A Mechanism Study Of Anti-PD-L1/TGF-βR In Potentiating The Efficiency Of Immunotherapy In Lung Cancer With Poor Lymphocyte Recovery

BackgroundLung cancer is the main cause of cancer-related death in the world.The five-year survival rate of patients with advanced non-small cell lung cancer(NSCLC)is only 10%-20%.With the rise of tumor immunotherapy,this relatively high-efficiency and low-toxicity treatment has given new hope to NSCLC patients.Immunotherapy kills cancer cells by activating our body’s own immune system and relying on its own immune function.While the success of immunotherapy is encouraging,the effective rate of programmed cell death protein-1(PD-1)monoclonal antibody in patients with NSCLC is only about 20%.According to our previous statistics,for patients with longterm lymphopenia,immunotherapy did not provide a significant improvement in prognosis compared to standard therapy.Therefore,we define this part of the population as "poor lymphocyte recovery" patients:before receiving immunotherapy,absolute lymphocyte count(ALC)in peripheral blood had been lower than normal level for more than 3 weeks,and it didn’t ever return to a normal value during immunotherapy.At present,how to improve the efficacy of PD-1 inhibitors,especially in patients with poor lymphocyte recovery,still needs further study.In order to improve the response rate,many studies have evaluated the combination of anti-PD-(L)1 drugs and other immunotherapy.However,these combinations have certain limitations,including additional toxic side effects,reduced ease of administration,etc.Bifunctional antibody is a new type of second-generation immunotherapy antibody,and the representative drugs include M7824,SHR-1701,etc.SHR-1701 is a fusion of monoclonal antibody against programmed death ligand-1(PDL1)and transforming growth factor-β receptor Ⅱ(TGF-βRⅡ).Compared with monoclonal antibodies,the efficacy of bifunctional antibodies in patients with poor lymphocyte recovery has not been reported,and the specific mechanism needs to be clarified.This research utilized a variety of biological means to study in two parts,including the mechanism of immunotherapy resistance to lung cancer caused by poor lymphocyte recovery and the application basis of bifunctional antibody in mouse models,which provides new ideas for the selection of applicable patients for immune drugs and future development of drug research.Part Ⅰ:The mechanism of poor lymphocyte recovery inducing resistance to immunotherapy in lung cancerObjective:(1)To reveal the significance of the number of lymphocytes in peripheral blood to immune efficacy and prognosis;(2)To establish an in-situ mouse model of lung cancer and explore the function of CD8+T/Treg in peripheral blood and peripheral lymphoid organs under the condition of poor lymphocyte recovery;(3)To clarify the efficacy/drug resistance mechanism of PD-1 inhibitor in mice with poor lymphocyte recovery.Materials and methods:(1)The clinical data of 205 patients with lung cancer who accepted anti-PD-1/PD-L1 antibody were collected retrospectively and followed up.Multivariate Cox regression analysis was applied to patients’ characteristics,and Kaplan-Meier(K-M)survival curve was drawn;(2)Collect the peripheral blood of normal and poor lymphocyte recovery lung cancer patients,respectively.Analyze the difference in number of CD8+T and Treg cells by flow cytometry;(3)Fireflyluciferase lentivirus was constructed to infect CMT167 cell line of mouse lung cancer,and stable cell line expressing luciferase gene(CMT167-luc)was obtained after puromycin screening;(4)The CMT167-luc cell line was used to establish a mouse model of lung tumor in situ,and then cisplatin(DDP)was administered at 4 mg/kg for 4 days to establish a mouse model of lung cancer with poor lymphocyte recovery.Flow cytometry and immunohistochemistry(IHC)were used to detect the contents of CD8+T and Treg cells in peripheral blood/tumor microenvironment(TME),and the changes of downstream signal pathways of CD4+/CD8+T cells were analyzed by RNA-seq.Hematoxylin-Eosin(HE)staining was used to observe the morphological changes of thymus and spleen;(5)According to five treatment modes of clinical lung cancer,mice were divided into five groups.Then the tumor growth rate under different treatment modes was observed by bioluminescence imaging applications,and the survival time of mice was also recorded;(6)Using flow cytometry and IHC staining technology,clarify the potential mechanism of lymphocyte recovery ability regulating the efficiency of immunotherapy;(7)The potential intervention targets of IL-2 and TGF-βwere screened by IHC staining and enzyme linked immunosorbent assay(ELISA).Result:(1)There is a significant correlation between lymphocyte recovery and PFS.The small number of lymphocytes leads to a decrease of neutrophil-to-lymphocyte ratio(NLR)and platelet-to-lymphocyte ratio(PLR),suggesting a poor prognosis.Further subgroup analysis showed that the recovery ability of lymphocytes was positively correlated with PFS.