Abstract 2943: 14g2a-based GD2-specific chimeric antigen receptors (CARs) constitutively signal, leading to rapidly induced T-cell exhaustion and poor antitumor efficacy in vivo
Authors: Long Adrienne , Orentas Rimas J. , Mackall Crystal L. 
Published: May 2015

Abstract
Chimeric antigen receptors (CARs) provide a promising new approach for generating T cells for the adoptive immunotherapy of cancer. In both pre-clinical and clinical studies, we have observed potent anti-tumor effects using CD19 CAR T cells (CART) to treat B cell malignancies. However, we observe less success when targeting solid tumors with GD2 CART. We hypothesized that the observed differences could be due to a more hostile microenvironment in solid tumors, and/or variable CAR potency. To address these issues, we created a CD19+GD2+ osteosarcoma (143B-CD19). This double positive solid tumor line allowed us to directly compare the in vivo efficacy of the GD2 and CD19 CARs, normalized for tumor type. In vitro 51Cr release demonstrated that CD19 and GD2 CART were equally active against 143B-CD19. However, in vivo models showed a significant difference in anti-tumor efficacy. NSG mice were injected with 143B-CD19 and treated with control, GD2 or CD19 CART 14d later. GD2 CART failed to persist in mice and did not inhibit tumor outgrowth. However, CD19 CART did persist and cleared the mice of all CD19+ solid tumor disease. Therefore, we conclude that the solid tumor microenvironment is not a barrier to effective CART therapies, and hypothesize that poor GD2 CAR potency leads to poor in vivo efficacy.
Further comparison of GD2 and CD19 CART demonstrated that excessive, rather than insufficient, signaling by the GD2 CAR contributes to the poor in vivo efficacy. GD2 CART have increased expression of activation makers (CD25, CD127, CD137) and increased size compared to CD19 CART following in vitro expansion. Despite this increased activation, GD2 CART expand poorly, have high rates of apoptosis, produce IL2, TNFa and IFNg poorly, and express high levels of PD1, TIM3, LAG3, and BLIMP1. This phenotype is consistent with T cell exhaustion, a state of poor effector function due to chronic antigen stimulation. This phenotype is prevented by knocking out GD2 CAR signaling through point mutations in the CD3 zeta and CD28 domains of the receptor, implicating tonic signaling through the CAR during in vitro expansion as the source of T cell exhaustion. Interestingly, we did not identify GD2 expression in the culture system. Point mutations in the CAR antigen-binding site, though blocking GD2 binding, did not prevent exhaustion. Thus, we postulate that constitutive receptor signaling may occur via interactions between the framework regions of the CAR, leading to exhaustion. This exhausted phenotype was recapitulated with CD19 CART when expanded in the presence of a crosslinking anti-idiotype antibody, suggesting that over-activation via other CARs also has the potential to limit efficacy. Ongoing experiments focus on elucidating the molecular pathways that link CAR signaling and induction of T cell exhaustion, as well as identifying the structural features of the GD2 CAR that lead to excessive signaling.
Citation Format: Adrienne Long, Rimas J. Orentas, Crystal L. Mackall. 14g2a-based GD2-specific chimeric antigen receptors (CARs) constitutively signal, leading to rapidly induced T-cell exhaustion and poor antitumor efficacy in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2943. doi:10.1158/1538-7445.AM2014-2943