Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy

IL13R alpha 2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13R alpha 1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13R alpha 1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13R alpha 2-specific response as binding domains for CAR T cells. Despite IL13R alpha 1 and IL13R alpha 2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13R alpha 1 did not drastically change binding affinity for IL13R alpha 2. Micromolar affinity to IL13R alpha 1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13R alpha 1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CART cells, was demonstrated when cocultured with IL13R alpha 1/IL4R alpha-coexpressing cancer cells. While low-affinity interactions with IL13Ra1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13R alpha 1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13R alpha 2-expressing malignancies.

Automated summary

IL13R alpha 2 is overexpressed in various cancers, while its binding partner IL13R alpha 1 is widely expressed in healthy tissue. This study generated two variants with improved selectivity for IL13R alpha 2 response using structure-guided protein engineering, and demonstrated their potential application in treating systemic malignancies expressing IL13R alpha 2.