Development And Function Assay Of CCR8 Inhibitory Monoclonal Antibody

Finding tumor-specific targets that induce the weakest immune-related adverse reactions is a hot spot and frontier in the field of oncology research.Studies have found that human CC chemokine receptor 8(CCR8)is a drug target with great potential for cancer immunotherapy and autoimmune diseases.CCR8~+Tregs expression is upregulated in a variety of cancer tumors and is a driver of immunosuppression.CCR8 is minimally expressed in helper/cytotoxic T cells in the tumor microenvironment,as well as in T cells in adjacent normal tissues.CCR8 inhibitory antibody with cytotoxic activity that specifically recognizes and eliminates tumor associated Tregs.However,the mechanism of CCR8 in tumors needs to be further studied.Therefore,the study of CCR8 monoclonal antibody can explore the unknown mechanism of tumor immunotherapy and CCR8 dependence.Therefore,mRNA immunized mice were used to prepare hybridoma cells,and the monoclonal antibody 22H9,which was obtained by purifying and preparing CCR8-blocking monoclonal antibody,22H9.The main research of the paper is as follows.1.mRNA immunization mice to prepare hybridoma cells.In this project,mice were first immunized with LNP(Lipid nanoparticle)modified mRNA due to the transmembrane conformational properties of the target protein.After immunizing mice,ELISA and FACS detected serum titer,and all 10mice had effective valence,indicating that mRNA immunity was feasible,and the splenocytes of mice with the highest titer were fused with SP2/0 IL-6 to obtain 2800 hybridoma cells.The two strains with the highest titer of ELISA and FACS were screened for subclone plating,and 42 monoclonal hybridoma cells were obtained,and the 22H9 with the highest expression of ELISA and FACS was expanded for culture.2.Visualization HEK293-cAMP-biosensor-CCR8 cell dynamic detection model established.The cAMP-biosensor,CCR8 gene was constructed into the HEK293 cell line by lentivirus infection.The CCR8 protein receptor regulates the cAMP signaling pathway,and the dynamic detection of intracellular cAMP content is visualized by Glo Sensor technology.3.Preparation and functional study of CCR8 inhibitory monoclonal antibody.The monoclonal antibody 22H9,which is CCR8-blocking,was obtained by protein purification cell supernatant,and the SDS-PAGE detection was of high purity,and the ELISA and FACS detection showed high specificity.The experimental results of ADCC and ADCP showed that the antibody had excellent anti-tumor activity.In summary,mRNA immunity was achieved for the first time in mice,and a new inhibitory monoclonal antibody was developed for CCR8,and the biological activity of the antibody was verified by preliminary exploration.A visual dynamic monitoring model was constructed to realize the dynamic detection of antibody functions.To explore the relationship between CCR8 target and tumor immunity,further development of this target in tumor treatment provides more options.