Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

Abstract

Background: Primary human T cells engineered with chimeric antigen receptors (CARs) ex vivo can be adoptively transferred to treat cancer. CD19-targeting CAR with CD28 costimulatory domain and CD3ζ activation domain have been approved by the US FDA for treating B cell malignancies. Methods: Here we generated mutation of immunorecpetor tyrosine-based activation motifs (ITAMs) in CD3ζ, namely 1XX CAR, which altered the balance of activation and costimulation. Next we investigated whether 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their WT counterparts. Results: Even though 1XX CARs decreased cytotoxicity against tumor cells in vitro, they showed better anti-tumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells proliferated more in response to antigen stimulation in vitro, persisted longer in vivo and had higher percentage of central memory cells. As 1XX modification directly calibrates CAR activation potential, we utilized fluorescence resonance energy transfer (FRET)-based biosensor to monitor signaling dynamics downstream of CARs. Decreased ITAM numbers in 1XX resulted in similar ZAP70 activation, while 1XX induced higher Ca2+ elevation and faster Erk activation than WT CAR, which may contribute to the better therapeutic potency of 1XX. Conclusions: Our results established the surpiosity of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical application of 1XX CARs against solid tumors.

Joining Forces for Cancer Treatment: From “TCR versus CAR” to “TCR and CAR”

“…It has been postulated that increasing the number of ITAMs can lead to higher sensitivity and potency, thereby minimizing the required number of engaged receptors for an equivalent response [ 26 , 67 ]. However, in vivo studies demonstrated that CD19 CARs harboring a mutated CD3ζ signaling domain with only one functional ITAM located proximally to the membrane and ablation of the second and third ITAMs (1XX) outperformed the standard CARs containing a full CD3ζ chain, while inactivation of the two N-terminal ITAMs (XX3) demonstrated decreased efficiency [ 68 , 69 ]. Despite those advances regarding optimal ITAM positioning and number in the CD3ζ chain, the diverse roles of the other CD3 chains are currently extensively studied, not only in regard to the TCR but also as additional or alternative domains in the CAR [ 70 ].…”