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

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.

High level prokaryotic expression of anti-Müllerian inhibiting substance type II receptor diabody, a new recombinant antibody for in vivo ovarian cancer imaging.

Prescription of therapeutic antibodies has radically modified the prognosis of some important diseases. However, the very high cost of these new drugs is a problem for public health organizations, which require assessment of the effectiveness of the antibody for each patient before beginning or during the treatment. In vivo immunoimaging is particularly well adapted to meet this demand. However, full-length antibodies are unsuitable for in vivo imaging due to their persistence in the serum and must be engineered in smaller formats to improve their pharmacokinetic properties without modifying their affinity and specificity. The small bivalent antibody fragment called diabody perfectly meets these in vivo imaging requirements. However, obtaining diabodies is laborious, time-consuming and sometimes unsuccessful. Using a diabody derived from a monoclonal antibody (12G4) directed against the human anti-Müllerian hormone receptor, a biomarker of ovarian cancers for which therapeutic antibodies are already undergoing clinical trials, we describe here a new diabody refolding protocol with various reducing conditions. Diabody functionality was checked in vitro and ex vivo with, respectively, a new immunoassay involving the epitopic peptide as a tracer and flow cytometry experiments with cells expressing recombinant anti-Müllerian hormone receptors. Our optimized protocol allows us to find the best refolding conditions for each diabody and to obtain large amounts of functional diabodies.

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

Endothelin B receptor (ETBR) is a G protein-coupled receptor able to bind equally to the three identified human endothelin peptides. It is expressed primarily on vascular endothelial cells and involved in various physiological processes including vascular tone homeostasis, enteric nervous system development, melanogenesis and angiogenesis. Furthermore, overactivation or overexpression of ETBR have been associated with the development of various diseases such as cardiovascular disorders and cancers. Therefore, ETBR appears to be relevant target for the therapy or diagnosis of highly prevalent human diseases. In this study, we report the in vitro characterization of rendomab-B1, a monoclonal antibody (mAb) obtained by genetic immunization, which selectively recognizes the native form of human ETBR (hETBR). Rendomab-B1 is the first-reported mAb that behaves as a potent antagonist of hETBR. It recognizes an original extracellular conformational epitope on the receptor, distinct from the endothelin-1 (ET-1) binding site. Rendomab-B1 not only blocks ET-1-induced calcium signaling pathway and triggers rapid receptor internalization on recombinant hETBR-expressing cells, but also exerts pharmacological activities on human vascular endothelial cells, reducing both cell viability and ET-1-induced hETBR synthesis. In addition, binding experiments using rendomab-B1 on different melanoma cell lines reveal the structural and functional heterogeneity of hETBR expressed at the surface of these cancer cells, strongly suggesting the existence of tumor-specific receptors. Collectively, our results underscore the value of rendomab-B1 for research, therapeutic and diagnostic applications dealing with hETBR.

Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor.

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.

Curative properties of antibodies against prion protein: a comparative in vitro study of monovalent fragments and divalent antibodies.

Prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans, are a group of devastating neurodegenerative disorders for which no therapy is yet available. However, passive immunotherapy appears to be a promising therapeutic approach, given that antibodies against the cellular prion protein (PrPc) have been shown in vitro to antagonize deposition of the disease-associated prion protein (PrPSc). Nevertheless, in vivo deleterious side effects of injected anti-PrP antibodies have been reported, mainly due to their Fc fragments and divalence. In this context, we examined here the ability of five Fabs (monovalent fragments devoid of the Fc part), prepared from antibodies already characterized in the laboratory, to inhibit prion replication in infected neuronal cells. We show that all Fabs (which all retain the same apparent affinity for PrPc as their whole antibody counterpart, as measured in EIA experiments) recognize quite well membrane bound-PrP in neuronal cells (as shown by flow cytometry analysis) and inhibit PrPSc formation in infected cells in a dose-dependent manner, most of them (four out of five) exhibiting a similar efficiency as whole antibodies. From a fundamental point of view, this report indicates that the in vitro curative effect of antibodies i) is epitope independent and only related to the efficiency of recognizing the native, membrane-inserted form of neuronal PrP and ii) probably occurs by directly or indirectly masking the PrPc epitopes involved in PrPSc interaction, rather than by cross-linking membrane bound PrPc. From a practical point of view, i.e. in the context of a possible immunotherapy of prion diseases, our data promote the use of monovalent antibodies (either Fabs or engineered recombinant fragments) for further in vivo studies.

