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

Abstract

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.

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

“…Hybridoma supernatants were first screened using the living cell-based ELISA that we described previously. 29 Then, the specificity of positive hybridoma was further confirmed by flow cytometric tests (data not shown) performed on three cell types (CHO cells transfected or not with hETBR, or transfected with an irrelevant GPCR, the NK1 human receptor for the neuropeptide substance P). These differential screenings, followed by the limiting dilution step, led to the selection of 27 pure clones (out of 169 initially selected by the ELISA test).…”

“…As we demonstrated in a previous paper, 29 DNA immunization is a powerful approach to generate specific polyclonal antibodies against hETBR, and more generally against GPCRs. Therefore, the same method of immunization was used in the present study to generate antihETBR mAbs (Fig.…”

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

“…MAb production, by DNA immunizations, was performed as previously described; 25,61 collected splenocytes of the 2 bestresponding mice were fused to NS1 mouse myeloma cells. Hybridoma supernatants were screened for production of anti-ETB specific antibodies by a living cell-based ELISA test, using untransfected CHO cells and CHO-ETB cells as targets, prior to confirmation of specificity and reactivity of antibodies by flow cytometry.…”

“…Rendomab-B4 epitope mapping was performed according to a protocol previously described, 61 incubating the membrane in 1 mg/mL of rendomab-B4 for 90 min at room temperature. The hybridization was revealed using an anti-mouse IgG labeled with alkaline phosphatase (Sigma-Aldrich).…”

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VHCDR3 targeting the Class A GPCR formyl-peptide receptor 1

“…11 Other successful examples of the use of peptides as antigens include the isolation of single chain Fv fragment (scFv) antibodies that are able to recognize the native cholecystokinin-B receptor expressed on cells by panning of a phage display antibody library using a synthetic peptide corresponding to the second extracellular loop of the receptor, 12 and isolation of a competitive antagonistic antibody for the class B GPCR glucosedependent insulinotropic polypeptide receptor using phage display and ribosome display selections against a peptide comprising the receptor N-terminus. 13 In cases where the use of extracellular domains or peptides as surrogate GPCR immunogens is not feasible, approaches that avoid the need for a purified GPCR have been used, such as the immunization of animals with DNA encoding the GPCR of interest 14 or the use of GPCR overexpressing cell lines for the immunization of animals. The latter is a widely used solution to identify antibodies targeting GPCRs, and by this route neutralizing antibodies to the CXCR4 receptor, the rat sphingosine 1-phosphate receptor and the CCR5 receptor among others have been derived.…”