title: IgG Fab fragments forming bivalent complexes by a conformational mechanism that is reversible by osmolytes.
abstract: Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes.
authors.affiliation: Department of Immunology, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
authors.forename: Alfreda D
authors.initials: AD
authors.lastname: Nelson
authors.forename: Michele M
authors.initials: MM
authors.lastname: Hoffmann
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Parks
authors.forename: Surendra
authors.initials: S
authors.lastname: Dasari
authors.forename: Adam G
authors.initials: AG
authors.lastname: Schrum
authors.forename: Diana
authors.initials: D
authors.lastname: Gil
mesh_terms: D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology*; D002364:Caseins / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D008055:Lipids / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D014674:Plant Proteins, Dietary / Q000494:pharmacology*; D011392:Proline / Q000494:pharmacology; D011487:Protein Conformation; D055550:Protein Stability / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology
chemical_list: D002241:Carbohydrates; D002364:Caseins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008055:Lipids; D014674:Plant Proteins, Dietary; D017260:Receptor-CD3 Complex, Antigen, T-Cell; C080652:Osmolite; D011392:Proline