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In this study, we describe the ability of hybrid mouse monoclonal antibody (mAb) to induce monocyte-mediated cytotoxicity towards human IgA1-coated E (HuIgA1-E), and the effect of mAb binding valency on Fc gamma RI-mediated ADCC. All hybrid monospecific (ms) anti-HuIgA1 and bispecific (bs) anti-HuIgA1/HRP mAb were capable of inducing monocyte-mediated lysis of HuIgA1-E, in spite of differences in mAb densities essential for optimal lysis. The cytotoxicity induced by hybrid mAb which consist of one or more mIgG2a H chains was predominantly mediated via Fc gamma RI, as shown by inhibition studies on monocytes with Fc gamma RI-blocking mAb TB-3 (approximately 80% inhibition). However, partial inhibition of mIgG1-2a and mIgG2a-2b-induced cytotoxicity (20-50%) was observed by using Fc gamma RII-blocking mAb IV.3 or CIKM5. For hybrid mIgG1-1 mAb the opposite was true; the cytotoxicity was predominantly mediated via Fc gamma RII (70-80%) and less via Fc gamma RI (20-30%). Comparing the hybrid ms anti-HuIgA1 mAb-induced cytotoxicity with the cytotoxicity induced by hybrid bs anti-HuIgA1/HRP mAb of the same isotype, we observed a decrease in cytotoxicity towards HuIgA1-E sensitized with univalently bound bs anti-HuIgA1/HRP mAb. This decrease was only found for Fc gamma RI-mediated ADCC (mIgG2a-2a, mIgG1-2a and mIgG2a-2b). This diminished recognition of univalently bound IgG relative to bivalently bound IgG by Fc gamma RI was also observed with U937 effector cells. In conclusion, this work shows that hybrid mAb are able to induce monocyte-mediated cytotoxicity towards E-HuIgA1 and that there appears to be an effect of Ag-IgG binding valency on Fc gamma RI-mediated cytotoxicity.

We studied the interaction of bispecific mouse mAb with human IgG Fc receptors, and assessed their ability to activate the monocytic cell line U937. Binding of monomeric hybrid anti-HuIgA1/HRP mAb to the high-affinity IgG receptor, Fc gamma RI, on U937 cells was only observed when mAb with one or more mIgG2a H chains (hybrid mIgG1-2a, mIgG2a-2b, and mIgG2a-2a) were used. These Fc gamma RI-bound hybrid mAb were capable of enhancing the internal free cytosolic Ca2+ concentration ([Ca2+]i) in U937 cells only when bound mIgG were cross-linked using F(ab')2 fragments of goat anti-mIg antibody. A hybrid mIgG1-2a mAb were cross-linked using goat anti-mIgG1 antibody, showing that the hybrid mAb themselves mediate the induction of Ca2+ increase. Remarkably, anti-Fc gamma RII mAb IV.3 was able to inhibit the Ca2+ increase induced via mIgG2a-1 or mIgG1-2a hybrid mAb completely, despite the fact that we could not detect any effect of IV.3 on binding of monomeric hybrid mIgG1-2a or mIgG2a-1 mAb to U937. The hybrid mAb were also able to induce lysis of HuIgA1-coated E using U937 effector cells. This lysis was completely inhibited by preincubation of U937 cells with mIgG2a mAb TB-3, which blocks Fc gamma RI via its Fc-part ("Kurlander phenomenon"). In contrast, Fc gamma RII-blocking mAb IV.3 and CIKM5 caused a significant enhancement of the antibody-dependent cellular cytotoxicity (ADCC) activity mediated by hybrid mIgG1-2a and mIgG2a-2b mAb. This enhancement did not occur when the parental anti-HuIgA1/2a or the hybrid anti-HuIgA1/HRP/2a-2a mAb were evaluated for ADCC activity. These findings suggest that hybrid mAb not only can bind to Fc gamma RI, but can mediate functional activation of myeloid cells. Given the effect of mAb IV.3 on [Ca2+]i changes and ADCC triggered through IgG1-2a mAb, we suggest that Fc gamma RII may have a role in the regulation of Fc gamma RI-triggered functions or signaling.