Abstract 549: The IgG4 hinge with CD28 transmembrane domain mediates CAR dimerization and improves the activity of glypican 1-targeted CAR T cells in pancreatic cancer

Abstract

Abstract Low antigen density has been suggested as a major cause of insufficient antitumor activity of chimeric antigen receptor (CAR) T cells. The impact of the hinge and the transmembrane (TM) domain on modulating CAR T cell reactivity is important to investigate. Here, we isolated a camel nanobody D4 recognizing glypican 1 (GPC1), a cell surface proteoglycan that is expressed in pancreatic cancer. We constructed a series of D4 4-1BBζ CARs differing only by their hinge (CD8 or IgG4) and TM (CD8 or CD28). D4 CARs containing a mutated IgG4 hinge and the CD28 TM, but not CD8 hinge with either TM, formed dimers in human T cells and resulted in improved stability of the CAR extracellular domain in a 3D structure model. The CAR dimerization increased phosphorylation of T cell signaling molecules, activated non-canonical NF-κB signaling after antigen stimulation. Consequently, the D4-IgG4 hinge-CD28TM CAR T cells triggered the most cytokine secretion and showed the best reactivity against low GPC1-expressing pancreatic tumor cells among all the D4 CARs in vitro and in vivo. Conversely, a CAR with mutations of the two cysteine residues in the IgG4 hinge disrupted CAR dimerization, damaged the formation of TM, and resulted in the loss of improved reactivity. Furthermore, adding extra spacers of CH3 or CH2CH3 between IgG4 hinge and CD28TM CAR attenuated the CAR activity. By optimizing the hinge and TM, our data indicated that 4-1BBζ CAR T cell activity can be optimized against low antigen density tumors. Citation Format: Nan Li, Dan Li, Jiajia Pan, Raul Cachau, Mitchell Ho. The IgG4 hinge with CD28 transmembrane domain mediates CAR dimerization and improves the activity of glypican 1-targeted CAR T cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 549.