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.
Highlights
Natural IgM antibodies play an important role in the innate immune response where they are involved in the early detection of foreign particles as well as the detection of modified self-structures including chemically modified proteins and amyloid fibrils [1,2,3]
PAT-SM6 was originally isolated from a gastric cancer patient and shown to bind to cell membrane-associated GRP78 in tumour cells [9] as well as oxidized plasma low density lipoproteins [8]
The present study shows that the interaction of purified PAT-SM6 with GRP78 is mediated through an aviditybased mechanism based on the multivalency of the antibody
Summary
Natural IgM antibodies play an important role in the innate immune response where they are involved in the early detection of foreign particles as well as the detection of modified self-structures including chemically modified proteins and amyloid fibrils [1,2,3]. The recent development of human hybridoma technology [5] has led to the isolation of a large number of monoclonal antibodies of the IgM class from the tumours of cancer patients [6]. A number of these antibodies kill malignant cells by inducing apoptotic pathways [7], highlighting the potential use of monoclonal IgM antibodies in the development of new anti-cancer treatments. The human IgM monoclonal antibody, PAT-SM6, induces the death of tumour cells via an apoptotic pathway accompanied by intracellular lipid accumulation [8]. PAT-SM6 targets tumour cells, by binding to the protein GRP78 which is over-expressed externally on the cell surface of tumour cells [9]. PAT-SM6 binds low density lipoprotein (LDL) and oxidized LDL [8] leading to a working model for the tumour-specific apoptotic activity of PAT-SM6 whereby PAT-SM6 delivers excess lipid in the form of bound LDL or oxidized LDL into tumours by binding to modified GRP78 present on the surface of tumour cells [8]
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