Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ET A and ET B

Wijkhuisen

et al. 2016

mAbs

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.

Section: Discussionsupporting

confidence: 88%

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

Borrull

Wijkhuisen

et al. 2016

mAbs

“…40,41 Collectively, these findings highlight the value of rendomab-B1 in investigating hETBR structure and in gathering more information about the location of the endothelin binding site, whose precise delineation is still unclear despite several recent publications tackling the issue. 42,43 Furthermore, this original noncompetitive inhibitory mechanism, rarely found for small inhibitory molecules like BQ788 that generally compete with natural ligands for the receptor orthosteric site, underlines the value of developing therapeutic antibodies that offer the possibility of targeting original epitopes on GPCRs, potentially linked to new pharmacological activities and better receptor selectivity. 22 To our knowledge, this is the first time that an antagonist mAb targeting hETBR has been described.…”

Section: Discussionmentioning

confidence: 99%

Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor

Wijkhuisen

Borrull

et al. 2013

mAbs

“…Since the major linear epitopes of our antibodies (at least those present in the two selected B3 sera) were delineated, we wondered whether these antibodies could be used to give any new insight into the localization of the ligand-binding site in hET B R, which is still a subject of great controversy (Lee et al, 1994;Wada et al, 1995;Boivin et al, 2004;Aubin et al, 2008;Lä ttig et al, 2009). For this purpose, we investigated whether endothelin-1 could compete with these antibodies for receptor binding.…”

Section: Et-1 Competes With Antibodies For Et B Receptor Bindingmentioning

confidence: 98%

Electroporation-Aided DNA Immunization Generates Polyclonal Antibodies Against the Native Conformation of Human Endothelin B Receptor

Priam

Deshayes

et al. 2011

DNA and Cell Biology

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.