Abstract 3746: Plasma membrane spanning and linker-domains from tumor necrosis factor receptor superfamily (TNFRSF) proteins provide novel functionality to chimeric antigen receptors (CARs) expressed in human T cells
Authors: Schneider Dina , Xiong Ying , Wu Darong , Dropulic Boro , Orentas Rimas 
Published: July 2017

Abstract
Chimeric antigen receptor T cells redirected to the B cell antigen CD19 (CAR19) have shown efficacy in the treatment of B-lineage acute lymphocytic leukemia, and are being evaluated in other hematologic malignancies as well. The prevalent configuration of CAR19 for clinical application is comprised of the extracellular single chain targeting domain (scFv) derived from the FMC63 antibody, a CD8 linker and transmembrane domain, a CD137 signaling domain, and a CD3-zeta signaling domain. Transmembrane domains from other immunologically-active proteins, including CD3, CD28, or CD4, and spacer domains from IgG have been employed as structural components of CARs as well. The relationship between the composition of the CAR linker and transmembrane domain and CAR T function is, however, poorly understood. We hypothesized that CAR transmembrane and linker domains derived from proteins of the tumor necrosis factor receptor superfamily (TNFRSF), whose expression is associated with normal T cell functions, may optimize the anti-tumor activity of CAR19. Therefore, primary human T cells were transduced with lentiviral expression vectors (LV) expressing new combinations of linker and transmembrane sequences encoded by various TNFRSF members. Specifically, the linker:transmembrane domain combinations were: CD8:CD4, CD8:TNFRSF19, TNFRSF19:TNFRSF19, truncated TNFRSF19:TNFRSF19, TNFRSF9:TNFRSF9 and TNFRSF16:TNFRSF16.
We found that CAR T cells comprised of transmembrane domains derived from either TNFRSF19 or TNFRSF9 were superior to the CD8 transmembrane CAR 19 in tumor killing, and in the induction of IFN gamma, TNF alpha, IL-2, and GM-CSF cytokine secretion in vitro, upon co-culture with leukemia cell line targets. Moreover, TNFRSF19-expressing CAR19 T cells were superior to the CD8 transmembrane CAR19 in the elimination of engrafted Raji cells (a Burkitt’s lymphoma line) in an NSG leukemia mouse model. On the other hand, the transmembrane domain derived from TNFRSF16/LNGFR, a low affinity neurotrophin receptor, created a CAR19 construct that had no functional activity either in vitro or in vivo, despite high levels of surface CAR expression in human T cells.
In conclusion, the magnitude of CAR T response can be enhanced by the use of novel linker and transmembrane domains derived from TNFRSF19 and TNFRSF9. Thus, the transmembrane domain can play a determining role in CAR T function beyond a direct topographical connection between the extracellular and the intracellular domains of the chimeric protein. Studies are underway to elucidate the mechanistic role of TNFRSF-derived linker and transmembrane domains in CAR T function.
Citation Format: Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. Plasma membrane spanning and linker-domains from tumor necrosis factor receptor superfamily (TNFRSF) proteins provide novel functionality to chimeric antigen receptors (CARs) expressed in human T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3746. doi:10.1158/1538-7445.AM2017-3746