Construction And Functional Study Of Chimeric Antigen Receptor T Cells With IL-12R Intracellular Domain

The current common treatments for cancer include surgery,radiation therapy,and chemotherapy.But these methods have limited effects in treating advanced cancer.Therefore,it is urgent to seek new treatments.Base on the remarkable effects of chimeric antigen receptor T?CAR-T?cells in treating hematological malignancies,CAR-T cells become research hotspot in treating solid tumors recently.However,a variety of factors including the tumor immunosuppressive microenvironment limited the application of CAR-T cells in the treatment of solid tumors.Therefore,a large amount of research is devoted to optimize the structure of CARs to improve the anti-tumor efficacy of T cells.Optimal activation of T cells requires the participation of three signals,including 1^st signal provided by T cell receptor?TCR?,2^nd signal provided by co-stimulatory molecules,and 3^rd signal provided by cytokines.However,the intracellular domain of CAR used in the clinic does not include a structure capable of transmitting 3^rd signal,and it only contains inhibitory signals such as TGF-?and IL-10in the tumor microenvironment.Therefore,one of the optimization methods of CAR structure is focusing on providing additional activated cytokine signals for CAR-T cells.As a cytokine that promotes Th1 cells differentiation,IL-12 is a classic activating 3^rdd signal during T cell activation and differentiation,and is considered to be an important effector molecule involved in restricting tumorigenesis.For this reason,the combination of CAR T cell therapy with constitutive or inducible IL-12 expression has been extensively explored for the treatment of several malignancies in preclinical stage.Meanwhile,the CAR has been armored with the ability to secrete interleukin-12?IL-12?.However,the possible lethal toxicities due to the unpredictable high levels of IL-12 required additional modifications for further application in the treatment of cancer patients.The biological functions of IL-12 are mainly due to its receptor mediated signal transduction.IL-12R?1 and IL-12R?2 subunits are closely related to the differentiation process of T cells.Therefore,in order to reduce the toxicity caused by the excessive secretion of cytokines and provide an activating 3^rd signal in the inhibitory microenvironment to overcome the inhibitory effect of tumor microenvironment.We tried to integrate the intracellular domains of IL-12R?1 or IL-12R?2 based on the second generation of CARs.The antigen-specific activation of this receptor simultaneously triggers TCR?through the CD3?domain?,co-stimulatory?through the4-1BB domain?and cytokine?through the IL-12R domain?signaling.These signals effectively provide all three synergistic signals required physiologically to drive full T cell activation.In this study,we aimed to determine whether the delivery of IL-12signaling only upon antigen engagement can enhance antitumor effects of CAR-T cells.So that CARs,encompassing the IL-12R intracellular signaling domain,can improve the anti-tumor activity of T cells in immunosuppressive microenvironment.In order to reflect the effectiveness of cytokine signals for CAR-T treatment of solid tumors,we selected CAR-T containing co-stimulatory signal 4-1BB for modification,which is currently the most widely used CAR-T cell in clinical practice.We constructed a novel PSMA/CD19-CAR-T cell containing the intracellular segment of IL12R?1 or IL12R?2 and targeting both the prostate cancer antigen PSMA and CD19,and then systematically evaluated their function.Compared with a CAR containing only the 4-1BB costimulatory signal domain,our findings have proven that CAR-T cells containing IL12R?1 or IL12R?2 intracellular signaling domain can promote the phosphorylation of STAT4 and the expression of CD69 and CD25 without affecting the proliferation of CAR-T cells,and transmit IL-12 activation signals for CAR-T cells.Based on the effect of IL-12 on the differentiation of T cells,we also explored the effect of CAR with IL12R?1 or IL12R?2 intracellular domain on T cell differentiation.The results showed that the integration of the intracellular domain of IL12R?1 or IL12R?2,especially the integration of IL12R?2 intracellular fragments,support CD45RO⁺memory T cells formation and maintenance,without affecting the proportion of CD4+/CD8+T cells and the exhaustion of CAR-T cells.Based on the above data,we evaluated the effect of integrating IL12R?1 or IL12R?2 intracellular segment on the effector function of CAR-T cells.Our results also suggested that by adding intracellular domains of IL12R?1 or IL12R?2 receptors to the CAR intracellular signaling domain,CAR-T cells can enhance IFN-?and TNF-?production and cytotoxic capabilities upon antigen stimulation.In addition,the integration of IL12R?1 or IL12R?2 intracellular domain can induce CAR-T cells to play anti-tumor effect in a short time.At the same time,under the multiple stimulation of the antigen,the continuous activation of IL-12activation signal can bring superior antitumor activity to CAR-T cells containing the IL12R?1 or IL12R?2 intracellular domain.What's more,compared with CAR-T cells integrating IL-12R?1 intracellular domain,CAR-T cells containing IL-12R?2intracellular domain have better performance in cytokine release and anti-tumor effect.Overall,our results clearly demonstrated that the integration of the cytokine receptor intracellular domain induces IL-12 activation signals upon antigen stimulation,and showed a novel strategy to enhance the antitumor efficacy of CAR-T cell therapy by integrating the intracellular segment of IL-12R,especially IL-12R?2,effectively improve CAR-T function in vitro.This study shows that the use of the cytokine receptor intracellular domain for CAR construction is a potent and feasible immunotherapy strategy worthy of being translationally developed in the clinical.This strategy is meaningful for use of cytokine activation signals to solve the problems of CAR-T cells in treating solid tumors.