Investigation on the Suppressive Mechanisms of Novel Anti-Glypican3 Antibody Drugs

Heparan sulfate proteoglycans (HSPGs) comprise a specific small group of proteins covalently linked to HS GAG chains. Changes in the expression of PGs and HS biosynthetic enzymes as well as the effect of these specific signatures play important roles on promoting invasion and metastasis in cancer pathologies. Glypican-3 (GPC3) is a member of the HSPG family, which acts as a tumor suppressor gene in some tissues, while acting as an ontofetal protein in other tissues. GPC3 messenger RNA and protein levels are increased in hepatocellular carcinoma (HCC), detectable in 74.8% of primary and recurrent HCCs, but in only 3.2% of normal livers. GPC3 over-expression was not detected in focal nodular hyperplasia and cirrhotic liver. Results from the tissue microarrays showed positive GPC3 expression in 55.7% of HCC patients, 9.6% of lung carcinoma and 5.7% of ovary adenocarcinoma. Intrahepatic cholangiocellular carcinoma, renal cell carcinoma, prostate acinar adenocarcinoma, breast carcinoma, gastric carcinoma and colorectal carcinoma showed negative GPC3 expression. All the results suggested that GPC3 protein is useful as a diagnostic marker. GPC3 has emerged as a candidate therapeutic target HCC. Several monoclonal antibodies (mAbs) against different epitopes of GPC3 have been evaluated their anti-tumour activity on cancer cell proliferation and survival. The combination of a humanized anti-GPC3 antibody (hGC33) and sorafenib is more potent in inhibiting tumour growth than sorafenib alone in the HepG2 xenograft model. The combination regimen may be clinically useful as an anti-liver cancer therapy. More recently, a human heavy-chain variable domain antibody recognizing a conformational epitope that requires both the amino and carboxy terminal domains of GPC3 inhibited proliferation of GPC3-positive cells and exhibited significant inhibition of HCC xenograft tumor growth in nude mice. Taken together, future work leading to the generation of new anti-GPC3 mAbs that target various functional domains and inhibit tumor growth by blocking the ligand–receptor interactions involved in different signaling pathways are important for understanding the mechanisms of tumorigenesis of HCC and other tumors. Combined together, all the antibodies will be useful for the development of therapeutic antibody drugs and diagnostic reagents for HCC. Thus, the objectives of this research proposal are as the following: 1. Expression and purification of recombinant human GPC3 proteins in variety of truncated fragments. 2. Characterization of polyclonal anti-GPC3 IgG and IgY antibodies in mice, chickens and human respectively. 3. Production and characterization of monoclonal anti-GPC3 scFv antibodies using phage display antibody technology. 4. Investigation of the inhibitory effect of these antibodies on tumorigenesis. 5. Study of the mechanisms involved in the inhibitory function of these antibodies. 6. Development of a highly sensitive method for detecting the presence of GPC3 protein in the patient sera. 7. Modification of anti-GPC3 scFv antibodies into chimeric or humanized antibodies with high affinity