Immune Checkpoint Blockade Immunotherapy Combined With Integrin α_vβ₃-targeted Radionuclide Therapy Synergistically Enhances Anti-tumor Efficacy

Objective:Immunotherapy target PD-1/PD-L1 pathway has been proven more effective than traditional therapies in several cancers.Preclinical studies and ongoing clinical trials have demonstrated that combining immunotherapy with radiotherapy could yield better outcomes.Targeted radionuclide therapy(TRT),a branch of radiation therapy,is being increasingly studied in the treatment of advanced tumors with multiple metastases.Integrin αvβ3,which is highly expressed on the surface of neovascular endothelial cells and various tumor cells,is one of the hot targets for tumor diagnosis and treatment.In the present study,in vivo distribution and efficacy of targeted integrinαvβ3 radionuclide therapy were evaluated in the patient-derived xenografts(PDX)of non-small cell lung cancer(NSCLC-PDX).Then we proposed a novel therapeutic regimen that combined PD-L1-based immunotherapy with 177Lu TRT in the MC38 murine colon cancer model,and investigated potential mechanisms subsequently.Methods:Integrin αvβ3-targeted molecule RGD was conjugated with the albumin binding moiety,Evans blue(EB),and then labeled with radionuclide 177Lu.177Lu-EBRGD SPECT imaging,biodistribution study,and in vivo therapy were subsequently performed in NSCLC-PDX.177Lu-EB-RGD TRT,immunotherapy,concurrent and sequential combine therapy were conducted in MC38 murine colon cancer model.18FFDG PET imaging,immunohistochemistry,flow cytometry and multiplexed immunofluorescence staining were used to evaluate therapy response and alterations in tumor microenvironment.Independent-samples t test was used for statistical analysis.Results:Regarding the SPECT imaging,intense tumor to background ratio were observed from 4 h to 120 h post-injection in PDX model,and the biodistribution study further confirmed the findings from SPECT imaging.Regarding targeted radiotherapy,177Lu-EB-RGD treated groups(Group C:18.5 MBq;Group D:29.6 MBq)had significant anti-tumor efficacy compared with saline/177Lu-RGD treated groups in PDX model.In MC38 murine colon cancer model,the difference of efficacy results between each treatment group had statistically significant.The average tumor volume from small to large were the concurrent combination therapy group,sequential combination therapy group,immunotherapy group,TRT group and control group.The concurrent combination therapy showed the most outstanding anti-tumor efficacy and best survival rate.Four of the mice in the concurrent combination therapy group with complete tumor regression were re-challenged with 106 MC38 cells on day 80 to investigate whether immunologic memory was generated,and all 4 mice were able to completely reject re-challenged tumors.The different therapy efficacy was further confirmed by FDG PET imaging and immunohistochemistry.In the results of multiplexed immunofluorescence staining and flow cytometry,TRT up-regulated the PD-L1 expression significantly on days 4 and 7,and combination therapy dramatically stimulated the infiltration of CD8+T cells,whereas no significant difference was observed in PD-L1 expression and infiltration of CD8+T cells on day 14.Conclusions:177Lu-EB-RGD targets αvβ3 positive NSCLC-PDX with intense tumor to background ratio and strong tumor inhibition efficacy.Concurrent rather than sequential blockade of the PD-1/PD-L1 axis combined with TRT improves overall survival and long-term tumor control in MC38 murine colon carcinoma model.Mcrecver,our data revealed that the time window for this combination therapy may be critical to outcome.