PDI-1 As An Immune-checkpoint Inhibitor For NSCLC And Melanoma

Background and PurposeIn recent years,cancer immunotherapy research has brought new hope for human beings to conquer cancer.Immune checkpoint proteins are a series of co-stimulatory or co-inhibitory signaling molecules,which play an extremely important role in cancer immunotherapy,among which PD-1/PD-L1 has attracted the most attention.When the immune checkpoint signal of the body is abnormal,cancer cells can weaken the immune response of the body,and then escape the monitoring and killing of the immune system,so that they can survive and proliferate in the body.According to this principle,in order to enhance the characteristics of immune system to recognize and kill cancer cells,the research and development of costimulatory signal agonists or coinhibitory signal antagonists has become an important means in cancer immunotherapy.Effective cancer immunotherapy can make use of the small difference between tumor cells and normal cells in appearance,so as to produce effective immune response and overcome tumor immunosuppression.In recent years,some approved monoclonal antibodies have achieved remarkable results in the immune checkpoint therapy of cancer: for example,the PD-1 or PD-L1 antibodies approved by the US Food and Drug Administration have shown good clinical applications.Its anti-tumor activity has become a new milestone in cancer immunotherapy,such as the anti-PD-1 monoclonal antibody Nivolumab.Nivolumab is a PD-1 targeted antibody drug developed by Bristol-Myers Squibb.It was approved in December 2014 for the treatment of NSCLC,melanoma and other malignant tumors.By binding to the PD-1 receptor on the surface of T cells,Nivolumab blocks the negative signal regulation pathway mediated by PD-L1,releases exhausted but activated effector T cell populations and reduces regulatory T cell functions,thereby restoring T cell anti-tumor function.In clinical,only a small fraction of cancer patients benefits from Nivolumab.At the same time,some patients have side effects of immunity after treated with these monoclonal antibodies.So far,the PD-1/PD-L1 antibody has achieved great success in cancer immunotherapy,but its high price,poor tumor penetration,and high autoimmune side effects have restricted its application in cancer patients.Compared with monoclonal antibodies,small molecule compounds have significant advantages in drug design: higher oral bioavailability;more exposure in the tumor microenvironment,easily crossing the physiological barrier,low cost,no need for refrigeration,and easier access for patients.In order for immunotherapy to achieve its full potential in cancer treatment,the cost of drugs must be reduced so that all those who might benefit can benefit.Methods1.Screen small molecule inhibitors of PD-1/PD-L1Using computer docking model,several candidate compounds were obtained.After screening,the candidate PDI-1 was finally obtained and the effect of PDI-1 was verified.Competitive enzyme-linked immunosorbent assay was used to determine the ability of PDI-1 to block the binding of PD-1 and PD-L1,and the IC50 value was obtained.Use surface plasmon resonance to analyze the affinity of PDI-1 with human or mouse PD-1 and/or PD-L1 protein in vitro.2.Explore the effect of PDI-1 on non-small cell lung cancer and melanoma cells in vitroThe MTS method was used to detect the effect of PDI-1 on the viability of NSCLC,melanoma cells and activated CD3+T cells.The effect of PDI-1 compound on the percentage of tumor cell apoptosis in the case of non-small cell lung cancer or melanoma cells co-cultured with activated CD3+T cells was detected by flow cytometry.ELISA was used to detect the changes of immune-related cytokines in the cell culture supernatant after PDI-1 in the case of non-small cell lung cancer or melanoma cells co-cultured with activated CD3+T cells.3.Study the action pathway and mechanism of PDI-1Construct a luciferase reporter gene system and use the luciferase reporter gene experiment to detect the effect of PDI-1 acts on changes in the signal pathways of PD-1/PD-L1 when h PD-1-NFAT-Jurkat cells are co-cultured with h PD-L1-TCR-HEK293 T cells or TCR-HEK293 T cells.Use MTS method to detect whether PDI-1 itself is toxic to h PD-1-NFAT-Jurkat cells.4.Explore the function of PDI-1 in vivoThe CRISPR/Cas9 gene knockout method was used to construct PD-L1 humanized B16F10 and KLN205 mouse tumor cell lines,and constructed a humanized PD-L1 syngenetic mouse model.Effect of PDI-1 on tumor infiltration of related immune cells was detected by HE staining.Multiple immunofluorescence histochemistry was used to detect the infiltration changes of related immune cells in solid tumors under the action of PDI-1.The multi-cytokine experiment was used to detect the changes of PDI-1 related immune cytokines in mice.Results1.PDI-1 can bound with human PD-1 and PD-L1,and can also bound with mouse PD-1 and PD-L1,and block the interaction between PD-1 and PD-L1.2.PDI-1 restores T cell activity,and enhances its ability to kill tumors.3.PDI-1 increases T cell toxicity and increases the production of related cytokines.4.PDI-1 not only enhances the function of effector T cells,but also inhibits the function of regulatory T cells in vivo,thereby inhibiting tumor growth.ConclusionsPDI-1 binds with human and mouse PD-1 and PD-L1,and block the interaction between PD-1 and PD-L1,thereby disrupting the PD-1/PD-L1 signaling pathway and restoring the activity of T cells.PDI-1 further enhances the toxicity of T cells and increases the production of immune-related cytokines,and enhances the tumor-killing capacity of T cells.PDI-1 enhances the tumor killer capacity of T cells,inhibits the function of regulatory T cells,and inhibits tumor growth in vivo.