Abstract
Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer.
Highlights
Glypican-3 (GPC3) is a glycophosphatidylinositol (GPI)-anchored cell surface heparan sulfate proteoglycan that is expressed during early development, and expression can be detected in human embryo, fetus and placental tissues[1], but not in normal adult tissue[2]
The complementarity determining region (CDR) identified by Kabat et al is based on the sequence variability of human, mouse and rabbit antibodies[15,16], whereas the IMGT CDR takes into account both the sequence variability calculated by Kabat and the antibody structure or antibody-antigen structure complex[17]
Sequencing of the mouse anti-GPC3 monoclonal antibodies revealed that the Fv regions of YP7, YP8, YP9 and YP9.1 were homologous to each other (Fig. 1a)
Summary
Glypican-3 (GPC3) is a glycophosphatidylinositol (GPI)-anchored cell surface heparan sulfate proteoglycan that is expressed during early development, and expression can be detected in human embryo, fetus and placental tissues[1], but not in normal adult tissue[2]. We initially tested one of these antibodies (YP7) and found that it had very specific binding towards HCC tumor cells in patient tissues and inhibited the growth of a hepatoblastoma xenograft tumor in nude mice[12]. We recently humanized a rabbit monoclonal antibody (YP218) by grafting dual CDRs (KABAT and IMGT) to the most similar human germline sequence without the need for back-mutation[18]. We tested whether the same dual CDR grafting method could be used to humanize the mouse anti-GPC3 antibodies. We found a non-CDR residue, the proline at position 41 in VH, is important in humanization of mouse antibodies and should be retained during humanization for the best activity and antigen binding affinity. We found that the hYP7 antibody inhibited Hep3B (an HCC cell line) xenograft tumor growth in nude mice
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