Screening Of Fully Human PD-L1 Antibody, Antitumor Effect And Mechanism Of Its Combination With Radiotherapy

Radiotherapy as one of most important traditional cancer therapy can lead tumor regress significantly, but shortly most tumor will relapse. In the recent years, several study show the immune infiltration status in tumor tissues is directly correlated with prognosis. Radiotherapy, especially, SBRT, can induce immune activation, but meanwhile might lead immune inhibitory molecules up-regulated on tumor cells, such as PD-L1, which can bind to PD-1 expressed on T cells and induce T cell exhaustion,and can’t kill the tumor cells completely. So we propose that SBRT combine PD-1/PD-L1 pathway antagonist antibody could achieve much better anti-tumor effect,and large likely can lead tumor complete shrinkage and protect from relapse. In this project, we aim to develop novel fully human PD-L1 antagonist Ab by yeast display technology. Meanwhile, setup breast cancer SBRT induced dormancy mouse model,investigate the therapeutic effect of SBRT and PD-L1 antagonist combine treatment and study the corresponding immune mechanisms.Taking advantage of yeast display technology, by screening the yeast display library with biotin labeled PD-L1 protein., we got 8 ideal yeast clones with distinct sequences. The positive binding yeast clones were sequenced and the Sc Fv part were cloned into eukaryotic expression system to get Sc Fv-Fc proteins. They are further analyzed by FACS and Elisa for the binding property of antigen. For the specifically binding clones, antagonist test were carried out by FACS and Elisa. We finally get two antibodies bind specifically to PD-L1 meanwhile block PD-1/PD-L1 binding, one of them can also bind corresponding mouse antigen, which can be used in future radiotherapy combine treatment study. The function of PD-L1 antagonist antibodies were analyzed both in vitro and in vivo. In vitro MLR assay show the PD-L1 blocking antibody can promote T cell activation and IFNγ secretion in a dose dependent manner.The antibodies showed comparable in vivo anti-tumor effect with the advanced stage PD-L1 antagonist MEDI4736 from Med Immune. This part of work will not only provide a good PD-L1 antagonist antibody used in further radiotherapy combine treatment study, but also play an important role in PD-L1 novel drug development.Patent have been applied and will be further used for clinical translation. It has the potential to be the first PD-L1 antagonist drug that breaks the Zero record in China.On the other side, a cancer dormancy mouse model has been setup by inoculating mouse breast cancer cell line Tubo subcutaneously into Balb/c mouse and giving different dose of radiation. The dormancy status were further analyzed which show that SBRT induce immune activation depend on CD8 T cells, but also lead PD-L1 up regulation on tumor cells. SBRT combine PD-L1 blocking antibody lead subcutaneous tumor shrink much better than the SBRT treatment group. In immunological mechanism study, for those tumor completely regressed mice, when they were rechallenged with 10 times more tumor cells to mimic the relapse, the mice can reject the tumor completely.That means the SBRT combine anti-PD-L1 blocking Abs treatment have induced effective memory response, and all these effects are CD8 T cell dependent. Besides,combine treatment leads CD8 T cell produce more IFNγ and TNFα. This study provides useful information, new ideas and base for radiotherapy and immune therapy combine treatment.