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

Abstract

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ETA and ETB) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ETB receptors (hETB) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ETB receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ETA receptor (hETA). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hETB receptor only some of new antibodies directed against ETA receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hETA is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

Integrative Analysis of Proteomics and Transcriptomics Reveals Endothelin Receptor B as Novel Single Target and Identifies New Combinatorial Targets for Multiple Myeloma

“…28 Rendomab B49 is a murine IgG1kappa mAb that recognizes such an epitope near the N-terminal of the receptor. 25 We obtained the RB49 antibody conjugated to Alexa Fluor 488 and compared its binding to primary myeloma cells with the binding of a control IgG1k antibody. In 8 of the 10 (80%) tested primary MM samples, we could confirm the binding of RB49 to primary malignant plasma cells (results from one of these patients is illustrated in Figure 6E).…”

“…ET B is a G‐protein‐coupled receptor that changes its confirmation upon activation with new epitopes becoming exposed 28 . Rendomab B49 is a murine IgG1kappa mAb that recognizes such an epitope near the N‐terminal of the receptor 25 . We obtained the RB49 antibody conjugated to Alexa Fluor 488 and compared its binding to primary myeloma cells with the binding of a control IgG1k antibody.…”

“…The anti-ET B monoclonal antibody (mAb) Rendomab B49 was produced after DNA-immunization of C57BL/6 mice and its affinities and binding epitopes determined. 24,25 For our flow cytometry studies, Rendomab49 (RB49) was directly conjugated to Alexa Fluor 488. Flow cytometry analyses were performed on a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ) and data were analyzed using BD FACSDiva Software V10 (BD Biosciences) or Kaluza V2.1 (Beckman Coulter, Brea, CA).…”

Synthesis and Preclinical Fluorescence Imaging of Dually Functionalized Antibody Conjugates Targeting Endothelin Receptor-Positive Tumors

“…Flow Cytometry. We used the protocol of Herbet et al 30 Briefly, three cell lines were used to assess the apparent affinity of xiRA63 or xiRB49: Chinese hamster ovary wild-type (CHO-WT) cells and CHO cells were stably transfected with either human ET A (CHO-ET A ) or human ET B (CHO-ET B ). Apparent affinity was determined with a dilution range from 0.001 to 50 nM for xiRA63 and from 0.01 nM to 125 nM for xiRB49.…”

“…To circumvent this issue, two high-affinity antibodies�which do not compete with ET-1�named Rendomab A63 (RA63) and Rendomab B49 (RB49), directed, respectively, against ET A and ET B receptors have been patented. 8,9 We have recently demonstrated that a chimeric version of RA63 called xiRA63 could be randomly labeled with zirconium-89 ( 89 Zr, t 1/2 = 3. ing. 10 Likewise, a chimeric version of the latter, xiRB49, was modified with a cytotoxic molecule to obtain an antibody− drug conjugate showing a very high efficacy in treating ET B + melanoma with no notable side effects on mice (Herbet et al, in preparation).…”