Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

Abstract

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

Acute lymphoblastic leukemia relapse after CD19-targeted chimeric antigen receptor T cell therapy

“…CAR19 stimulation yields many of the same signal transduction events that are observed in TCR stimulation [35], which suggests that some mechanisms of CTL dysfunction could also apply to CART19 cell. In addition, either diminished functions (Fig.…”

Third-generation CD28/4-1BB chimeric antigen receptor T cells for chemotherapy relapsed or refractory acute lymphoblastic leukaemia: a non-randomised, open-label phase I trial protocol

“…However, distinct signalling of CD28 or 4-1BB co-receptors have different influence on the metabolic characteristics of CAR-T cells and balance the response towards long-term persistence (long-lived memory) or immediate antitumour potency (short-lived effect cells). Moreover, a preclinical study showed that 3rd-G CAR-T cells combining CD28 and 4-1BB co-receptors had superior in vitro activation and proliferation capacity compared with 2nd-G CAR-T cells carrying CD28 signal domains, and both kinds of cells displayed in vivo comparable efficacy in eliminating CD19+ B cells 35. Another preclinical study demonstrated that in vivo persistence of 3rd-G CAR-T cells was inferior to that of 2nd-G ones 41.…”

“…Do the third-generation (3rd-G) CARs combining 4-1BB and CD28 signalling domains have these two different functional properties for T cells? Although preclinical study showed that 3rd-G CAR-T cells had a higher activation status than that of 2nd-G,35 there is no clinical evidence yet for using them in treating patients with B-ALL. Therefore, a phase I clinical trial will be conducted to treat r/r B-ALL using anti-CD19 3rd-G CAR-T cells.…”

CARs: Synthetic Immunoreceptors for Cancer Therapy and Beyond

“…It is worth noting that the specific effects of CAR expression on T-cell biology appear to correlate more strongly with the type of CAR expressed (e.g., CARs containing CD28 vs. 4-1BB) than with the genetic background of the T cells, as illustrated by transcriptional profiling of CD28 and 4-1BB CAR-T cells generated from multiple donors [10]. Furthermore, the number of costimulatory domains incorporated into CAR molecules has been shown to affect the basal phosphorylation levels of signaling proteins important in human T-cell activation (LAT, ZAP-70, SYK, ERK, and LCK) [13], suggesting that the specific composition of CAR molecules can profoundly influence T-cell biology independent of antigen stimulation. A recent study compared the function of T cells expressing a CD19 CAR that was either randomly integrated via retroviral transduction, or site-specifically integrated into the TCR α constant ( TRAC ) or β 2 -microglobulin loci, with, or without additional promoter/enhancer elements [12].…”