| Cancer is one of the major diseases that threaten human health.The immune system can recognize and eliminate cancer cells,but its efficacy can be affected by inhibitory receptors(IR)and ligands.Under normal circumstances,the immune checkpoint can maintain the immune tolerance by adjusting the intensity of the autoimmune response.However,when the body is affected by a tumor,tumor cells activate the immune checkpoint abnormally to suppress the body’s immunity to tumor cells.Monitoring and clearance to achieve tumor cell proliferation and escape.Drugs that block immune checkpoints can relieve the tumor’s immune suppression,activate the body’s own immune function,and then help patients suppress and kill tumor cells.In recent years,immune checkpoint blockers represented by PD-1(Programmed cell death protein 1)/PD-L1(Programmed cell death 1 ligand 1)have brought break through in the antitumor-therapeutic market.Currently,there are many onging clinical trials related to immune checkpoint inhibitors mediated antitumor therapy.The LAG-3 molecule is a membrane protein belong to member of the immunoglobulin superfamily discovered by Frédéric Triebel In 1990,and is expressed in a variety of immune cell subtypes,including T cells,natural killer cells(NK),dendritic cells(DCs),and B cells.The latest research shows that this molecule can negatively regulate the proliferation of CD8 + and CD4 + T cells to activate effector functions.It is the second-generation target of immune checkpoints at present,which has accumulated considerable clinical data and a significant effect in combination with PD-1.Antibodies to this target may become important antitumor drugs in the future.LAG-3 has structural homology with CD4,and both bind to the major histocompatibility complex class Ⅱ(MHC-Ⅱ).LAG-3 has much a higher bingding affinity with MHC-Ⅱ than CD4 due to an extra loop containing 30 amino acids present in its domain1 structure.In this thesis,we used the extra loop on LAG-3 molecule D1 as a target and synthesized a peptide as an immunogen to immunize New Zealand male rabbits.After the immunization,rabbit PBMC cells were isolated.B lymphocytes capable of recognizing the immunogen polypeptide were sorted by flow cytometry.LAG-3 rabbit monoclonal antibody was made using single B cell cloning technology,and the light and heavy chain variable region sequences of the antibodies were successfully retrieved.The retrieved sequence was constructed on the antibodies expression vector by homologous recombination.Expi293? eukaryotic expression system was purified,and 8 antibodies were characterized.The antibodies efficacy was evaluzted by ELISA using the specific LAG-3 peptide epitoes and the EC50 values of the antibodies were determined.The highest EC50 was 0.001224 and the lowest was0.00002479.Western blot analysis revealed that all eight strains of antibodies could recognize recombinant human-derived LAG-3 protein,and some could recognize the LAG-3 protein in activated PBMC cells,Flow cytometry results show that some endogenous LAG-3 can be recognized.The above results indicate that we have obtained the LAG-3 rabbit monoclonal antibodies with the high affinity and specificity which has lay a cornerstone for further antibody optimalization,humanization and clinical development. |
