Chimeric antigen receptor (CAR) design incorporates a binding moiety to direct T cell activation toward an individual cancer biomarker that a T cell would not normally recognize. Although antibody variable fragments are commonly used for their versatility in antigen recognition, naturally occurring ligands are also capable of directing specificity. One such ligand, IL13, has been incorporated as the binding moiety for CAR T cell therapy of IL13 receptor α2 (IL13Rα2) expressing glioblastoma multiforme (GBM). This IL13-CAR T cell therapy has shown notable safety and success in early stage clinical trials via local delivery. IL13Rα2 is an attractive target for many malignancies and has rare normal tissue expression, motivating application of systemic delivery of IL13-CAR T cells. However, IL13Rα1, the other receptor for IL13, has low level ubiquitous expression on normal tissue. Notably, IL13Rα1 and ILR4α, a high affinity receptor pair for IL13, are co-expressed in lung tissue. This normal tissue expression raises concerns for systemic application of IL13-CAR T cells including 1) impedance of CAR T cell trafficking, 2) early exhaustion of CAR T cells due to antigen experience on route to tumors, and 3) on-target off-tumor toxicity. Herein, we describe the engineering and investigation of CARs incorporating IL13 mutants (termed muteins) with markedly enhanced selectivity toward IL13Rα2-binding. Structure-guided mutagenesis of IL13 yielded two mutants that retain binding affinity for IL13Rα2 while diminishing affinity for IL13Rα1 &gt;1,000-fold (termed C4 and D7). CARs incorporating these muteins direct T cell-mediated cytotoxicity against IL13Rα2-expressing tumors identically to IL13-CAR T cells both in vitro and in an orthotopic GBM model. Importantly, both muteins display significantly diminished T cell activity against IL13Rα1-overexpressing tumors in vitro, but only the C4 variant spares IL13Rα1 and IL4Rα co-expressing tumors in vitro and in vivo. T cell trafficking studies illuminate potential advantages of decreased IL13Rα1 interactions in the mutein-CAR context.

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

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.