Selective IL13Rα2-Targeted Functionality of IL13-Ligand CARs is Enhanced by Inclusion of 4-1BB Co-Stimulation

Chimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand- based CAR targeting the cancer antigen IL13Rα2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13Rα2 versus the more ubiquitous unintended target IL13Rα1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3ζ- containing CARs (IL13ζ), second-generation 4-1BB- (CD137) or CD28-containing CARs (IL13- BBζ or IL13-28ζ), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BBζ). In vitro co-culture assays at high tumor burden establish that 2nd generation IL13-BBζ or IL13- 28ζ outperform first-generation IL13ζ and 3rd generation IL13-28BBζ CAR designs, with IL13- BBζ providing superior CAR proliferation and in vivo anti-tumor potency in human xenograft mouse models. IL13-28ζ displayed a lower threshold for antigen recognition, resulting in higher off-target IL13Rα1 reactivity both in vitro and in vivo . Syngeneic mouse models of GBM also demonstrate safety and anti-tumor potency of murine IL13-BBζ CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BBζ CARs for greater selective recognition of IL13Rα2 over IL13Rα1, higher proliferative potential, and superior anti-tumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition.. ### Competing Interest Statement Patents associated with IL13Rα2-CAR T have been licensed by Mustang Bio., Inc., for which S.J. Forman and C.E. Brown receive royalty payments.