Expression of the Extracellular Domain of Mouse PD-L1 and Production of Antibodies to PD-L1

Abstract

Objective: A number of molecules expressed on mammalian cells are involved in the formation of auto-tolerance. These primarily include CTLA-4/B7 and PD1-PD-L1 signaling pathways. Blockers of these signaling pathways, called checkpoint inhibitors (ICTs) 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 the treatment of melanoma. 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 ICT does not exceed 25–30%. 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 mouse PD-L1 protein (exPD-L1) and obtain antibodies to PD-L1. Methods: 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 antibody producers. Results and Discussion: 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 the mouse PD-L1. Conclusions: 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.