Synthesis And Immunological Study Of CD44 And EGFR Dual-Targeted Multivalent Antibody-Recruiting Complex

Malignant tumors seriously threaten the life and health safety of human beings.Currently,commonly used treatments such as surgery,chemotherapy and radiotherapy lack target specificity and prone to cause certain side effects on normal cells.Antibody-recruiting molecules composed of target binding terminus and antibody binding terminus have the dual functions of targeting tumor cells and recruiting endogenous antibodies,which have great application prospects in tumor immunotherapy.Endogenous antibodies are naturally occurring antibodies in the human serum,such as anti-rhamnose（Rha）antibodies,which account for about 2-8%of total human serum proteins.However,monovalent antibody binding terminus have limited ability to recruit endogenous antibodies,and tumor cells often lack specific targets,which can easily cause off-target problems at the target binding terminus and affect the effect of antibody-recruiting molecules.Since the multivalent antibody binding termini is an important direction to further improve the antibody recruitment ability,dual-targeting binding termini is an effective strategy to solve the off-target problem,and therefore the effect of antibody-recruiting molecules can be enhanced by constructing dual-targeting multivalent antibody-ecruiting molecules.In view of this,cyclodextrin-modified hyaluronic acid was used as a targeting ligand for CD44,and it was coupled with the antibody recruitment ligand Rha.At the same time,we used adamantane-modified peptide GE11 or nanobody 7D12 as the targeting ligand of Epidermal growth factor receptor（EGFR）,and coupled them to hyaluronic acid backbone through the host-guest interaction of cyclodextrin and adamantane to form two multivalent antibody-recruiting complexes with dual-targeting properties.Firstly,the targeting specificity and antibody recruitment ability of complexes were characterized by cellular immunofluorescence and flow cytometry experiments.Secondly,the complex-mediated immunological activity was assessed by complement-dependent cytotoxicity（CDC）and antibody-dependent cellular phagocytosis（ADCP）assays.The main studies and results are as follows:（1）Construction of the host molecule of multivalent antibody-recruiting complex.The amino-modified cyclodextrinβ-CD-EDA and amino-modified rhamnose derivative Rha-PEG₃-NH₂ were prepared chemically with yields of 33.13%and 55.29%,respectively.The host molecule Rha-HACD was obtained by coupling cyclodextrin and rhamnose derivative to the hyaluronic acid backbone by amide condensation reaction,and the degree of substitutions were17.25%and 38.96%,respectively.（2）Construction of guest molecules of multivalent antibody-recruiting complex.The guest molecule Ada-7D12 was obtained by modifying adamantane to the C-terminus of nanobody7D12 by an eSrtA enzyme-mediated fixed-site ligation reaction with yield of 70.03%,and another guest molecule,adamantane-modified peptide Ada-GE11,was prepared by solid-phase peptide synthesis in a yield of 57.85%.（3）Preparation of multivalent antibody-recruiting complexes.Two multivalent antibody-recruiting complexes were formed by linking the host and guest molecules through the host-guest interaction of cyclodextrin and adamantane.（4）Activity verification of the multivalent antibody-recruiting complex Rha-HACD/Ada-GE11.The ability of the complex to specifically recognize tumor cells and recruit endogenous rhamnose antibodies to the surface of target tumor cells was demonstrated by flow cytometry and cellular immunofluorescence assays.Tumor cell killing assays showed that the complex could kill tumor cells by recruiting anti-rhamnose antibodies to exert CDC and ADCP activities,with CDC and ADCP activities of 56.87%and 42.75%,respectively.（5）Activity verification of the multivalent antibody-recruiting complex Rha-HACD/Ada-7D12.Experimental analyses such as cellular immunofluorescence and flow cytometry showed that the complex could selectively recognize tumor cells and recruit anti-rhamnose antibodies.The tumor cell killing assays showed that the complex was able to mediate a certain degree of CDC and ADCP effects,with cell killing rates of 58.71%and 45.41%,respectively.