Anti-Human EDNRB Recombinant Antibody (Rendomab-BI) (CAT#: TAB-013WM)

Figure 1 Rendomab-B1 recognizes native hETBR in recombinant cell lines with a subnanomolar affinity.

(A) Cells were incubated with 50 nM rendomab-B1 and signals were revealed with R-PE -labeled goat anti-mouse polyclonal antibodies (green histograms, lower panel). Pink histograms stand for hETBR siRNA transfected cells. To check hETBR expression at cell surfaces, cells were exposed to ET-1-FAM (green histograms, upper panel). Shaded violet histograms correspond to cells incubated with buffer alone (upper panel) or with 50 nM of an isotype control antibody (lower panel). (B and C) To evaluate rendomab-B1 affinity, increasing concentrations of rendomab-B1 were incubated with CHO-hETBR (B), HEK-hETBR (C) or WT HEK cells (C, inset), for 24 h at 4°C. Rendomab-B1 binding was measured by flow cytometry, and resulting binding curves, corresponding to mean fluorescence intensity (MFI) as a function of antibody concentrations, were plotted and fitted using GraphPad Prism software.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.

Figure 2 Rendomab-B1 inhibits ET-1 binding to hETBR and conversely.

(A) CHOhET BR cells were incubated simultaneously with 10 nM ET-1-FAM and varying concentrations of rendomab-B1, BQ-788 or isotype control antibody. ET-1-FAM binding was measured using flow cytometry. (B) CHO-hETBR cells were incubated simultaneously with 3 nM rendomab-B1 and varying concentrations of ET-1, ET-2, ET -3, sarafotoxin 6c (S6c) or 21-amino-acid control peptide. Rendomab-B1 binding was quantified by flow cytometry. The inhibition binding curves(A and B) are plots of MFI values vs. inhibitor concentrations. Curves were fitted using GraphPad Prism with the adequate dose-response equation and IC50 ± s.d. of three independent tests are presented. (C) Confocal experiments: viable CHO-hETBR cells were stained with 50 nM rendomab-B1 (left picture) or with a combination of 50 nM rendomabB1 plus 1 μM ET-1 (right picture). Nuclei were counterstained with DRAQ5 (blue). White bars = 10 μm.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.

Figure 3 Rendomab-B1 behaves like a non-competitive inhibitor of ET-1 toward hETBR.

(A and B) Both ET -1 and rendomab-B1 concentrations were varied and added simultaneously to CHO-hETBR cells. After 24 h incubation at 4°C, rendomab-B1 binding (using FITC-labeled antibody) in the presence of increasing concentrations of unlabeled ET-1, or ET-1 binding (using FAM-labeled peptide) in the presence of unlabeled rendomab-B1, were measured by flow cytometry. Resulting binding curves are presented in(A) and (B), respectively.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.

Figure 4 Rendomab-B1 blocks ET-1-induced intracellular calcium mobilization by competing with ET -1 and triggering rapid internalization of h-ET BR.

(A) CHO-hETBR cells were loaded with a calcium-sensitive dye for 1 h. One hour before dye loading, cells were treated with various concentrations of rendomab-B1, BQ788 or isotype control antibody. At the end of the dye incubation period, cells were stimulated with 2 nM ET -1 and intracellular calcium fluxes were monitored with a FLEXStation 2 multi-mode reading system. The picture illustrates ET-1-induced calcium flux variations obtained in cells treated with increasing concentrations of rendomab-B1, from 2.5 nM to 500 nM. Calcium flux variations obtained in cells treated with BQ-788 or isotype control antibody are not illustrated here. (B) Data obtained from the FLEXStation(Area under curve) were plotted vs. inhibitor concentrations and fitted with an adequate dose-response equation (GraphPad Prism). IC50 ± s.d. stand for three independent assays. (C) Confocal experiments: viable CHO-hETBR cells were stained with 100 nM of fluorescently labeled rendomab-B1 (green signal). Red fluorescence corresponds to early endosomal antigen 1 (EE A1) staining. Merged images are presented. Nuclear staining with DRAQ5 is not displayed. (D) CHO-hETBR cells were treated as in A except that the preincubation period of inhibitors was varied and only one inhibitor concentration was used (200 nM). Error bars stand for mean ± s.d. of three independent experiments. (E) Flow cytometry experiments: internalization of hETBR induced by rendomab-B1. CHO-hETBR cells were pre-incubated for 2 h at 37°C in the presence of rendomab-B1, or isotype control antibody, or ET-1(All compounds at 100 nM), or kept in the buffer alone. After rinsing, CHO-hETBR cells were incubated for 30 min at 4°C with 10 nM ET-1-FAM before fluorescence measurement. Pre-incubations in the presence of rendomab-B1 (orange histogram), or ET-1 (blue histogram), or control antibody (pink histogram), or in the buffer alone (green histogram). Basal fluorescence (in the absence of ET-1-FAM): black histogram.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.

