Preparation Of PD-L1 Monoclonal Antibody And Its Antitumor Activity In Humanized Mouse Model

Cancer is usually caused by the loss of normal regulatory functions of the cells,resulting in the excessive cell proliferation and abnormal tissue formation.The occurrence and development of cancer have many biological characteristics such as uncontrollability,invasion and metastasis.Many new advances regarding cancer treatments and mechanistic understandings have been achieved in recent years,which provides many new options for tackling on cancer.Under normal circumstances,immune cells can identify and eliminate abnormal cells in the body.Immune checkpoints maintain immune tolerance by controlling the strength of the immune response,ensuring that the body’s immune system is activated at the appropriate time,and preventing immune cells from being over-activated and wanton attack normal tissue cells,further leading to the occurrence of autoimmune diseases.Tumor cells can transmit inhibitory signals to the immune system through abnormal activation of immune checkpoints,resulting in impairment of the immune system’s monitoring and clearance functions,inducing immune cell apoptosis,and causing the tumor cell immune escape.Programmed apoptosis ligand（PD-L1）,also known as B7-H1,CD274,is a 40 k Da type I transmembrane protein.Tumor cells can inhibit the activity of cytotoxic T cells through high expression of PD-L1 and bind to PD-1 on the surface of T cells,thereby inhibiting the immune response of the immune system to tumor cells and achieving immune escape.PD-L1 antibody drug is an important targeted drug in tumor immunotherapy.Its mechanism of action is to fully activate immune cells by blocking the combination of tumor cell PD-L1 and T cell PD-1,thereby awakening the immune system to kill tumors.Clinically,PD-L1 antibody drugs have shown outstanding therapeutic effects.However,the efficacy of PD-L1 antibody drugs in different patients varies,which still requires the development of novel PD-L1 antibody drugs.The tumor innoculated in humanized mice is a attractive model for evaluating the efficacy and safety of antibody drugs in the preclinical stage.In recent years,it has been widely used to evaluate the anti-tumor efficacy of immunosuppressants.In this paper,we first constructed a cell line MC38-hu PD-L1 that highly expresses human PD-L1 protein,and injected it subcutaneously into humanized PD-1 mice.After the tumor is formed,the PD-1 antibody expressing the sequence of Nivolumab is used as a therapeutic drug to verify the feasibility of the humanized mouse tumor model,and then the eukaryotic protein expression system is used to prepare high-purity human PD-L1 extracellular structural protein.Subsequently,this protein was used as an immunogen to immunize New Zealand male rabbits and we isolated PBMC cells from rabbit blood.PBMC cells were labeled with various antibodies,and B lymphocytes that could recognize PD-L1 protein were sorted by flow cytometry.Then a single B cell cloning technique is used to retrieve the sequences of the variable regions of the light chain and heavy chain of the PD-L1 antibody,and these sequences are constructed on the corresponding antibody expression vector by the method of homologous recombination.Then we also used Expi293F^TM eukaryotic expression system to express and purify rabbit anti-human PD-L1 monoclonal antibody.In order to identify and verify the affinity of the antibody,we conducted the enzyme-linked immunosorbent assay and treatment of humanized mouse tumor models.So as to screen out the PD-L1 monoclonal antibody cell line with the advantages of VH/VL natural pairing,good specificity,high affinity,and rich gene diversity,we analysed the blocking ability of antibodies in the process of killing tumors,which lays the foundation for the subsequent improvement of the effectiveness of antibody detection and the construction of bispecific antibody drugs.