| Immune checkpoint molecules play an important role in maintaining immune tolerance and immune homeostasis.When T cells are activated,they can inhibit the activity of T cells,thereby avoiding excessive activation of the immune system.Cancer cells inhibit the activity of the immune system by abnormally activating immune checkpoints during the proliferation process.For instance,the highly expressed programmed death receptor-ligand 1(PD-L1)on the surface of tumor cells binds to the programmed death receptor 1(PD-1)on the surface of T cells,which inhibits the activity of T cells.Immune checkpoint blockade therapy based on PD-1 and PD-L1 restores T cell activity by blocking the combination of the two with either the PD-1 or PD-L1 antibodies.However,PD-1/PD-L1 antibody drugs show generally only 20% to 30% efficacy against solid tumors.Continuing to find new immune checkpoints and developing corresponding inhibitory antibodies is one of the main goals of current tumor therapy.Sialic acid-binding Ig-like lectin 15(Siglec-15)is a member of the Siglec family.Similar to PD-L1,it can significantly inhibit the activity of T cells after interacting with unknown molecules on the surface of T cells.Therefore,it is also considered an immune checkpoint.The expression of Siglec-15 in tumor cells is up-regulated,which is related to the immune escape of tumors,so antibody drugs targeting Siglec-15 are likely to become an important anti-tumor drug in the future.Monoclonal antibody drugs have the characteristics of strong targeting,high efficacy and small side effects.The preparation of Siglec-15 monoclonal antibody is of great significance for the subsequent development of antibody drugs.Here,we have developed the specific Siglec-15 monoclonal antibodies by using the single B cell antibody preparation technology.The human Siglec-15 extracellular domain protein was initially expressed and purified in the Expi293 eukaryotic expression system,and used as an antigen to immunize rabbits.After three times of immunolization,Single B cells in rabbit PBMCs were isolated.The light and heavy chain variable region fragments of antibodies in B cells were extracted by nested PCR.Next,we extracted 40 antibody light chain variable regions and 23 heavy chain variable region fragments,and successfully cloned 19 Siglec-15 rabbit monoclonal antibodies.We then carried out a series of functional activity identification of the two most specific antibodies,and the results showed that these two antibodies could recognize and bind human Siglec-15 protein as analyzed by Western blotting,ELISA,Flow Cyto Metry(FCM)and Immunofluorescence(IF).These two antibodies show better specificity than the commercial polyclonal antibodies tested.Therefore,we have successfully developed the novel Siglec-15 monoclonal sntibodies,which will provide a solid foundation for the subsequent development of Siglec-15 antibody drugs and testing kits. |
