TRAIL-R1-targeted Chimeric Antigen Receptor T Cells Exhibit Dual Antitumor Effects

Background:Malignant tumor seriously endangers human life and health because of its high morbidity and mortality.The commonly used treatment methods include surgery,chemotherapy and radiotherapy in the present,but for patients with advanced,the treatment effect is poor and easy to relapse.In recent years,immunotherapy which uses the body’s immune system to attack tumor cells has become one of the promising research directions in the treatment of malignant tumors.Chimeric antigen receptor T cells（CAR T cells）have become a new treatment method after surgery,chemotherapy and radiotherapy.CAR-T has shown promising clinical results in the treatment of CD19-targeted B-cell lymphomas.However,the therapeutic effect in solid tumors is limited due to the difficulty in finding ideal targets for solid tumors,immunosuppressive tumor microenvironment,poor infiltration of CAR-T cells and poor persistence after entering solid tumors.Tumor necrosis factor related apoptosis-inducing ligand（TRAIL）is a member of Tumor necrosis factor superfamily,and its receptor TRAIL-R1 is highly expressed in many Tumor cells.The expression of TRAIL in normal cells is low or absent,and TRAIL can induce tumor cell apoptosis by binding to its receptor TRAIL-R1.In previous study,we have successfully prepared the full human monoclonal antibody(TR1⁴¹⁹)and determined that it can target and kill tumor cells expressing TRAIL-R1.In this study,multiple CAR-T cells targeting TRAIL-R1 with different structures were prepared,the phenotypic characteristics of the CAR-T cells targeting TRAIL-R1 were detected,which could not only kill tumor cells through the extracellular region of CAR,but also induce apoptosis of tumor cells through the activation of T cells by CAR signal.This study provides a basis for tumor immunotherapy with CAR T cells.Objective:（1）This study aims to construct an optimized CAR-T cell that not only induces tumor cell apoptosis through the TRAIL-R1 pathway,but also activates T cells through CAR signaling to mediate the dual killing of tumor cells.（2）The sensitivity of the second-and third-generation CAR-T cells to target antigen and the proliferation after target antigen stimulation were compared,the functional and phenotypic effects of second-and third-generation CAR-T cells were compared.Methods:（1）TR1⁴¹⁹-CAR sequences was cloned into lentiviral expression vector PWPXL by using genetic engineering technology,the CAR plasmids of second generation TR1⁴¹⁹-28ζand TR1⁴¹⁹-BBζcontaining CD28 or CD137（4-1BB）co-stimulating domain were constructed respectively.The TR1⁴¹⁹-28BBζthird generation CAR plasmid containing both CD28 and 4-1BB costimulant domains was constructed,and the control plasmid TR1⁴¹⁹.Δζcontaining CD8αhinged region,CD28 transmembrane region and sc Fv truncated CD3ζwas constructed.The plasmids of TRAIL-R1 truncation（without the DD dead domain）and TRAIL-R1 full length were constructed.（2）TR1⁴¹⁹-28ζTR1⁴¹⁹-BBζand TR1⁴¹⁹-28BBζCAR T cells and TR1⁴¹⁹-BBζ-Jurkat cells were prepared by lentiviral containing CAR in the second generation lentiviral system and infected with activated T cells and Jurkat cells.TR1⁴¹⁹.Δζ-T cells and TR1⁴¹⁹.Δζ-Jurkat cells were prepared.