR T cell incorporating both 806-derived humanized anti-EGFR single-chain variable fragment (scFv) and human IL13 targeting IL13Rα2. In this study, humanized anti-EGFR Fabs with binding affinities comparable to murine 806 were generated. Anti-EGFR CARs were constructed from these humanized scFvs and CAR T cells function were analyzed. The resulting humanized CAR T (6T) showed reduced cytotoxic activity against EGFRwt-low-expressing cells and eliminated patient-derived GBM xenografts in immunodeficient mice as efficient as 806-CAR T cells. Several bispecific CAR T cells incorporating humanized 6T scFv and human IL13 in one CAR construct were then engineered and tested. To model GBM heterogeneity, a patient-derived GBM cell line PBT206 was transduced to stably express either IL13Rα2 or EGFRvIII and mixed equally. Notably, in vitro cytotoxicity assays using this tumor mixture showed that a bispecific CAR construct (T4) efficiently eliminated both tumor cells, with comparable efficacy to the combination therapy comprised of both monovalent CAR T cells specific for either EGFR or IL13Rα2. Ongoing studies will compare the in vivo antitumor efficacy of the T4 bispecific CAR T cells with monovalent and combination CAR T treatments across multiple mouse models. Overall, this bispecific CAR T cells offer a potential therapeutic strategy to overcome antigen heterogeneity and immunogenicity in GBM.