Recombinant Dimeric IgA Antibodies against the Epidermal Growth Factor Receptor Mediate Effective Tumor Cell Killing

Abstract

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti–His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.

Characterization of a Mutated IgA2 Antibody of the m(1) Allotype against the Epidermal Growth Factor Receptor for the Recruitment of Monocytes and Macrophages

“…The mutant IgA2m(1) heavy chain sequence was subcloned into the pEE14.4 glutamine synthetase-expression system and transfected into CHO-K1 cells (Lonza Biologics, Slough, UK). Single clones were additionally transfected with a plasmid encoding a His-tagged human J-chain, and transfectants were selected using puromycin (PAA) (12). Single clones were grown in disposable CELLine CL 1000 bioreactors (Sartorius, Goettingen, Germany) under serum-free suspension culture conditions.…”

“…225-IgA was affinity-purified using Capture Select Fab chromatography medium (Capture Select, Naarden, The Netherlands) and a prepacked Superdex 200 26 ϫ 600 column (GE Healthcare) (30). Dimeric IgA2 was additionally purified by the C-terminal His tag of the J-chain using His tag-specific columns (GE Healthcare) (12). All purification steps were run on an ÄKTAprime liquid chromatography system (GE Healthcare).…”

“…The major differences between IgA1 and IgA2 are located (i) in the hinge region, with IgA2 lacking the 13-amino acid elongation with up to five O-glycosylation sites existing in IgA1, (ii) in the N-glycosylation, with IgA2 being more than twice as heavily glycosylated than IgA1, and (iii) in the heavy and light chain linkage, where the Caucasian IgA2m(1) allotype lacks a covalent disulfide bond between heavy and light chains, which is present in IgA1 and in the IgA2m(2) allotype of those of African and Asian descent (14,20). In previous reports, we demonstrated that IgA antibodies were able to trigger polymorphonuclear cell (PMN)-mediated ADCC more efficiently than IgG antibodies, with IgA2 being more effective than IgA1 (11) and dimeric being more effective than monomeric IgA1 (12). However, the noncovalent linkage of heavy and light chains in the Caucasian IgA2m(1) makes this isotype less attractive for biotechnological production and for a therapeutic antibody as it might lead to an unstable recombinant molecule under certain conditions.…”

Staphylococcus aureus Formyl Peptide Receptor–like 1 Inhibitor (FLIPr) and Its Homologue FLIPr-like Are Potent FcγR Antagonists That Inhibit IgG-Mediated Effector Functions

“…2E, 2F). The two isoforms of FcgRIIIb, differing by six ectodomain amino acids from FcgRIIIa-F158 (and 5 aa from FcgRIIIa-V158), of which two are located in IgG-binding regions (23,24,28) binding of recombinant human IgG1 to FcgR was determined. FLIPr and FLIPr-like efficiently prevented binding of human IgG1 to the FcgRs (Fig.…”

“…ADCC assays against 51 Cr-labeled A1207 target cells were performed as described (23,24). In short, isolated neutrophils or NK cells (from PBMCs using The EasySep Human NK cell Enrichment Kit; STEMCELL Technologies) were preincubated with 3 mg/ml inhibitory protein.…”