Development of anti-PD-L1-scFv antibody for use in cancer diagnosis and treatment

Cancer is a serious health problem and one of the leading diseases that cause morbidity and mortality worldwide. In the fight against cancer cells, it has become possible to develop more specific and effective treatment agents by elucidating the mechanisms involved in antigen recognition and activation of T cells, the body's most important weapon. PD-1 found in activated T cells is a molecule that limits and inhibits immunological activation. Two main ligands (PD-L1 and PD-L2) enable this inhibition to occur by binding to PD-1 in T cells. These ligands are found on antigen-presenting cells, other stromal cells, and tumor cells. With the increase of PD-L1 expression in tumor tissue, tumors suppress the specific T-cell response developed against them and escape from the immune system and continue to grow. From this point of view, monoclonal antibody therapies produced to increase the T cell response against tumors by blocking the PD-1 receptor or PD-L1 ligand have revolutionized the field of oncology, as they have shown unprecedented success in the treatment of advanced cancer in the last 10 years. In clinical studies, it has been shown that a higher rate of treatment response was obtained in patients who were shown to have PD-L1 expression in tumor tissue by immunohistochemistry. PD-L1 expression in tumors is predicted to be a biomarker for therapies targeting this molecule. Predictive biomarkers are needed to determine patient groups that will benefit from immunotherapies because a significant proportion of patients do not benefit from PD-1/PD-L1 agents, the treatments cause immune-related toxicity in patients, and the high cost of treatment. The routine use of full antibodies is restricted due to their large molecular structure, causing problems in tissue penetration and the potential for serious side effects in patients due to the Fc part they carry. The scFv fragment of the antibody molecule is the smallest functional link of an antibody that binds antigen. The scFv antibody has a high potential for use in many fields such as research, diagnostics and clinical applications. The smaller scFv antibody structure compared to whole antibodies allows it to penetrate tumors and other tissues more quickly and evenly. Because these fragments can be removed from the blood faster and healthy tissue is less exposed to these antibodies, scFv antibodies can be combined with anti-cancer drugs and radionuclides to be used in cancer treatment and imaging. In this study, an scFv molecule was formed by obtaining and combining the immunoglobulin VH and VL variable regions from rat spleen cell cDNAs obtained by immunization against PD-L1 antigen using recombinant DNA technology. After the scFv antibody library obtained was expressed on the surface of the phages by means of the phage display method, affinity selection was made to obtain the anti-PD-L1-scFv antibody that binds to the PD-L1 antigen with the highest affinity. After expression of high-affinity anti-PD-L1-scFv antibody in HEK293 cells and purification by IMAC method, the antigen binding performances of the antibody were determined and the in vivo treatment efficacy was compared with the full anti-PD-L1 antibody. The unique anti-PD-L1-scFv antibody obtained can be used to activate the immune system directly, as well as have the potential to be used as a potent carrier in delivering other anti-tumor drugs and chemicals to tumor cells. In addition, by binding the obtained anti-PD-L1-scFv antibody with a radionuclide and detecting the instant PD-L1 expression rate of the patients, accurate PD-L1 expression level analysis can contribute to the elimination of the limitations in the immunohistochemical analysis of PD-L1.