Recombinant Chimeric IgA1 and IgA2 Antibodies for Tumor Immunotherapy.

The therapeutic potential of human IgA antibodies has hardly been explored, although IgA is the most abundantly produced antibody isotype in humans. Here, we describe the generation and functional characterization of human/mouse chimeric IgA antibodies against the epidermal growth factor receptor (EGF-R), which is a validated target antigen for tumor immunotherapy. Human IgG1, IgA1 and IgA2 variants of the C225 antibody were produced in CHO-K1 transfectomas using serum-free, non-adherent culture conditions. Productivity rates of selected IgA producing clones were approximately 5 pg/cell/day. A two-step purification procedure consisting of thiophilic agarose columns followed by gel electrophoresis on Superdex 200 yielded highly purified monomeric IgA antibodies, which were stable at 4° C. Silver staining and Western blots with human kappa-light- and human α-heavy chain antibodies confirmed the purity and correct assemby of IgA1 and IgA2 antibodies. Co-transfection of a His-tagged joining (J) chain significantly increased the amount of dimeric IgA antibodies, which were detected by anti-His antibodies. IgG1 and monomeric IgA antibodies demonstrated similar binding to EGF-R expressing A431 tumor cells (EC50 approx. 10 μg/ml) and to EGF-R transfectants. Furthermore, IgA1 and IgA2, but not IgG1 demonstrated binding to FcαRI (CD89)- transfected cells. All three isotypes were similarly effective in recruiting F(ab’)- mediated killing mechanisms such as blockade of EGF binding, inhibiting EGF-R phosphorylation, and mediating growth inhibition of EGF-R expressing cells. All three isotypes did not activate complement- mediated killing. Human IgG1 effectively triggered MNC- mediated ADCC, but activated PMN only weakly. Importantly, both human IgA isotypes were significantly more effective than IgG1 in recruiting neutrophils for ADCC (EC50 approx. 0.25 μg/ml), and triggered ADCC at effector to target cell ratios as low as 5:1. Since neutrophils constitute the most abundant effector cell population in human blood, IgA antibodies triggered higher levels of tumor cell killing in whole blood assays than human IgG1 antibodies - particularly when blood from G-CSF- primed donors was compared to healthy donor blood. Together, these data suggest that recombinant human IgA antibodies can be produced in reasonable amounts, and that they may constitute promising reagents for immunotherapy.