PMID- 23028685 OWN - NLM STAT- MEDLINE DCOM- 20130305 LR - 20240313 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 7 IP - 9 DP - 2012 TI - The anti-cancer IgM monoclonal antibody PAT-SM6 binds with high avidity to the unfolded protein response regulator GRP78. PG - e44927 LID - 10.1371/journal.pone.0044927 [doi] LID - e44927 AB - 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. FAU - Rosenes, Zachary AU - Rosenes Z AD - Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia. FAU - Mulhern, Terrence D AU - Mulhern TD FAU - Hatters, Danny M AU - Hatters DM FAU - Ilag, Leodevico L AU - Ilag LL FAU - Power, Barbara E AU - Power BE FAU - Hosking, Chris AU - Hosking C FAU - Hensel, Frank AU - Hensel F FAU - Howlett, Geoffrey J AU - Howlett GJ FAU - Mok, Yee-Foong AU - Mok YF LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20120919 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Antibodies, Monoclonal) RN - 0 (Antineoplastic Agents) RN - 0 (Endoplasmic Reticulum Chaperone BiP) RN - 0 (HSPA5 protein, human) RN - 0 (Heat-Shock Proteins) RN - 0 (Immobilized Proteins) RN - 0 (Immunoglobulin M) RN - 0 (Solutions) SB - IM MH - Antibodies, Monoclonal/*immunology MH - *Antibody Specificity MH - Antineoplastic Agents/*immunology MH - Endoplasmic Reticulum Chaperone BiP MH - Enzyme-Linked Immunosorbent Assay MH - Heat-Shock Proteins/*immunology MH - Humans MH - Immobilized Proteins/immunology MH - Immunoglobulin M/*immunology MH - Solutions PMC - PMC3446985 COIS- Competing Interests: This work is partially supported by Patrys Ltd, a company currently conducting clinical trials of PAT-SM6 as a potential anti-cancer treatment. EDAT- 2012/10/03 06:00 MHDA- 2013/03/06 06:00 PMCR- 2012/09/19 CRDT- 2012/10/03 06:00 PHST- 2012/06/21 00:00 [received] PHST- 2012/08/09 00:00 [accepted] PHST- 2012/10/03 06:00 [entrez] PHST- 2012/10/03 06:00 [pubmed] PHST- 2013/03/06 06:00 [medline] PHST- 2012/09/19 00:00 [pmc-release] AID - PONE-D-12-18562 [pii] AID - 10.1371/journal.pone.0044927 [doi] PST - ppublish SO - PLoS One. 2012;7(9):e44927. doi: 10.1371/journal.pone.0044927. Epub 2012 Sep 19.