| Antibody recruitment strategy is a novel tumor immunotherapy with great potential development in recent years.This strategy aims to recruit endogenous antibodies naturally present in the body or generated by preimmunization to the surface of target cells by means of antibody recruitment molecules,and then activate the immune system to kill the target cells through the interaction between the Fc end of the antibody and the Fc receptors on the surface of complement or effector cells.Classical antibody recruitment molecules consist of two functional domains,the target binding terminal and the antibody binding terminal,which are responsible for the recognition of target cells and the recruitment of endogenous antibodies respectively.However,it should be pointed out that the current selection of antibodies is mainly limited to anti-2,4-Dinitrophenol(DNP)antibody,anti-galactose-α-(1,3)-galactose(α-Gal)antibody and anti-rhamnose(Rha)antibody.However,the abundance of these three antibodies in human body is low and the individual variation is large.Therefore,exploring new endogenous antibodies can be used in tumor immunotherapy,for further development and application of the antibodies to raise strategy is of great significance.Hepatitis B caused by Hepatitis B virus(HBV)is one of the major infectious diseases threatening human health.The purpose of preventing HBV infection can be achieved by inducing the production of high titer HBV antibody by hepatitis B vaccine.Considering the mandatory hepatitis B vaccine and the popularization of hepatitis B vaccine booster,HBV antibody has become a widespread and abundant endogenous antibody in the population.Based on this,we used anti-epidermal growth factor receptor(EGFR)nanobody as the target binding end,HBV Peptide(139-147)as the antibody binding end.To prepare a series of antibody recruitment molecules in the form of nanobody-peptide conjugates,and to verify their tumor-specific targeting ability and HBV antibody recruitment ability.Finally,the potential of HBV antibody for tumor immunotherapy was evaluated by cytotoxicity test in vitro.The main experimental research contents and results are as follows:(1)Preparation and characterization of nanobody-peptide conjugates.Three anti-EGFR nanobodies(7D12,9G8 and 7D12-9G8)were constructed and synthesized.Sortase A mediated ligation reaction was used to modify the HBV epitope peptide derivatives on the C-terminus of the nanobody,and a total of 9 HBV antibody recruitment molecules were obtained.The results of SDS-PAGE and Western Blot showed that the preparation was successful.(2)Evaluation of tumor targeting ability and HBV antibody recruitment ability of nanobody-polypeptide conjugates.Flow cytometry results showed that nanobodies modified with HBV epitopes could specifically recognize and bind to EGFR high-expressing cells,and the modification of HBV epitopes did not significantly affect the ability of nanobodies to target tumors.Immunofluorescence and flow cytometry demonstrated the HBV antibody recruitment capacity of the nanobody-peptide conjugates,and all of them were found to selectively anchor HBV antibodies to the surface of tumor cells with high EGFR expression.(3)Assessment of the level of cell killing mediated by nanobody-peptide conjugates.Cytotoxicity assay results showed that the nanobody-peptide conjugates caused significant EGFR-overexpressing cell death compared with the control group,confirming the feasibility of using HBV antibody for tumor immunotherapy.Further structure-activity relationship results showed that optimized chain length increased tumor killing activity mediated by antibody recruitment molecules by up to 20.9% compared with no PEG chain optimization. |
