Study On The Molecular Mechanism Of Human Single-chain Antibody Against COX-2

COX-2(Cyclooxygenase-2)is a key enzyme which catalyzes the conversion of arachidonic acid(AA)into prostaglandins(PGs).It plays an important role in pathophysiological processes such as tumorigenesis,angiogenesis,inflammation,drug resistance of tumor cells.Therefore,COX-2 has been an important target for cancer therapy.In consideration of the toxic side effects of COX-2 inhibitors in clinical application,it is urgent to develop a new strategy with high specificity and efficiency to block the biological function of COX-2.ScFv(Single-chain variable fragments)has been emerging tremendous application prospect in cancer therapy and diagnosis because of its specific affinity to the antigen,high specificity,low immunogenicity and strong tumor tissue penetration.In previous studies,we have successfully developed an anti-COX-2 scFv which could specifically bind to COX-2 and block its biological activity in vitro and in vivo.Endoplasmic reticulum retained anti-COX-2 intracellular scFv effectively inhibits the growth,proliferation,migration and invasion of tumor cells,but induces tumor cell apoptosis.However,the molecular mechanism underlying the interaction between COX-2 and the scFv is still not clear.In order to explore the mechanism,this study was designed to apply the phage circular 7-mer peptides library display technology,homology modeling and molecular docking to investigate the interaction model and molecular basis between COX-2 and anti-COX-2 scFv.The main results were as follows:1.We successfully purified the recombinant human COX-2 and anti-COX-2 scFv expressed in E.Coli BL21 and E.Coli HB2151 with protein purity of>95%.2.ELISA results showed that the binding activity of COX-2 to anti-COX-2 scFv decreased with the increase of heat-treatment temperature,and was lost when the temperature reached 100?.The results indicated that COX-2 no longer has the ability to bind with anti-COX-2 scFv as the conformation of COX-2 protein has been completely destroyed at 100?.It suggested that epitope on COX-2 recognized by anti-COX-2 scFv is non-linear epitope.3.Using anti-COX-2 scFv as target protein,we got 36 positive phage clones which could specifically bind to anti-COX-2 scFv after four rounds of phage circular 7-mer peptides library biopanning.Target DNA of 36 clones were cloned into the pMD-18T vector and sequenced.Finally,19 effective circular 7-mer peptide sequences were obtained.4.Results from circular 7-mer peptide sequence alignment demonstrated that these peptides shared three motifs(KRTMREN,PGA and TSKG)and were homologous to the C-terminal from Lys-511 to Asn-537,Pro-514 to Ala-527,Thr-521 to Gly-533 of COX-2,respectively.As these motifs had an overlap when aligning with COX-2,we got a mimotope peptide KRPTMEGASKGN which were highly homologous to amino acids at the C-terminal from Lys-511 to Asn-537 of COX-2.So we supposed that KRPTMEGASKGN may be involved in the formation of the COX-2 epitope interacting with anti-COX-2 scFv and play an important role in the recognition between COX-2 and scFv.5.Homology modeling of anti-COX-2 scFv and molecular docking between circular 7-mer peptide and anti-COX-2 scFv showed that majority of peptides could bind to the CDRs of scFv including the amino acid residues 93-98,160-162,182-186 and 228-232,and these peptides shared the above conserved motifs.These results further support our opinion that KRPTMEGASKGN may be involved in the formation of the COX-2 epitope interacting with anti-COX-2 scFv.6.By sequence alignment the mimic epitope peptide with the key amino acids in the catalytic domain of COX-2,we found the mimic epitope peptide contains several key amino acids for COX-2 enzyme activity,indicating that anti-COX-2 scFv may interact with these amino acids and then inhibit the catalytic activity of COX-2.In summary,by using phage cirlular 7-mer peptides library display technology and comoputer simulation,the mimic epitope of COX-2 recognized by anti-COX-2 scFv and the possible mechanism that anti-COX-2 scFv inhibits the function of COX-2 were speculated.It laid a molecular foundation for anti-tumor antibody therapy targeting COX-2.