Antibody-dependent cellular cytotoxicity mediated by Cetuximab against colon cancer cell lines is inhibited by membrane expression of HLA-E on target cells

3996 Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in Western nations. Cytotoxic agents have increased the median survival of patients, and promising studies in CRC therapy have emerged using new agents such as Cetuximab, a chimeric IgG 1 monoclonal antibody (mAb) that binds to the extracellular domain of EGFR. Direct growth inhibition, apoptosis induction, complement-dependent cytotoxicity (CDC) and Ab-dependent cellular cytotoxicity (ADCC) are the modes of action of Ab therapies. In a previous work, we demonstrated that the expression of HLA-E, a non-polymorphic MHC class Ib molecule, is significantly increased in primary human CRC, perhaps contributing to tumor escape from immune surveillance. Accordingly, we found that CRC patients with a high percentage of tumor HLA-E positive cells had a shorter disease-free survival. To establish if HLA-E could be a factor that renders CRC cells less susceptible to Cetuximab-mediated ADCC, in the present study we performed a detailed analysis of ADCC activity against five CRC cell lines, expressing or not HLA-E at the cell surface. We first observed that CRC cells treated with Cetuximab (1 μ g/ml) were killed more efficiently by ADCC, as measured by calcein-AM release from target cells. Interestingly, recombinant human- β 2 microglobulin treatment of target cells inhibits Ab-mediated ADCC activity, probably through HLA-E membrane stabilization; this result confirms that HLA-E at the cell surface can reliably suppress the ADCC effect of the mAb. Furthermore, Cetuximab induced a direct growth inhibition (measured by MTT assay) but only at high antibody concentrations (at 100 μ g/ml inhibits proliferation 93.7 %); in contrast, CDC effect was quite moderate (observed only in one cell line at 100 μ g/ml), and we failed to observe a pro-apoptotic effect, as measured by the annexin-V assay. Taking into account that our findings suggest that ADCC activity is the main anti-tumour effect observed at clinically achievable concentrations of Cetuximab (1 μ g/ml) at the tumor site, we believe that the characterization of new biologic markers of potential responsiveness to ADCC-inducing agents could be relevant. Considering the immunomodulatory function assigned to HLA-E molecule, its expression in malignant cells may represent one of various mechanisms used by the tumor cells to escape immune surveillance and constitute a reliable determinant of ADCC inhibition. We believe that our data justify further studies using samples from Cetuximab-treated patients to establish if high HLA-E levels could be a bad prognosis marker for antibody therapy in primary CRC.