Studies On The Anti-tumor Efficacy Of The Combination Of Pingyangmycin With Anti-PD1 Antibody And Its Mechanisms

The anti-PD1 antibody,an immune checkpoint inhibitors(ICIs),can relieve the body’s inhibitory effect on immune cells and reactivate immune cells to clear the tumor cells.Some chemotherapy drugs can cause immunogenic cell death(ICD)of tumor cells,thereby enhancing the relevant antitumor immune response.As an antitumor chemotherapeutic drug,Pingyangmycin(PYM),an anti-tumor antibiotic developed in China,is noted for its capability of causing DNA breaks and its mild effect on hemopoiesis;moreover,showing no obvious immunosuppressive effect.The present study is set to investigate the capability of PYM to induce ICD in tumor cells and the enhanced antitumor efficacy of the PYM and anti-PD1 antibody combination.Firstly,we examined the ATP,HMGBI,and active oxygen in PYM-treated cancer cells,and tested the chenmotactic tendency of THP-1 cells,derived from human monocytic leukemia,toward the supernatant of PYM-treated cells through the Transwell experiment.As shown,PYM increased the ATP,HMGB1 and reactive oxygen levels in 4T1 breast cancer and B16 melanoma cells,indicating that PYM could induce immunogenic cell death.These results suggest that PYM may be used for cancer immunotherapy.The chemotactic tendency of THP-1 cells was increased by the supernatants of PYM-treated tumor cells,indicating that PYM might promote the antigen presentation process.Secondly,we further did in vivo studies to evaluate the therapeutic efficacy of the combination of PYM and anti-PD1 antibody in different tumor models including murine 4T1 breast cancer and B16 melanomas.The toxic effects of the combination on bone marrow and various organs and the immunological responses to the combination treatment were investigated in tumor-bearing animals.The in vivo imaging in tumor-bearing animals was used to observe the tumor growth-inhibitory effect of PYM and anti-PDl antibody in 4T1 tumor model.To investigate the effect of PYM and anti-PD1 antibody on various immunological parameters,peripheral blood and tumor samples were analyzed with flow cytometry.In the 4T1 breast cancer model,the tumor growth inhibition rates by PYM monotherapy,anti-PD1 antibody monotherapy and their combination therapy were 66.3%,16.1%and 77.6%,respectively(P<0.05),indicating that therapeutic efficacy of the combination was much higher than that of anti-PD1 alone.In vivo imaging showed that the tumor volume of the drug combination group was much smaller than that of PYM or anti-PD1 antibody treated group;furthermore,there is significant difference between the single drug group and the combination group against 4T1 breast cancer.By graphic analysis,the nucleated cell intensity in femur bone marrow remained unchanged after treatment with the combination of PYM and anti-PD1 antibody.In addition,histopathological examination showed no obvious toxico-pathological changes in various major organs,including the heart,lung,liver,spleen and kidney,indicating that the PYM and anti-PD1 antibody combination can achieve synergistic therapeutic efficacy at a well-tolerated dosage level.Flow cytometry analysis of the peripheral blood samples from the combination medication group revealed that the proportion of CD3+,CD4+,CD8+lymphocytes remained unchanged;however,the proportion of peripheral blood NK cells and NK-like T cells increased significantly compared with the control group.Moreover,flow cytometry analysis of spleen single cell suspension showed that the proportion of spleen DC cells increased.Notably,significant changes occurred in tumor infiltrating lymphocytes:the proportion of CD8+TIL cells increased,while the proportion of CD4+cells decreased.In addition,an increase of CD8+cell proportion and a decrease of CD4+cell proportion in tumor infiltrating lymphocytes were found in PYM monotherapy group.Studies have demonstrated that the combination of PYM and anti-PD1 antibody exerts a synergistic antitumor effect in the 4T1 breast cancer model.To our knowledge,this is the first report on the synergistic antitumor efficacy of PYM and antiPDl antibody combination.The results of this study suggest that the combination of pingyangmycin and anti-PD1 antibody might be promising in clinical application for cancer therapy.In addition,we have preliminarily coupled PYM with human serum albumin(HSA)as well as its nanoparticles,and performed coupling ratio determination and passive targeted detection in vivo.The results of site-specific coupling reaction based on reactive sulfhydryl groups in albumin molecules showed low efficiency and low coupling ratio,indicating that this method is not suitable for the coupling of PYM.In the case of PYM coupled to albumin nanoparticles,the efficiency of PYM coupling can be significantly improved by increasing the number of sulfhydryl groups.The in vivo imaging results showed that the albumin nanoparticles could passively targeted at the tumor site of tumor-bearing mice.