（3）Flow cytometry was used to detect the infection rate,phenotypic and the expression of inhibitory molecules of TR1⁴¹⁹-CAR.Real-time Cellular Analysis（RTCA）and calcein-AM（CAM）staining were used to detect the cytotoxicity of TR1⁴¹⁹-CAR extracellular regions to tumor cells.Enzyme-linked Immunosorbent Assays（ELISA）was used to detect the secretion of cytokines IFN-γand Granzyme B in the killing supernatant.The proliferation of TR1⁴¹⁹-CAR-T cells after co-incubation with tumor cells was detected by flow cytometry using CFSE staining.（4）Annexin V/7-AAD and intracellular Caspase-3 in tumor cell were detected by flow cytometry after co-incubation of TR1⁴¹⁹-28Bζ-Jurkat cells and TR1⁴¹⁹.Δζ-Jurkat cells with target cells respectively.Results:（1）The chimeric antigen receptor expressing plasmid PWPXL-TRAIL-R1-CAR and related plasmids were successfully constructed.The targeting TRAIL-R1 second generation TR1⁴¹⁹-28ζ、TR1⁴¹⁹-BBζCAR-T cells and the third generation TR1⁴¹⁹-28BBζCAR-T cells were prepared.TR1⁴¹⁹.Δζ-T cells and TR1⁴¹⁹-28BBζ-Jurkat cells,and TR1⁴¹⁹.Δζ-Jurkat cells were constrcted.Further,demonstrated that TR1⁴¹⁹-28BBζCAR T cells can kill TRAIL-R1-expressing tumor cells with significant effector cytokine IFN-γand granulosin B secretion.（2）TR1⁴¹⁹.Δζ-Jurkat cells and TR1⁴¹⁹-28BBζ-Jurkat cells could detect the expression of Annexin V/7-AAD in target cells after co-incubation with target cells（24.9%and 25.3%）,respectively.And the expression of intracellular apoptotic protein caspase-3（46.9%and 40.9%）.The apoptosis was inhibited by the addition of TRAIL-R1-Fc fusion protein.（3）TR1⁴¹⁹-28BBζCAR-T cells and TR1⁴¹⁹.Δζ-T cells could not induce apoptosis of TRAIL-R1-negative 293T cells and did not secrete effector cytokines.TR1⁴¹⁹.Δζ-T cells had no significant killing effect on 293T cells expressing TRAIL-R1 truncation and without secretion of cytokines,but TR1⁴¹⁹-28BBζCAR-T cells expressing TRAIL-R1 truncation had significant killing effect on the secretion of IFN-γand granzyme B,whereas TR1⁴¹⁹-28BBζCAR-T cells expressing TRAIL-R1 truncation had significant killing effect on IFN-γand granzyme B.TR1⁴¹⁹-28BBζCAR-T cells and TR1⁴¹⁹.Δζ-T cells can effectively kill 293T cells expressing the full length of TRAIL-R1,while TR1⁴¹⁹-28BBζCAR-T cells show higher killing specificity than TR1⁴¹⁹.Δζ-T cells could not detect cytokine secretion,while TR1⁴¹⁹-28BBζCAR-T cells had higher cytokine secretion.（4）Compared with the second generation TR1⁴¹⁹-28ζand TR1⁴¹⁹-BBζCAR-T cells,the third generation TR1⁴¹⁹-28BBζCAR-T cells proliferated faster after target antigen stimulation and showed higher expression of activated molecule CD137 at the same concentration of target antigen（0.05ug/m L）.The expression of Programmed death-1（PD-1）is slightly increased（10%）.There was no significant difference in cytotoxicity and phenotypic distribution between the two second-generation TR1⁴¹⁹-28ζand TR1⁴¹⁹-BBζCAR-T cells.Conclusion:（1）In this study,it was found that TR1⁴¹⁹-CAR-T cells targeting TRAIL-R1had a strong apoptosis-inducing effect on tumor cells.The mechanism of action is that TR1⁴¹⁹-CAR T cells not only mediate tumor cell apoptosis by activating TRAIL-R1 death receptor-dependent apoptosis signaling pathway,but also activate T cells to mediate target cell killing by CAR signaling.（2）Compared with the second generation TR1⁴¹⁹-CAR T cells,the third generation TR1⁴¹⁹-28BBζCAR-T cells showed higher sensitivity to target antigen increased proliferation and increased expression of the immunosuppressive molecule PD-1 after target antigen stimulation.This study provides a research basis for subsequent CAR-T cell therapy targeting TRAIL-R1,and provides a new idea for the optimal design of other CAR-T cells.