(2)Effect of cisplatin on lymphocyte recovery and regulation of immune microenvironment.Cisplatin induced poor lymphocyte recovery,and CD8+T/Treg ratio decreased in peripheral blood/thymus/spleen of mice.CD3/CD2 8 co-stimulation experiment confirmed that the proliferation and activation of CD8+T cells were inhibited.RNA-seq showed that CD4+T cells were enriched with genes related to Treg differentiation,and the expression of genes related to CD8+T cell function was down-regulated,indicating the immunosuppressive state of tumor microenvironment.(3)The first generation of PD-1 inhibitors is not effective in mice with poor lymphocyte recovery,and TGF-β may become a potential target for future research.Experiments in mice with lung cancer show that the first generation of PD-1 inhibitors is not effective in mice with poor lymphocyte recovery,regardless of synchronous or sequential mode.Furthermore,IL-2 and TGF-β were screened by IHC and ELISA,and finally TGF-β was identified as the potential intervention target for the next step.Conclusion:According to clinical data and animal models,previous chemotherapy broke the balance between CD8+T and Treg cells in some populations,destroyed the recovery of lymphocyte homeostasis,and led to poor response to PD-1 inhibitors.TGF-β could be a potential target for the next step of research to overcome immune resistance.Part Ⅱ:Basic exploration of the application of immune bifunctional drugs in lung cancer models with poor lymphocyte recoveryObjective:(1)To verify the efficacy of SHR-1701(PD-L1/TGF-βRⅡ fusion protein)in mouse model,and explore its mechanism of reversing immunosuppressive microenvironment;(2)In the peripheral blood of patients with lung cancer,the efficacy of the first-and second-generation monoclonal antibody was compared under different lymphocyte recovery status;(3)To explore the regulation of dual-antibody drugs on PI3K/Akt/Erk and TGF-β signaling pathways in Treg and CD8+T cells.Materials and methods:(1)On the basis of the first part of this study,the sixth treatment mode was added:SHR-1701 was given after cisplatin,and the record of tumor size and survival time of mice were analyzed by bioluminescence imaging applications and K-M survival curve;(2)The effect of SHR-1701 on the proliferation of CD8+T and Treg cells in tumor/thymus/spleen of mice with poor lymphocyte recovery were investigated by flow cytometry and IHC staining;(3)SHR-1701 was used to treat the peripheral blood of patients with lung cancer,and the expression of activation-related protein on CD8+ T cells was detected by flow cytometry.What’s more,CD4+T cells and CD8+T cells in patients’ peripheral blood were sorted,and the regulation of TGF-β and PI3K/Akt/Erk signaling pathways by different treatment was detected by Western Blot.Result:(1)SHR-1701 overcomes acquired resistance to PD-1 monoclonal antibody.In vivo experiments in mice confirmed that SHR-1701 applied to poor lymphocyte recovery mice compared to PD-1 monoclonal antibodies produced more significant tumor regression and longer survival time.(2)Regulation of SHR-1701 on immune microenvironment of tumors/spleens.IHC staining indicated that after SHR1701 was used in mice with poor lymphocyte recovery,the number of CD8+T cells in tumor microenvironment increased and Tregs decreased.CD8+T cells from tumors and spleens were extracted and given CD3/CD28 co-stimulation.It was found that the number of GITR+/IFN-y+/CD69+CD8+T cells in mice treated with SHR-1701 increased significantly,suggesting that CD8+T cells entered an active stage of proliferation and differentiation.(3)SHR-1701 promotes the proliferation and differentiation of peripheral CD8+T cells in patients with poor lymphocyte recovery.SHR-1701 was used to treat peripheral blood of lung cancer patients with poor lymphocyte recovery.It was found that SHR-1701 could effectively promote the production of IFN-y and the expression of Ki-67 in peripheral CD8+T cells.(4)Regulation of SHR-1701 on signaling pathways of peripheral lymphocytes in patients.SHR-1701 inhibited TGF-β signaling pathway,activated PI3K/Akt/Erk signaling pathway,and saved the anti-tumor function of CD8+T cells in peripheral blood of lung cancer patients with poor lymphocyte recovery.Conclusion:Cisplatin induces the imbalance between CD8+T and Treg cells,which could be reversed by TGF-βR inhibitor.Synchronous blocking of TGF-βR and PD-L1(SHR-1701)achieved better tumor control in the model with cisplatin-induced poor lymphocyte recovery.