WO2024209089A1

WO2024209089A1 - Use of antibody against the endothelin receptor b for diagnostic and therapeutic applications

Info

Publication number: WO2024209089A1
Application number: PCT/EP2024/059388
Authority: WO
Inventors: Didier Boquet; Amaury HERBET; Narciso COSTA; Frédéric Ducancel; Jean-Yves Couraud
Original assignee: Commissariat A L'energie Atomique Et Aux Energies Alternatives; Université Paris Cité
Priority date: 2023-04-07
Filing date: 2024-04-05
Publication date: 2024-10-10

Abstract

The present invention relates to novel antibodies, in particular murine monoclonal antibodies, chimeric and humanized, that are able to recognize specifically the human Endothelin B receptors expressed on melanoma cells, as well as the amino and nucleic acid sequences coding for such antibodies. The invention also comprises the use of such antibodies or of fragments thereof as diagnostic agents or as medicaments for treating patients suffering from melanoma and other cancers in which the human endothelin B receptor is overexpressed, such as glioblastoma, bladder cancer, lung cancer, kidney cancer, or vulvar cancer.

Description

USE OF ANTIBODY AGAINST THE ENDOTHELIN RECEPTOR B

FOR DIAGNOSTIC AND THERAPEUTIC APPLICATIONS

SUMMARY OF THE INVENTION

BACKGROUND OF THE INVENTION

Endothelins (ETs) constitute a family of 3 21 -amino acid peptides, ET-1 , ET-2 and ET-3, which bind to two distinct 7-transmembrane domain receptors: ETA (standing for “Endothelin subtype A receptor”) and ETB (standing for “Endothelin subtype B receptor”), these two receptors belonging to the G protein-coupled receptor (GPCR) family. The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in the regulation of physiological smooth muscle motility, but ET-1 is also implicated in a large variety of pathologies, including hypertension, heart failure, kidney disorders and infectious diseases. In addition, the ET axis is overexpressed in cancer of different organs contributing to tumor growth by acting on cell proliferation, survival, migration, differentiation, angiogenesis and inflammatory cell recruitment. ETA are upregulated in prostate, ovary and breast cancers while ETB is deregulated in melanoma, lung, renal and vulvar cancers (Rosano et al, 2013).

Melanoma is an aggressive cancerthat presents an increased incidence rate. This cancer is characterized by its capacity to metastasize promptly, leading to an increase in mortality rates in many countries. Somatic mutations have been found in BRAF and N-RAS genes in about 50% and 20% of melanomas, respectively, resulting in constitutive activation of ERK1/2 MARK pathway. Moreover, gene expression profiling and targeted approaches have demonstrated that ETB expression is upregulated in melanoma. The upregulation of ETB is involved in proliferation, migration and angiogenesis associated with tumor growth and invasiveness. In melanoma, ET-1 via ETB expressed on cancer cells modulates migration and formation of vasculogenic mimicry via the upregulation of HIF/ VEGF/VEGFR pathway. These data implicate ETB as a potential driver of melanoma progression and an important marker of aggressive phenotype. Hence new therapeutic molecules targeting ETB need to be developed in order to inhibit the ET pleiotropic effects on melanoma cells.

An ETs-specific peptidic antagonist (BQ788) has been used in basic research to reduce the proliferation of cancer cells. Preclinical trial confirmed the efficacy of BQ788 on melanoma growth (Lahav R. 2005) but with poor clinical interest (Wouters J. et al, 2015).

The dual receptor antagonist bosentan was also successfully used in melanoma cell lines (Berger Y et al., 2006), yet the two Phase 2 studies in patients with stage IV metastatic melanoma were disappointing (Maguire JJ et al, 2015; Kefford RF et al, 2010). Used as a monotherapy, bosentan stabilized less than 20% of the patients, and no additional effect on temporal progression of tumors was observed when bosentan was combined with a chemotherapeutic agent (dacarbazine). Macitentan was recently tested for the treatment of recurrent glioblastoma, but the results show little effect on the tumoral progression (Weathers SP et al, 2021).

In this context, the development of other therapeutic molecules targeting ETB is needed to block the upregulated signaling pathways that occur in melanoma upon ET induction. The development of monoclonal antibodies (mAbs) for the treatment of cancers is an alternative to conventional therapeutic agents and is currently a rapidly expanding sector. In the particular case of metastatic melanoma treatment, the anti-CTLA-4 blocking antibody ipilimumab and the anti-PD-1 antibodies pembrolizumab and nivolumab that modulate the immune checkpoints have been recently approved (Russo A. et al, 2014; Turneh PC et al, 2014; Topalian SL et al, 2014). However, despite the improvement of overall survival observed with these mAbs, they do not lead to remission of the disease.

Compared to small pharmacological molecules, mAbs can detect fine antigenic differences between normal and pathologic cells, inhibiting different functions involved in cell growth, migration, angiogenesis or metastasis. Moreover, mAbs display various cytotoxic actions through the immune system, and they can be coupled to several imaging tracers and markers or cytotoxic molecules. For example, the mAb Trastuzumab, which is directed against the human epidermal growth factor receptor HER-2 often overexpressed in breast cancer, has been shown to significantly improve the overall survival of HER2-positive cancer patients (Recondo G. et al, 2014).

