Engineering γδT cells limits tonic signaling associated with chimeric antigen receptors

Abstract

Despite the benefits of chimeric antigen receptor (CAR)–T cell therapies against lymphoid malignancies, responses in solid tumors have been more limited and off-target toxicities have been more marked. Among the possible design limitations of CAR-T cells for cancer are unwanted tonic (antigen-independent) signaling and off-target activation. Efforts to overcome these hurdles have been blunted by a lack of mechanistic understanding. Here, we showed that single-cell analysis with time course mass cytometry provided a rapid means of assessing CAR-T cell activation. We compared signal transduction in expanded T cells to that in T cells transduced to express second-generation CARs and found that cell expansion enhanced the response to stimulation. However, expansion also induced tonic signaling and reduced network plasticity, which were associated with expression of the T cell exhaustion markers PD-1 and TIM-3. Because this was most evident in pathways downstream of CD3ζ, we performed similar analyses on γδT cells that expressed chimeric costimulatory receptors (CCRs) lacking CD3ζ but containing DAP10 stimulatory domains. These CCR-γδT cells did not exhibit tonic signaling but were efficiently activated and mounted cytotoxic responses in the presence of CCR-specific stimuli or cognate leukemic cells. Single-cell signaling analysis enabled detailed characterization of CAR-T and CCR-T cell activation to better understand their functional activities. Furthermore, we demonstrated that CCR-γδT cells may offer the potential to avoid on-target, off-tumor toxicity and allo-reactivity in the context of myeloid malignancies.

Engineering Solutions for Mitigation of Chimeric Antigen Receptor T-Cell Dysfunction

“…EF1a promoter resulted in bright CAR surface expression accompanied by non-antigen specific signalling, while this was not observed using CMV or truncated PGK promoters. Importantly, this observation with EF1a promoter was made using FMC63-based CD19 CAR previously described as free from tonic signalling [43,45]. Eyquem and colleagues further showed attenuation of CAR expression by targeting the CAR into the T-cell receptor alpha constant (TRAC) locus [69].…”

“…By expressing chimeric co-stimulatory receptors against target antigens to provide co-stimulation in trans, the engineered cells can mount effective responses against tumour cells expressing the target antigen whilst ignoring normal tissues expressing the same target antigen at the same levels [133]. Indeed, single cell signalling studies performed with this model system has demonstrated a lack of tonic signalling, which is linked with the absence of expression of exhaustion markers [45].…”

“…In first generation CARs, signal 1 of T cell activation is affected by a single signalling domain most commonly CD3zeta (CD3ζ), whilst second and third generation CARs have an additional one or two co-stimulatory domains, respectively ( Figure 1 B). Poor CAR design can lead to suboptimal T-cell activation, on-target off-tumour toxicities and antigen-independent (tonic) signalling; the latter resulting in an exhausted phenotype [ 43 , 44 , 45 , 46 , 47 , 48 ]. All regions of the CAR are important in determining function, and hence, are substrates for engineering solutions ( Figure 1 C).…”

Chimeric Antigen Receptor beyond CAR-T Cells

“…This precise discrimination provides a safeguard for solid tumours that lack tumour-specific antigens. Additionally, separating co-stimulatory signals can abrogate tonic signalling, mirrored by potent effector functions and lower exhaustion marker expression [ 25 ]. This feature may prolong the in vivo persistence of the gene-modified T cells.…”

Engineering the Bridge between Innate and Adaptive Immunity for Cancer Immunotherapy: Focus on γδ T and NK Cells

“…Using “AND gate” system, which divides the signals, CCR γδ T cells were activated and released cytokines only when both CD3 and CCR receptors were engaged, whereas in CAR γδ T cells only CAR activation was sufficient. Using single-cell signaling analysis and mass cytometry (CyTOFF), which allowed a detailed characterization of the CAR signaling, they demonstrated that CCR in γδ T cells induced minimal baseline activity of CD3ζ-associated kinases in the absence of the antigen, meaning that tonic signaling decreased and the network plasticity increased [ 100 ]. This approach can ameliorate engineered T cells exhaustion and off-target cytotoxicity, features that affect consistently the clinical efficacy of CAR-T cells [ 40 ] ( Figure 1 ).…”