Abstract 5513: Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape
Authors: Peralta Eigen , Lu Dan , Landon Mark , Chu Hui-Yi , Mehta Amit , Chu Philip , Witty Alec , Lee Tom , Valamehr Bahram 
Published: June 2022

Despite success in hematologic malignancies, chimeric antigen receptor (CAR) T cells have been less effective in solid tumors, in part because of the heterogeneous expression of the CAR-specific target antigen (1° Ag) found within the tumor mass. In combination with a CAR, bispecific T cell engagers (BiTEs) can target additional tumor antigens (2° Ag) while engaging CD3 signaling molecules on T cells, providing a unique way to address tumor heterogeneity, mitigate antigen escape and further potentiate durable effector function. However, in generating off-the-shelf universal CAR-T and NK cells, a combination strategy is not feasible as neither modified cell product is compatible to specifically engage with a BiTE. In developing allogeneic CAR-T cells, the T cell receptor (TCR) surface expression must be eliminated to prevent graft versus host disease, but the absence of surface TCR expression leads to loss of surface CD3 expression and BiTE compatibility. Similarly, NK cells naturally lack TCR expression and have no surface CD3 molecules for BiTE engagement.

Here we discuss the development of a novel chimeric CD3ε fusion receptor (CD3-CFR) with a modified transmembrane and endodomain enabling surface expression in TCR-less T or NK cells, allowing for a novel combinatorial solution between universal CAR-T or NK cells with BiTEs in an allogeneic setting.

CD3-CFR signal transduction was initially investigated in TRAC knockout NFAT reporter (T-KO) Jurkat cells engineered with CD3-CFR and co-cultured with EpCAM+ target cells. When soluble EpCAM-BiTE was added to the co-culture, the observed 6.9-fold increase in NFAT activity indicated successful engagement associated with the interaction of CD3-CFR, target cells and the BiTE. CD3-CFR T-KO Jurkat cells were further modified to secrete the EpCAM-BiTE and showed increased NFAT activity, demonstrating the feasibility of self-secreting BiTE-mediated targeting through CD3-CFR signaling.

We next engineered CD3-CFR expressing iPSC-derived CAR-T (iT) cells to show preserved T cell differentiation kinetics and maintained CAR activity against 1° Ag positive targets (&gt;90% cytolysis). Importantly, while CAR-iT cells showed only background lysis of 1° Ag negative tumor cells, CD3-CFR+ CAR-iT cells were able to lyse ~99% of 1° Ag negative tumor cells with the addition of soluble EpCAM-BiTE targeting the 2° Ag. When CD3-CFR+ CAR-iT cells were further engineered to secrete the EpCAM-BiTE, CD3-CFR+ BiTE+ CAR-iT cells demonstrated superior killing of heterogenous tumors compared to CD3-CFR+ BiTE- CAR-iT cells (70% vs. 5% cytolysis). Taken together these data demonstrate for the first time that CD3 expression and BiTE engagement can be combined in a universal iPSC-derived CAR-iT cell product to overcome antigen heterogeneity and enhance efficacy in solid tumor settings.

Citation Format: Eigen Peralta, Dan Lu, Mark Landon, Hui-Yi Chu, Amit Mehta, Philip Chu, Alec Witty, Tom Lee, Bahram Valamehr. Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape [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 5513.