Expression of extracellular fragment of murine PD-L1 and production of antibodies to PD-L1

A number of molecules expressed on mammalian cells are involved in the formation of autotolerance. These primarily include CTLA-4/B7 and PD1-PD-L1 signaling pathways. Blockers of these signaling pathways, called checkpoint inhibitors (CPIs) of immunity, are used in the clinic for the treatment of various forms of cancer. Antibodies to CTLA-4 cause systemic toxicity and are approved only for some tumors. Antibodies against PD1 or PD-L1 have been successfully used for the treatment of various forms of cancer and are characterized by low toxicity. However, the response to therapy using CPIs is not always observed. The development of more effective approaches to cancer therapy based on PD1/PD-L1 inhibitors requires additional research. The aim of this work was to express the extracellular part of the murine PD-L1 protein (exPD-L1) and obtain antibodies to PD-L1. The mouse exPD-L1 protein was obtained and characterized in the bacterial expression system. exPD-L1 protein was used to immunize mice in order to produce anti-PD-L1 antibodies. Using hybridomic technology, 5 clones expressing antibodies to exPD-L1 were obtained. Antibodies of the B12 clone were developed in the ascitic fluid of BALB/c mice and purified by affinity chromatography. The ELISA method for purified antibodies showed specific binding to the exPD-L1 protein and the commercial protein of the extracellular part of murine PD-L1. Experiments using flow cytometry and confocal microscopy have shown that the antibodies obtained bind the intracellular form of the PD-L1 protein, unlike commercial antibodies binding the membrane form.