Abstract
Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.
Highlights
Engineered antibodies and variable region (V-region)1 fragments are established as indispensable tools for scientific and clinical uses
Because ligand binding can stabilize recombinant VH-VL pairing in vitro [22, 23], we tested whether addition of the hapten NPPC (5 mM) to cultured cells would increase the secretion of Ig into the surrounding medium
APPC binds with ϳ12-fold lower affinity as compared with NPPC [24], suggesting that binding affinity may correlate with efficiency of rescue
Summary
V-region, variable region; CDR, complementarity determining region; ER, endoplasmic reticulum; VH, variable region of Ig heavy chain; VL, variable region of Ig light chain; PC, phosphocholine; NPPC, p-nitrophenylphosphocholine; APPC, p-aminophenylphosphocholine; WT, wild type; H, heavy; L, light; ELISA, enzyme-linked immunosorbent assay; BFA, brefeldin A; PAGE, polyacrylamide gel electrophoresis. The mechanism(s) by which chemical chaperones function are not fully understood but are thought to include stabilization of improperly folded proteins [10], reduction of aggregation [13], and prevention of nonproductive interactions with ER proteins [11]. Chemical chaperones such as glycerol and trimethylamine N-oxide appear to be nonspecific as they are able to rescue diverse types of proteins [16], whereas other chemical chaperones only function with a narrower set of proteins [13, 17]. The capacity of trafficking-impaired Ig to bind antigen correlated with ligand-mediated rescue of secretion These data indicate that the mechanism of rescue involves the intracellular stabilization of VH-VL pairing. Hapten ligands may find application for regulating or increasing assembly and secretion of anti-hapten Ig from hybridomas
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