Molecular Basis For The Interaction Between Human Single-chain Antibody Against COX-2 And Its Antigen

COX-2(Cyclooxygenase-2)is a key rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostanoids.Since COX-2 is involved in many pathophysiological processes of the body including promoting the progress of inflammation,promoting angiogenesis,promoting the occurrence and development of tumors,and enhancing the drug resistance of tumor cells,it has become an important target for tumor treatment.Given that the high risk of COX-2 inhibitors in serious toxicities and side effects in clinical applications,it is of great significance to explore a new strategy for targeting,safe,and efficient tumor treatment targeting COX-2.The single-chain antibody has been applied in many forms in clinic because of its advantages of high affinity,high specificity,strong tissue penetration,and low immunogenicity,and has unique advantages in the diagnosis and treatment of tumors.Our laboratory has successfully prepared a human single-chain antibody that specifically binds to COX-2 in vitro and in vivo and inhibits cell growth,proliferation,migration,and invasion ability of tumor cells,and can promote the apoptosis of tumor cells.In addition,the mechanism of interaction between COX-2 single-chain antibody and COX-2 was studied using phage random peptide library screening and computer simulation.The possible molecular mechanism underlying anti-COX-2 intrabody mediated anti-tumor effects was preliminarily presumed.However,it still needs further experimental evidence.Therefore,based on our previous results from phage random peptide library screening and computer simulation,this study conducts mutation analysis to determine the pattern and key amino acids for antigen-antibody interaction,and reveal the molecular basis of antigen-antibody interaction.The details are as following:1.Prokaryotic expression vectors for 5 kinds of COX-2 deletion mutants were constructed successfully.We expressed and purified these mutants with the purity of95% or more in E.coli BL21(DE3)cells.2.ELISA results of deletion mutants showed that the interaction epitope between COX-2 and anti-COX-2 single-chain antibody was located in amino acids 466-604.3.Prokaryotic expression vectors for 4 kinds of COX-2 site-directed mutantswere successfully constructed.We expressed and purified these site-directed mutants.They were trCOX-2 V523 A,trCOX-2 S530 A,trCOX-2 L534 A and trCOX-2 N537 A.4.ELISA was used to identify the binding activity between site-directed mutant and OX-1.The results showed that all of Val-523,Ser-530,Leu-534 and Asn-537 amino acid residues involved in the formation of COX-2 epitope.Ser-530 played a crucial role in the interaction between COX-2 and anti-COX-2 single-chain antibody.In summary,we preliminarily detemined the key amino acid segments and key sites in the interaction between COX-2 and anti-COX-2 single chain antibody by deletion mutagenesis and site-directed mutagenesis assay.It laid a molecular foundation for the treatment of tumors with antibodies targeting COX-2.