Abstract
Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.
Highlights
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The availability of a series of affinity mutants derived from a single human single-chain Fv (scFv) makes this a valuable resource because analysis is not confounded by the uncertainties associated with Abs that bind to distinct epitopes on a targeted Ag, or possess differing isotypes that may influence their interactions with Fc receptors or complement
We show in this study that affinity for the target Ag clearly influences the extent and efficiency of Ab-dependent cellular cytotoxicity (ADCC), and that the relationships hold true in tumor cell lines with widely disparate levels of target Ag expression
Summary
ADCC, Ab-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; MFI, mean fluorescence intensity; scFv, singlechain Fv; ECD, extracellular domain; CHO, Chinese hamster ovary. We have previously designed and characterized the binding properties of a series of single-chain Fv (scFv) molecules that target an identical epitope on the extracellular domain (ECD) of HER2/neu. The regulation of ADCC promotion by the affinity of IgG mAbs has not been examined previously using a rigorously derived panel of Abs that bind to an identical epitope of the targeted www.jimmunol.org. We demonstrate that high intrinsic binding affinity variants promote more ADCC, even when the affinity mutants possess essentially identical apparent affinities. These findings have important implications for the development of clinically relevant unconjugated Abs that mediate ADCC
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