title: Simultaneous binding of the anti-cancer IgM monoclonal antibody PAT-SM6 to low density lipoproteins and GRP78.
abstract: The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and 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: Yee-Foong
authors.initials: YF
authors.lastname: Mok
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
authors.forename: Terrence D
authors.initials: TD
authors.lastname: Mulhern
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: Frank
authors.initials: F
authors.lastname: Hensel
authors.forename: Geoffrey J
authors.initials: GJ
authors.lastname: Howlett
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D003300:Copper / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004797:Enzyme-Linked Immunosorbent Assay; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008863:Microspheres; D010084:Oxidation-Reduction / Q000187:drug effects; D011137:Polystyrenes / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000941:Antigens; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008077:Lipoproteins, LDL; D011137:Polystyrenes; C044203:anti-IgM; C099252:oxidized low density lipoprotein; D003300:Copper