Epitope Mapping of Series of Monoclonal Antibodies Against the Hepatocellular Carcinoma‐associated Antigen HAb18G/CD147

Abstract

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.

Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma

“…The protein pellet was resuspended in cold acetone and the precipitation step was repeated. Samples were analyzed by immunoblotting with antibodies against extracellular CD147 (HAb18 antibody, prepared by our laboratory) [21] or intracellular CD147 (C-19 antibody, Santa Cruz Biotechnology, CA, USA).…”

An epitope-specific novel anti-EMMPRIN polyclonal antibody inhibits tumor progression

“…19 However, in contrast to these findings, another study found that the sequence AAGTVFTT-VEDLGSKILLTCSLNDSATEV (positions 22-50 in the human EMMRIN sequence), which does not include our epitope at all, was responsible for the EMMPRIN MMP induction activity and association with tumor invasion. 20 We suggest that because we used an antibody to target a specific epitope of 11 amino acids, we could use a shorter sequence than the two previous studies that used peptides to directly generate a steric hindrance or to compete with the homophilic binding to EMMPRIN. More importantly, all of these epitope-mapping studies focused only on the MMP-induction activity of EMMPRIN, and showed that it might span over a stretch of about 40 amino acids, but no data so far revealed the epitope responsible for the VEGF-induction activity of the protein.…”

Characterization of Basigin Isoforms and the Inhibitory Function of Basigin-3 in Human Hepatocellular Carcinoma Proliferation and Invasion

“…HAb18 is a monoclonal antibody (MAb) against the Ig-C2 domain (specific to basigin-2) (20). Recombinant protein B23ID-64P (IgI domain, consisting of 64 residues from T121 to C185 of basigin-2) and synthetic polypeptide B4N-11P (11 residues from the N terminus of basigin-4, MKQSDASPQER-C) were used as antigens for the preparation of rabbit polyclonal antibodies B23ID and B4N11, respectively, against the IgI domain (specific to both basigin-2 and basigin-3) and basigin-4.…”