The role of epidermal growth factor receptor (EGFR)-directed recombinant dimeric IgA antibodies in tumor cell killing.

Abstract

e13014 Background: The epidermal growth factor receptor (EGFR) is a validated target for personalized cancer therapy with IgG antibodies. IgA antibodies contribute to the humoral part of the mucosal immune system, but their immunotherapeutic potential has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA antibodies against EGFR. Methods: Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGFR-specific monomeric 225-IgA (mIgA) with a vector coding for His-tagged human J-chain. Dimeric IgA (dIgA) was purified by two different affinity (anti-human-κ and anti-His-tag) and one size exclusion chromatography. The resulting homogenous preparation of highly pure IgA dimers was biochemically and functionally characterized. Results: Functional studies demonstrated dIgA to be at least as effective as mIgA in triggering antibody-dependent cellular cytotoxicity of A431 tumor cells by isolated monocytes (EC50 1.9/0.8 nM for mIgA/dIgA), by isolated PMN (EC50 5.6/3.7 nM for mIgA/dIgA) and in human whole blood 51chromium-release-assays (EC50 1.8/1.4 nM for mIgA/dIgA). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells and furthermore downmodulation of EGFR by dIgA was achieved at significantly lower concentrations than by mIgA (EC50 14.9/124.5 nM, 76.2/335.2 nM, respectively). In addition, growth of EGFR-expressing DiFi cells was inhibited more effectively by dimeric than by monomeric IgA (EC50 6.2/49.7 nM). Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported via the pIgR through an epithelial cell monolayer in a transcytosis assay with polarized MDCK cells stably transfected with human polymeric immunoglobulin receptor (pIgR). Conclusions: These studies demonstrate that recombinant dimeric IgA antibodies against EGFR trigger a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies - suggesting that dimeric IgA antibodies may constitute an interesting antibody format for tumor therapy.