IgG isotype, glycosylation, and EGFR expression determine the induction of antibody-dependent cellular cytotoxicity in vitro by cetuximab.

PURPOSE: To evaluate the antibody-dependent cellular cytotoxicity (ADCC) of cetuximab, an anti-epidermal growth factor receptor (EGFR) IgG1 antibody, in vitro. METHODS: Binding to human Fc receptors was measured by ELISA. ADCC against a panel of tumor cell lines was evaluated using peripheral blood mononuclear cells or NK cells as effectors and lactate dehydrogenase release as a marker of cell killing. Cetuximab was compared with two glycan variants of cetuximab and with panitumumab, an anti-EGFR IgG2. RESULTS: Cetuximab bound with high affinity to FcγRI (EC50 = 0.13 nM) and FcγRIIIa (EC50 = 6 nM) and effectively induced ADCC across multiple tumor cell lines. Panitumumab and aglycosylated cetuximab did not bind to FcγRI or FcγRIIIa nor have ADCC activity even at high effector-target cell ratios, even though the EGFR-binding affinity of cetuximab and panitumumab were shown to be comparable (KD = 87 pM and 83 pM, respectively). The extent of cetuximab-elicited ADCC was associated with the level of EGFR expression on tumor cells. CONCLUSIONS: Cetuximab elicits effective ADCC activity against a wide range of tumor cells in vitro. This activity is dependent on antibody glycosylation and IgG1 isotype as well as tumor-cell EGFR expression. These findings suggest that ADCC may contribute to the antitumor activity of cetuximab.

Generation and characterization of a therapeutic human antibody to melanoma antigen TYRP1.

TYRP1 (tyrosinase-related protein 1) is a melanoma antigen expressed in melanosomes and on the surface of melanoma cells. Previous studies have shown that mouse antibodies to TYRP1 localized to melanomas in vivo and inhibited tumor growth and metastasis. Here, we describe the characterization of a novel fully human anti-TYRP1 MAb (20D7) generated by immunizing HuMAb mice (Medarex). 20D7 recognized recombinant and native human TYRP1 by Western blotting and ELISA, and native TYRP1 in melanoma cells as determined by flow cytometry analysis. 20D7 cross-reacted with mouse TYRP1. The binding affinity to human TYRP1 for the human MAb was in the low nM range as determined by surface plasmon resonance kinetics. 20D7 can bind to human and mouse Fc receptor and induce a strong ADCC response against human melanoma cells in vitro. The antitumor activity of 20D7 was tested in human melanoma xenografts and mouse metastatic melanoma models in athymic nude mice. Growth of s.c. human melanoma tumors and metastatic nodules of murine B16 tumor were significantly suppressed by 20D7 compared to human IgG control. These results suggest that human anti-TYRP1 MAb may be a potent therapeutic for the treatment of malignant melanoma.