Pegylated Granulocyte Colony Stimulating Factor (Peg-G-CSF) Enhances Mapatumumab-Mediated Antibody Dependent Cellular Cytotoxicity (ADCC) Against Non-Hodgkin's Lymphoma (NHL) Cell Lines Independent of NADPH Oxidase-Derived Reactive Oxidant Intermediates (ROIs).

Abstract

ADCC is an important mechanism of action of rituximab and other monoclonal antibodies (mAb). In an attempt to improve the efficacy of rituximab while minimizing its toxicity, our group have explored the use of cytokines that target neutrophil maturation and activation in combination with rituximab in pre-clinical and clinical models. In response to a growing need to develop alternative biological therapies, a new generation of humanized antibodies are under development. Mapatumumab is a humanized mAb targeting the death receptor-4 (DR4), active against NHL. A variable degree of ADCC has been observed in mapatumumab-treated NHL cells, raising the possibility that its biological activity may be augmented by cytokines. To this end, we studied the effects of cytokine-priming of neutrophils in the activity of mapatumumab and the role of free radical species in mAb-mediated ADCC. We used Raji cells, a CD20 and DR4 expressing Burkitt's lymphoma cell line, as target cells in a standardized 51Cr release assay. Effector cells consisted of peripheral blood mononuclear cells (PBMC) and neutrophils collected from healthy volunteers or lymphoma patients who had received peg-G-CSF for the prevention of chemotherapy-induced neutropenia. In addition, thiolglycollate-elicted peritoneal neutrophils and a bone marrow-derived macrophage cell line from C57BL/6 and genetically engineered chronic granulomatous disease (CGD) mice were used to evaluate the role of NADPH oxidase-derived ROIs in mAb-mediated ADCC. For ex vivo priming, healthy volunteer PBMC/neutrophils were exposed to peg-G-CSF (1mcg/ml) or placebo for 24hrs at 37oC,5%CO2. Subsequently, effector cells were co-cultured with 51Cr-labeled Raji cells in the presence of mapatumumab or isotype control at an effector:target ratio of 40:1. Following a period of 6 hrs of incubation, the supernatant was collected and the percentage of lysis was calculated. For in vivo cytokine priming, PBMC/neutrophils were collected from lymphoma patients who had received peg-G-CSF (6mg sq) at the time of white cell count recovery and used as effector cells in ADCC assays as described above. Finally, to establish the role of ROIs in mapatumumab-associated ADCC, we conducted standard 51Cr release assays with mapatumumab plus C57BL/6 or CGD murine neutrophils or macrophages as effector cells. Ex vivo cytokine-primed killing (34.54% +/− 1.69) and in vivo cytokine-primed killing (26.17% +/−0.28) resulted in an improvement in mapatumumab-associated ADCC as compared to controls (10.26% +/−0.79; P = 0.002, and P = 0.003, respectively). In murine studies, mapatumumab resulted in significant neutrophil and macrophage-mediated ADCC against Raji cells (20% and 15%, respectively). No differences were observed in mapatumumab anti-tumor activity between C57BL/6 versus CGD effector cells suggesting that NADPH oxidase-derived ROIs do not influence mapatumumab-induced ADCC. In summary, our data strongly suggest that peg-G-CSF can potentiate the anti-tumor activity of mapatumumab and support additional translational and clinical research of the combination of cytokine-primed effector cells plus mAbs targeting the death receptor pathway.