Recombinant Dimeric IgA Antibodies as Tumor-Specific Agents.

Abstract

AbstractAbstract 1488Dimeric IgA antibodies contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA, exemplarily directed against the epidermal growth factor receptor (EGF-R). Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGF-R specific monomeric IgA with a vector coding for the His-tagged human J-chain. For purification of J-chain-containing dimeric IgA two different affinity (anti-human-kappa and anti-His-tag) and one size exclusion chromatography were combined, resulting in a homogenous preparation of highly pure IgA dimers, as determined by gel electrophoresis under denaturing and native conditions using silver stain, Coomassie blue or Western blots for protein detection, respectively. Functional studies demonstrated dimeric IgA (dIgA) to be at least as effective as monomeric IgA (mIgA) in triggering ADCC of A431 tumor cells by isolated monocytes (EC50 0.35 and 0.30 μg/ml for mIgA and dIgA, 0.23 μg/ml for the corresponding IgG1; maximal killing 51.2 vs 43.3 vs 56.3 %), by isolated PMN (EC50 0.96, 1.20 and 0.8 μg/ml for mIgA, dIgA and IgG1; maximal killing 52.6 vs 50.6 vs 19.9 %) and in human whole blood assays (EC50 0.32 and 0.32 μg/ml for mIgA and dIgA; maximal killing 7.9 vs 12.5 %; IgG1: maximal killing 0.7 %, EC50 not determinable). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells by dimeric IgA was achieved at significantly lower concentrations than by monomeric IgA (EC50 7.1 vs 18.1 μg/ml, respectively). In addition, growth of EGF-R expressing DiFi colon carcinoma cells was inhibited at significantly lower concentrations by dimeric than by monomeric IgA (EC50 1.6 vs 3.7 μg/ml). Both IgA isoforms and their IgG1 variant were similarly effective in triggering apoptosis of DiFi cells, as analyzed by PARP cleavage. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric immunoglobulin receptor (pIgR) through an epithelial cell monolayer, as analyzed by an experimental transcytosis assay with polarized MDCK cells stably transfected with human pIgR. Together, these studies demonstrate that recombinant dimeric IgA antibodies recruit a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies, trigger important biological functions and constitute an interesting antibody format for future tumor therapies. Disclosures:van de Winkel:Genmab: Employment, Membership on an entity's Board of Directors or advisory committees. Valerius:Genmab: Consultancy, Research Funding.