Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Abstract

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer. We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB. These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.

Antibodies Targeting Human Endothelin-1 Receptors Reveal Different Conformational States in Cancer Cells

“…Binding of rendomab B49 and rendomab A63 to their respective endothelin receptors are not suppressed in presence of ET-1 The binding curve of RB49, determined by flow cytometry, clearly shows that RB49 binding is not modified in the presence of excess ET-1 (100 nM) ( Fig. 1A), as previously observed for RB4 (Borrull et al 2016). The calculated K D values are similar: 1.6±0.01 nM for RB49 and 1.9±0.01 nM for RB49 with ET-1.…”

“…Chinese hamster ovary (CHO) cells expressing CHO-ET B , CHO-ET A and melanoma cells lines were cultured as previously described (Borrull et al 2016). Glioblastoma stem cells (GSC) were provided by Dr. J.P. Hugnot, INSERM U1051 (Guichet et al 2014.…”

“…Glioblastoma stem cells (GSC) were provided by Dr. J.P. Hugnot, INSERM U1051 (Guichet et al 2014. Flow cytometry experiments were performed using a previously described protocol (Borrull et al 2016). Briefly, to determine the affinity of RB49 and RA63, increasing concentrations of monoclonal antibody were incubated with CHO-ET B or CHO-ET A cells for 2 h at 4 °C.…”

“…The location of the two epitopic peptides of RB4 is indicated in red in Figure 2C (Borrull et al 2016). Following the same rule as for the peptide previously described, we named the peptides ERB4-R1 ( 28 ERGFPPDRATP 38 ) and ERB4-R2 ( 70 EVPKGDRT 77 ).…”

“…67 developed to target and inhibit the endothelin axis in different diseases, such as bosentan (Clozel et al 1993) for the treatment of pulmonary artery hypertension or atrasentan (Opgenorth et al 1996) in some cancers and diabetic nephropathy (Egido et al 2017). We have previously detailed the properties of rendomab B1 (RB1) and B4 (RB4), which are antibodies directed against human subtype B endothelin receptor (ET B ) obtained through genetic immunization (Allard et al 2013, Borrull et al 2016. Surprisingly, RB1 was unable to bind ET B at the surface of melanoma cell lines, in contrast to RB4.…”