Epitope mapping of series of monoclonal antibodies against the hepatocellular carcinoma-associated antigen HAb18G/CD147

The hepatocellular carcinoma-associated antigen HAb18G/CD147, a member of CD147 family, could promote tumour invasion and metastasis via inducing the secretion of matrix metalloproteinases (MMP). Anti-CD147 monoclonal antibodies (MoAb) have exhibited obvious inhibitory effect on MMP induction. However, none of the epitopes of these MoAb has been reported. We previously prepared five MoAb against HAb18G/CD147, named HAb18, 3B3, 1B3, 5A5 and 4D2. To map the epitopes of these MoAb, a series of truncated fragments of extracellular region of HAb18G/CD147 was expressed in Escherichia coli and the MoAb-binding affinity to these fragments was examined with an enzyme-linked immunosorbent assay and Western blot. The residues ³⁹LTCSLNDSATEV⁵⁰, ³⁶KILLTCS⁴² and ²²AAGTVFTTVEDL³³ were determined to be the epitopes of HAb18, 3B3 and 1B3, respectively, which were further proved by a dot-blot analysis with synthesized peptides and bioinformatics epitope prediction. The binding regions of MoAb 5A5 and 4D2 were located at residues E¹²⁰-R²⁰³. Then we constructed and expressed full-length HAb18G/CD147 and truncated HAb18G/CD147 without residues A²²-V⁵⁰ in COS-7 cells. Gelatin zymography and Boyden chamber assay showed that the COS-7 cells expressing truncated HAb18G/CD147 failed to induce MMP production and enhance the cells' invasive potential, compared with the cells expressing full-length HAb18G/CD147. Taken together with the obviously inhibitory effects of HAb18 on the function of full-length HAb18G/CD147, these findings suggest that residues ²²AAGTVFTTVEDLGSKILLTCSLNDSATEV⁵⁰ may play a critical role in the functions of HAb18G/CD147 on MMP secretion and tumour invasion. These key residues can be used as potential drug target in cancer therapy.