Fig.5.TR1419-28BBζ CAR-T cells showed greater antigen-specific expansion compared with TR1419-28ζ CAR-T cells and TR1419-BBζ CAR-T cells. (A) Expression of CD137 on TR1419CAR-T cells was detected by flow cytometry cultured overnight on plate-bound recombinant human TRAIL-R1 at varying protein concentrations. (B, C) Cytotoxic activity of TR1419-28ζ CAR, TR1419-BBζ CAR or TR1419-28BBζ CAR T cells against TRAIL-R1 positive tumor cell lines (SW480 and HCT116). The effector cells were co-cultured for 6 h with target cells at E:T ratio of 14:1, 7:1 and 3:1, respectively. Cytotoxicity was determined by Calcein AM release-based cytotoxic cell assay. The analysis was performed using Students’ t tests. Data reflected the mean ± SEM of three triplicate wells,*p < 0.05. (D) TR1419CAR T cell proliferation indicated by division of CFSE was assessed by flow cytometry. TR1419-28ζ CAR, TR1419-BBζ CAR and TR1419-28BBζ CAR T cells were stained with CFSE, respectively. Then, TR1419CAR T cells were cultured with SW480 for 7 d, and analyzed by flow cytometry. (E-H) Expression of PD-1 on TR1419CAR T cells was analyzed by FACS after 7 days of co-culture with tumor cells. TR1419CAR T cells were classified as PD-1 negative, PD-1low and PD-1high expressing sub-populations. The analyses were performed using Students’ t tests. Data reflected the mean ± SEM of three independent experiments,*p < 0.05, **p < 0.01.
Discussion
In the present work, we demonstrated that TR1419-based CAR-T cells had potent dual anti-tumor activities in vitro. On the one hand, TR1419CAR can mediate tumor cell apoptosis through activation of the death receptor-dependent apoptotic pathway. On the other hand, TR1419CAR can effectively induce T-cell mediated cytotoxicity by inducing multiple signals with the binding of TRAIL-R1. Meanwhile, by comparing phenotype and function between the second- and third-generation TR1419CAR-T cells, the third-generation TR1419-28BBζ CAR-T cells showed higher sensitivity to target antigen, proliferated more rapidly and expressed higher levels of PD-1 upon antigen stimulation.
Previously, we developed fully human TR1419-IgG with tumoricidal activity, but this antibody required additional crosslinking antibodies to induce apoptosis in the target cells [32]. We demonstrated that the scFv from TR1419 designed into CAR structure can directly mediate tumor apoptosis by binding to TRAIL-R1 in the absence of additional crosslinking antibodies. The underlying mechanism of cell apoptosis induced by scFv from TR1419 may be related to the spatial structure of the TR1419CAR. The specific mechanism still requires further research and investigation.
Currently, the clinical efficacy of CAR-T cells in hematological malignancies is rarely achieved in solid tumors, and the factors necessary for improving its efficacy are currently being determined [33,34]. Combinations with other treatments will be a promising strategy to improve the efficacy of CAR-T cells on solid tumors [35]. In our study, TR1419-based CAR-T cells had potent dual anti-tumor activities. The induction of apoptotic signals from TR1 on the target cells was equivalent to that of other therapies in this case. However, the apoptotic signals from TR1 are targeted, unlike chemotherapy and radiotherapy, without inducing localized immune cell damage [36]. Moreover, TR1-mediated tumor cell apoptosis can induce immunogenic cell death of tumor cells which enhances antigen presentation and antitumor immune responses.
Although, the role of costimulatory signals in CAR-T development is well-established, the optimal costimulatory domains for CAR-T cells remains to be defined, and should be evaluated case-by-case in order to fine-tune immunotherapy approaches [37,38]. Literature reported that the choice of 4-1BB signaling domain in CARs conferred improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype and reduced basal T-cell activation [39,40]. In our research, there was no significant difference in phenotypes and function between the TR1419-28ζ and TR1419-28BBζ CAR-T cells. However, the third-generation TR1419-28BBζ CAR-T cells showed higher sensitivity to target antigen and proliferated more rapidly upon antigen stimulation. The incongruent results may have the following reasons: 1) the various preparation methods and experimental conditions, 2) the various scFv domains, 3) the various other components of CARs.
There are some limitations in our study that requires further acknowledgement. Further and more comprehensive studies are required to confirm these findings. And additional in vivo experiments are needed to verify the results of the above in vitro. Lastly, the safety issues related to off-tumor toxicity also needs further studies to address. In conclusion, we designed and demonstrated that TR1419CAR-T cells can mediate dual tumor cell killing, and the third-generation TR141928BBCAR-T cells exhibited a higher sensitivity at low antigen, and proliferated more rapidly upon antigen stimulation, compared to the second-generation TR1419CAR-T cells. This dual killing mechanism provides a novel strategy for other CARs design.