Figure 5 Rendomab-B1 recognizes a discontinuous epitope on hETBR, involving N-terminal tail and E2 loop regions.

(A) Rendomab-B1 epitope was investigated using a Pepscan membrane spotted with dodecapeptides (frameshifted by one amino acid) covering the entire hETBR extracellular regions. Interacting peptides were revealed using an alkaline phosphatase conjugated secondary antibody. (B) Schematic localization of the regions recognized by rendomab-B1 on hETBR. (C) Spot signals obtained on the membrane were quantified by densitometry. Isolated (no more than two consecutive) spots, as well spot series not presenting any signal peak higher than three times the background, are not represented and were considered as irrelevant. (D) Table mentioning the sequence of most intensely stained spots on the membrane. Underlined motifs correspond to putative key residues for rendomab-B1 binding. This experiment was done twice with two independent membranes. An isotype control antibody did not generate any staining.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.

Figure 6 Rendomab-B1 triggers hETBR internalization in HUVECs, impairs cell viability and leads to hETBR mRNA downmodulation.

(A) Specific rendomab-B1 binding to HUVECs as revealed by flow cytometry. HUVECs were incubated with 100 nM rendomab-B1 (in green histogram) or isotype control antibody (violet histogram). Staining specificity was checked by pretreating cells for 1 h at 37°C with 500 nM sarafotoxin 6c (S6c). (B) Internalization of hETBR induced by rendomab-B1. HUVECs were pre-incubated for 2 h at 37°C in the presence of rendomab-B1, or isotype control antibody, or ET-1(All compounds at 100 nM), or kept in the buffer alone. After rinsing, HUVECs were incubated for 30 min at 4°C with 10 nM ET-1-FAM before flow cytometry analysis. Pre-incubations in the presence of rendomab-B1 (orange histogram), or ET-1 (blue histogram), or control antibody (pink histogram), or in buffer alone (green histogram). Basal fluorescence (in the absence of ET-1-FAM): black histogram. (C) HUVECs were grown for several days in a reduced-serum medium containing rendomab-B1, BQ788, anti-ET -1 antibody or isotype control antibody, all compounds at the same concentration (500 nM). Cell viability was then assessed using the MTT tetrazolium salt. Data are presented as percentage viability compared with untreated control cells. (D) HUVECs were treated for 24 h with rendomab-B1, BQ788 or isotype control antibody(All at 500 nM) in combination with 10 nM ET-1. Thereafter, hETBR mRNA was amplified by RT-PCR.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.

Figure 7 Rendomab-B1 poorly recognizes hETBR expressed by human melanoma cell lines.

(A and B) hETBR transfected cells and human melanoma cells (UACC257, SLM8, WM266-4) were incubated with varying concentrations of ET-1 FAM for 24 h and analyzed by flow cytometry. (C and D) Cells were stained with varying concentrations of anti-hETBR polyclonal sera before flow cytometry analysis. (E and F) Cells were stained with varying dilutions of rendomab-B1 and resulting fluorescence was measured by flow cytometry.

Allard, B., Wijkhuisen, A., Borrull, A., Deshayes, F., Priam, F., Lamourette, P.,... & Couraud, J. Y. (2013, January). Generation and characterization of rendomab-B1, a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. In MAbs (Vol. 5, No. 1, pp. 56-69). Taylor & Francis.