Abstract 2527: Antibody-dependent cellular cytotoxicity of cetuximab against colorectal cancer cells with wild-type and mutant KRAS

Abstract

Abstract Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). In addition to direct EGFR inhibition, antibody-dependent cellular cytotoxicity (ADCC) is considered to be an important mechanism of action of cetuximab. Recently, KRAS mutations in tumors have been shown to be a negative predictive factor for response to cetuximab treatment for colorectal cancer (CRC). KRAS activating mutations constitutively transduce RAS/RAF/MEK/ERK pathway signaling, which theoretically cannot be suppressed by inhibition of the upstream receptor through the use of cetuximab. Thus, KRAS mutations potentially contribute to the resistance to the direct inhibitory effect of cetuximab. However, the influence of KRAS mutations on cetuximab induced ADCC is not fully understood. In this study, we investigated cetuximab-mediated ADCC against human CRC lines, HCT116 and DLD-1, which carry mutated KRAS, and their derivative cell lines, HKh2 and DKO-4 respectively, in which the mutated KRAS allele was deleted by targeted disruption (KRAS wild-type). Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer cell line, exogenously expressing Fcα3A (CD16a) were used as effector cells. We first determined the growth-inhibitory effect of cetuximab alone using a WST-8 assay. Growth inhibition was observed only in KRAS wild-type cell lines, but its effect was only slight. In a standard short-term (4hr) ADCC assay that mainly evaluated lytic activity via perforin/granzyme, ADCC activities were exhibited to a similar extent against CRC cell lines regardless of the KRAS mutation status. We also examined a long-term (24hr) ADCC assay under the perforin-inhibited condition to evaluate the death receptor ligand-induced apoptosis, which is another major mechanism of ADCC. In this assay, cetuximab-mediated ADCC was induced in wild-type but not mutant KRAS cell lines. Similar to a previous report, Hkh2 and DKO-4 displayed a higher sensitivity to recombinant human Fas ligand (FasL) and TRAIL than did HCT116 and DLD-1, respectively. Furthermore, blocking experiments with neutralizing antibodies revealed that induction of apoptosis via Fas-FasL interaction is involved in perforin-independent cetuximab-mediated ADCC against KRAS wild-type cells. In conclusion, these results suggest that mutated KRAS may contribute to the resistance to the antitumor effects of cetuximab not only through direct receptor inhibition but also through ADCC mediated by Fas-FasL interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2527. doi:1538-7445.AM2012-2527