Polyclonal anti-idiotypic antibodies which mimic an epitope of the human prion protein.

Immunization with anti-idiotypic (anti-Id) antibodies, used as surrogate antigens, has led to promising results, notably in active immunotherapy of cancers, essentially because it breaks immunological tolerance against self-tumor-associated antigens. The aim of the present study was to provide a proof-of-principle that this vaccination approach could be envisaged also in the field of prion diseases, caused by the accumulation of an aggregated pathological isoform of the highly tolerogenic self-prion protein (PrP), and for which no therapy is available. We investigated the possibility of raising anti-Id antibodies mimicking the human PrP (hPrP), using as immunogens either a peptide derived from the paratope of an anti-PrP mAb or the entire antibody. To this end, we cloned and sequenced SAF61 mAb, an anti-PrP antibody already produced in the laboratory, directed against a critical epitope of PrP involved in the aggregation process. A synthetic peptide (denoted CDR3L) was designed from the identification of a 17-amino-acid sequence encompassing the CDR3 region of the light chain whose hydropathic profile was opposed to that of PrP epitope. CDR3L peptide was directly demonstrated to bind hPrP, confirming the role of hydropathic complementarity in antigen-antibody interactions. When injected into rabbits, CDR3L generated anti-SAF61 anti-Id polyclonal antibodies that exclusively recognized SAF61 mAb but were unable to compete with hPrP for antibody binding. By contrast, immunizations with the entire SAF61 mAb generated anti-Id antibodies specifically competing with soluble or membrane-bound hPrP (in EIA or flow cytometry experiments, respectively) for binding not only SAF61 mAb but also other anti-PrP mAbs directed against similar epitopes, i.e. behaving as "internal images" of this disease-related PrP epitope. These results could open the way to raising PrP-like mAbs, which might serve as surrogate antigens in a new active immunotherapeutic approach to prion diseases.

Enhancement of antibody responses in DNA vaccination using a vector encoding a universal T-helper cell epitope.

DNA vaccination appears as a very promising approach to raise protective antibodies against a variety of proteins from pathogens or tumor cells, but is often hindered by the low immunogenicity of the genetic vectors used for the immunizations. To enhance the humoral response through improvement of the antigenic presentation of newly synthesized proteins upon vaccination, we engineered a plasmid coding for a low immunogenic protein (an scFv, i.e. the single-chain Fragment variable of a well-characterized antibody) fused to a small-size universal T-helper cell epitope derived from tetanus toxin, whose efficiency in classical protein-based immunization protocols has already been demonstrated. We found that immunization of C57Bl/6 mice using this vector greatly enhanced the production not only of specific antibodies recognizing essentially conformational epitopes on the undenatured scFv protein but also of antibodies against linear epitopes on the denatured protein. Since this T-epitope is known to be accommodated by several haplotypes of H-2 molecules in mice, as well as by various class II MHC molecules in humans, the results reported here allow us to conclude that this method could be of general interest for future applications of genetic immunization, including DNA-based vaccinations in humans.

Pharmacological properties of peptides derived from an antibody against the tachykinin NK1 receptor for the neuropeptide substance P.

Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.

Anti-allergen antibodies can be neutralized by antibodies obtained against a peptide complementary to the allergen: towards a new peptide therapy for allergy.

The concept of specific immune treatment against allergic diseases requires the development of antibodies capable of specifically neutralizing anti-allergen antibodies. The aim of the present study was to investigate whether a novel approach, consisting in raising anti-idiotypic blocking antibodies through peptide immunization, could be envisaged in the field of allergy. Using allergy to cow's milk as a model, we prepared polyclonal antibodies against a peptide that is complementary (i.e. hydropathically opposed) to a major epitope of bovine beta-lactoglobulin (BLG), one of the main allergens of bovine milk. Anti-complementary peptide antibodies were found to neutralize in vitro both well-characterized anti-BLG monoclonal antibodies from mice sensitized to BLG and anti-BLG IgE from two patients suffering from milk allergy. These results suggest a new strategy for the functional inhibition of specific disease-associated IgE that may be applicable to the specific treatment of various allergic disorders.