title: The anti-cancer IgM monoclonal antibody PAT-SM6 binds with high avidity to the unfolded protein response regulator GRP78.
abstract: The monoclonal IgM antibody PAT-SM6 derived from human tumours induces apoptosis in tumour cells and is considered a potential anti-cancer agent. A primary target for PAT-SM6 is the unfolded protein response regulator GRP78, over-expressed externally on the cell surface of tumour cells. Small angle X-ray scattering (SAXS) studies of human GRP78 showed a two-domain dumbbell-shaped monomer, while SAXS analysis of PAT-SM6 revealed a saucer-shaped structure accommodating five-fold symmetry, consistent with previous studies of related proteins. Sedimentation velocity analysis of GRP78 and PAT-SM6 mixtures indicated weak complex formation characterized by dissociation constants in the high micromolar concentration range. In contrast, enzyme-linked immunosorbant assays (ELISAs) showed strong and specific interactions between PAT-SM6 and immobilized GRP78. The apparent binding constant estimated from a PAT-SM6 saturation curve correlated strongly with the concentration of GRP78 used to coat the microtiter tray. Experiments using polyclonal antiGRP78 IgG antibodies or a monoclonal IgG derivative of PAT-SM6 did not show a similar dependence. Competition experiments with soluble GRP78 indicated more effective inhibition of PAT-SM6 binding at low GRP78 coating concentrations. These observations suggest an avidity-based binding mechanism that depends on the multi-point attachment of PAT-SM6 to GRP78 clustered on the surface of the tray. Analysis of ELISA data at high GRP78 coating concentrations yielded an apparent dissociation constant of approximately 4 nM. We propose that the biological action of PAT-SM6 in tumour cell apoptosis may depend on the multivalent nature of PAT-SM6 and the high avidity of its interaction with multiple GRP78 molecules clustered on the tumour cell surface.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Rosenes
authors.forename: Terrence D
authors.initials: TD
authors.lastname: Mulhern
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: Leodevico L
authors.initials: LL
authors.lastname: Ilag
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Power
authors.forename: Chris
authors.initials: C
authors.lastname: Hosking
authors.forename: Frank
authors.initials: F
authors.lastname: Hensel
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Howlett
authors.forename: Yee-Foong
authors.initials: YF
authors.lastname: Mok
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000276:immunology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004797:Enzyme-Linked Immunosorbent Assay; D006360:Heat-Shock Proteins / Q000276:immunology*; D006801:Humans; D055767:Immobilized Proteins / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology*; D012996:Solutions
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D055767:Immobilized Proteins; D007075:Immunoglobulin M; D012996:Solutions