It should be noted that mAbs with the highest binding signals for the peptide in a standard ELISA have very weak or no binding to cell-surface associated GPC3 (data not shown)

It should be noted that mAbs with the highest binding signals for the peptide in a standard ELISA have very weak or no binding to cell-surface associated GPC3 (data not shown). and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, KD = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy. Keywords: cell-surface glycoproteins, flow cytometry, heparan sulfate proteoglycans, hepatocellular carcinoma, high-throughput screening, hybridoma technology, peptide immunization Introduction Liver cancer is the fifth most common malignant tumor worldwide.1 Hepatocellular carcinoma (HCC) is the most common form. Daptomycin Cholangiocarcinoma (CCA) is usually another major form of primary liver malignancy. Although surgical resection offers a standard method for treatment of the disease, only a small portion of patients are eligible for the procedure. Liver cancer does not respond to most chemotherapy drugs. There is a critical need for novel immunotherapy such as antibody therapy. In view of this, it has been suggested that glypican-3 (GPC3) represents a stylish target for liver malignancy therapy because it is usually highly expressed in HCC.2,3 The GPC3 gene encodes a 70-kDa precursor core protein that can be cleaved by furin to generate a 40-kDa amino (N) terminal protein and a 30-kDa membrane-bound carboxyl (C) terminal protein.4,5 The C-terminus is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. GPC3 has been suggested as a target for antibody and cell-based immunotherapies.6-10 However, GPC3 expression is usually highly heterogeneous in HCC and other cancers, e.g., ovarian clear cell carcinoma, melanoma.11 Ideal therapeutic monoclonal antibodies (mAbs) should eliminate tumor cells expressing low levels of target antigen. Research in the area has been hampered by the lack of high-affinity mAbs that could be used to detect low expression of GPC3 in tumor cells for cancer therapy and diagnostics. Filmus and colleagues developed the widely-used 1G12 mAb specific for the C-terminus of GPC3 and established an ELISA method to detect serum GPC3 in HCC patients.2 On the other hand, Hippo et al. developed mAbs specific for the N-terminus of GPC3.12 While both studies detected soluble GPC3 protein in HCC culture supernatant or in the circulating blood of cancer patients, it is not clear whether the N-terminal or C-terminal subunit represents the soluble GPC3 format.3 Our unpublished work (and that of others) indicates that concentrations of serum Daptomycin GPC3 are generally not high in patients and that none of the readily available mAbs can be used to measure serum GPC3 due most likely to low affinity. Thus, new high-affinity antibodies for GPC3 may be useful to analyze and measure serum GPC3 in patients and to study liver cancer. Here, we describe a panel of novel antibodies that bind with high affinity to cell surface and Daptomycin soluble GPC3 proteins. A high-throughput flow cytometry method using the Guava EasyCyte system was developed for primary screening of a large number of mAbs recognizing the native form of GPC3 around the cell surface. The best mAb may be a very promising candidate for liver malignancy diagnostics and therapy. Results Design of the peptide as an immunogen for mouse immunization To design a peptide for immunization, we analyzed the primary structure of the GPC3 protein because a 3D structure is not available. The mature GPC3 protein around the cell surface is usually altered by heparan sulfate chains and Rabbit Polyclonal to OR52E2 attached to the cell membrane by a GPI anchor (Fig.?1A). The GPC3 gene is located on human X chromosome (Xq26) and transcribed and alternatively spliced into four mRNA.