Abstract
Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to determine whether PMN ADCC also would be increased by the addition of an antibody/GM-CSF fusion protein and whether this would be associated with the up-regulation and activation of Mac-1 (CD11b/CD18) and with azurophil granule exocytosis. ADCC against LA-N-1 human neuroblastoma cells was evaluated with 4-hour calcein acetoxymethyl ester (calcein-AM) microcytotoxicity assay, electron microscopy, and multi-parameter flow cytometry. With the calcein-AM assay, LA-N-1 cell survival was 10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside (anti-GD2) immunocytokine hu14.18/GM-CSF, by monoclonal antibody (mAb) hu14.18 mixed with GM-CSF, and by hu14.18 alone. Function-blocking mAbs demonstrated that FcgammaRII and FcgammaRIII were required for ADCC with hu14.18 alone or mixed with GM-CSF, but that only FcgammaRII was required for ADCC with hu14.18/GM-CSF. ADCC mediated by hu14.18 and hu14.18/GM-CSF was Mac-1 dependent. Electron microscopy demonstrated the greatest PMN adhesion, spreading, and lysis of targets with hu14.18/GM-CSF. Monoclonal antibodies blocking Mac-1 function allowed the tethering of PMN to targets with hu14.18/GM-CSF but prevented adhesion, spreading, and cytolysis. Flow cytometry showed that hu14.18 with or without GM-CSF and hu14.18/GM-CSF all mediated Mac-1-dependent PMN-target cell conjugate formation but that GM-CSF was required for the highest expression and activation of Mac-1, as evidenced by the mAb24-defined beta(2)-integrin activation epitope. Hu14.18/GM-CSF induced the highest sustained azurophil granule exocytosis, almost exclusively in PMNs with activated Mac-1. Thus, hu14.18/GM-CSF facilitates PMN ADCC against neuroblastoma cells associated with FcgammaRII and Mac-1-dependent enhanced adhesion and degranulation.
Highlights
Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF)
10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside immunocytokine hu14.18/GMCSF, by monoclonal antibody hu14.18 mixed with GM-CSF, and by hu14.18 alone
PMN ADCC activity of hu14.18, hu14.18 mixed with GM-CSF, and hu14.18/GM-CSF was determined using LA-N-1 neuroblastoma cell targets at levels achieved in serum in phase 1 studies of the closely related human–mouse chimeric 14.18 antibody (Figure 1).[27,28,29,30]
Summary
Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). Polymorphonuclear leukocytes (PMNs) constitute the largest population of white blood cells, and they can mediate antibody-dependent cellular cytotoxicity (ADCC) against tumor cells.[5,6,7,8,9] In vitro studies demonstrated the strong facilitation of PMN ADCC against neuroblastoma cells by mixing granulocytemacrophage colony-stimulating factor (GM-CSF) with an antiGD2 antibody and by a human–mouse chimeric anti-GD2/GMCSF immunocytokine.[5,10] Fc receptors (FcR) involved in PMN ADCC may include Fc␥RII (CD32) and Fc␥RIII (CD16), which are constitutively expressed, and Fc␥RI (CD64), which is induced by interferon-␥ and G-CSF.[11,12] In addition, Fc␣RI of PMNs may be activated by constructing bispecific monoclonal antibodies (mAbs) that recognize a target cell molecule and Fc␣RI.[13] Binding of an antibody-cytokine fusion protein to FcR may be affected by the physical presence of the cytokine or by the cytokinemodulating FcR expression.[14,15] FcRs used by PMNs in ADCC with anti-GD2/GM-CSF immunocytokines have not yet been determined.
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