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Antibody therapy of cancer is increasingly used in the clinic and has improved patient's life expectancy. Except for immune checkpoint inhibition, the mode of action of many antibodies is to recognize overexpressed or specific tumor antigens and initiate either direct F(ab') 2 -mediated tumor cell killing, or Fc-mediated effects such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors. All antibodies used in the clinic are of the IgG isotype. The IgA isotype can, however, also elicit powerful anti-tumor responses through engagement of the activating Fc receptor for monomeric IgA (FcαRI). In addition to monocytes, macrophages and eosinophils as FcαRI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized tumor cells. However, with IgG as single agent it appears almost impossible to activate neutrophils efficiently, as we have visualized by live cell imaging of tumor cell killing. In this study, we investigated Fc receptor expression, binding and signaling to clarify why triggering of neutrophils by IgA is more efficient than by IgG. FcαRI expression on neutrophils is ~2 times and ~20 times lower than that of Fcγ receptors FcγRIIa and FcγRIIIb, but still, binding of neutrophils to IgA- or IgG-coated surfaces was similar. In addition, our data suggest that IgA-mediated binding of neutrophils is more stable compared to IgG. IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG as indicated by measuring the p-ERK signaling molecule. We propose that the higher stoichiometry of IgA to the FcαR/FcRγ-chain complex, activating four ITAMs (Immunoreceptor Tyrosine-based Activating Motifs) compared to a single ITAM for FcγRIIa, combined with a possible decoy role of the highly expressed FcγRIIIb, explains why IgA is much better than IgG at triggering tumor cell killing by neutrophils. We anticipate that harnessing the vast population of neutrophils by the use of IgA monoclonal antibodies can be a valuable addition to the growing arsenal of antibody-based therapeutics for cancer treatment.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy., (©2015 American Association for Cancer Research.)

Fc receptors (FcR) are expressed on immune cells and bind to the Fc tail of antibodies. This interaction is essential for FcR-mediated signaling and triggering of cellular effector functions. FcR activation is tightly regulated to prevent immune responses by non-antigen bound antibodies or in the absence of 'danger signals'. FcR activity may be modulated at the plasma membrane via cross-talk with integrins. In addition, cytokines at the site of infection/inflammation can increase FcR avidity, a process referred to as inside-out signaling. This regulatory mechanism has been described for FcγRI (CD64), FcγRIIa (CD32a), and FcαRI (CD89) and is also well-known for integrins. Key cellular events during inside-out signaling are (de)phosphorylation, clustering, cytoskeleton rearrangements, and conformational changes. The latter can be studied with antibodies that specifically recognize epitopes exposed by the active (high affinity) or inactive (low affinity) state of the FcR. These antibodies are important tools to investigate the role of FcR activation in disease settings. Research on FcR has gained momentum with the rise of monoclonal antibodies (mAb) entering the clinic for the treatment of cancer and other diseases. The clinical outcome of mAb therapy may be improved by increasing FcR avidity by cytokine stimulation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)