Like HER-2 in breast cancer, the ETB receptor is overexpressed at the surface of melanoma cells and it is therefore tempting to try to antagonize this receptor with mAbs. Yet the ETB receptor has a complex three-dimensional structure, and it is difficult to obtain a native and functional form of the polypeptide, outside its membrane context. Moreover, ETB as ETA are allosteric receptors, i.e. capable to have conformational modifications after the ligand binding (Shihoya et al. 2016). Several mAbs have been nevertheless proposed to target ETB:

Kondoh et al, 1990 ("Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization", BBRC, vol. 172, pages 503-510) describe the binding properties of 4 monoclonal antibodies (A2, G9, E7 and G10) to solubilized complexes of endothelin receptors. Yet, these authors do not provide any information about the fine specificity of these antibodies (against the receptors ETA and/or ETB), as regards the recognition of human origin receptors, nor as regards a possible antagonistic property. Yamaguchi et al, 2004 ("Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells", Biotechnology Letters, vol. 26, pages 293-299) showed the binding properties of 5 mouse monoclonal antibodies obtained after protein immunization with recombinant human ETB produced in insect cells. Four of them had a similar affinity (in the nanomolar range) for ETB, whereas the fifth one was 10 times less affine. The epitope analysis of 3 of them (N-6, N-3 and N-1) revealed that they recognized the ETB N- terminal domain and more particularly the sequence corresponding to amino acids 27-35 of the ETB (for N-6); the sequence corresponding to amino acids 27-41 of the ETB (for N-3) and the sequence corresponding to amino acids 71-85 (for N-1). Finally, these three antibodies were capable of recognizing COS cells over-expressing human ETB.

Patent application JP 2012111706 related to a monoclonal antibody called hB07. This was an lgG2a/lambda isotype mouse immunoglobulin, which was specific to human ETB. hB07 was capable of competitively blocking endothelin 1 (ET-1) binding to ETB with an efficiency (IC50) calculated of 1 .7 10^-7M.

Another monoclonal antibody antagonist to human ETB, called Rendomab-B1 , has been described in (Allard B. et al, 2013). Rendomab-B1 was the first-reported mAb behaving as a potent antagonist of human ETB, blocking ET-1-induced signaling in CHO cells overexpressing ETB. Nevertheless, rendomab B1 displayed a very low binding for ETB overexpressed at the surface of some human melanoma cells (for example: A375; WM-266-4), suggesting either a structural heterogeneity among ETB receptors expressed by different cell types, or a high level of ETB receptors complexed with endothelin ligand preventing the rendomab B1 binding (Figure 4D).

After that, a therapeutic antibody called 5E9 has been obtained, targeting the human ETB over-expressed at the surface of melanomas (WO 2013/063001). This antibody 5E9 however cross-reacted with the rodent ETB as well as with the non-human primate receptor (Asundi J. et al, 2011). Moreover, it had no inhibitory effect by itself. It was nevertheless conjugated with monomethyl auristatin E, and showed good efficacy against human melanoma cell lines and xenograft tumor models. Finally, three antibodies specific of the subtype B of the human endothelin receptor (“rendomab-B49”, “rendomab-B41 ”, and “Rendomab-B36”) were generated (WO2017/220739). These antibodies recognized the human ETB receptor expressed by glioblastoma cells with a strong affinity, but they were not able to antagonize efficiently this receptor; i.e., they were not capable of inhibiting or blocking the binding of the endothelin ligands (ET1 , ET2 or ET3) on same.

Altogether, it appears from the analysis of all these prior art documents that they propose no monoclonal antibody recognizing i) the epitopes of SEQ ID NO:9 and SEQ ID NO:10 on human ETB receptor specifically, ii) when said receptors are expressed at the surface of human melanoma cells, iii) with very high affinity and iv) capable to inhibit the PLC signaling pathway and melanoma migration.

DETAILED DESCRIPTION OF THE INVENTION

Antibodies of the invention

The present inventors herein report the identification of a novel anti-human ETB mAb (clone Rendomab-B4, hereafter referred to as the “antibody of the invention”), that has been selected on its capacity to recognize specifically the human ETB receptor (herein called ETB) at the surface of melanoma cells. The Complementary Determining Regions (CDRs) of this mAb were obtained by cloning and sequencing. These CDRs can be used to generate a mouse-human chimeric antibody, a humanized or a fully human antibody that will be able to treat human patients suffering from a melanoma cancer.

To obtain the antibody of the invention, the inventors have used a particular selection immunisation strategy, coupled with a hybridoma screening procedure in ELISA-cell and then by flow cytometry that favours the obtention of monoclonal antibodies specific to ETB in its native conformation (Allard B. et al, 2013). By using this genetic immunization, the present inventors identified a particularly interesting monoclonal antibody that : is able to recognize UACC-257 melanoma ETB with very high affinity subnanomolar (0.15 nM (figures 1 and 2), and to inhibit the phospholipase C activation, cf. figure 8A) from this receptor, does not recognize the other sub-type of endothelin receptor ETA (not shown), does not inhibit ERK1/2 pathway nor the p-arrestin-dependent signaling in melanoma cells, recognizes the epitopes of SEQ ID NO:9 and SEQ ID NO:10 on ETB R, promotes the ETB internalisation in melanoma cells (figure 7), completely inhibit the migration of melanoma cells induced by ET-1 (figure 9).

Importantly, the antibody of the invention is not capable of recognizing or inhibiting the other endothelin sub-type receptor ETA, nor the ETB receptor from murine species. Its effect is therefore opposite to the other ETB antibodies of the prior art.

The antibody of the invention is the first reported mAb to possess the ability to differentially affect ETs-coupled signaling pathways, behaving as a biased allosteric modulator inhibiting G protein-dependent processes (e.g., PLC activation) but not ERK1/2 and p-arrestin-dependent ones.

Importantly, the inventors herein showed (figure 9) that the antibody of the invention is able to strongly inhibit the migration of melanoma cells induced by ET-1 . It may therefore represent a valuable starting point for the development of new tools for treatment of melanoma.

Moreover, the inventors herein showed (figure 7) that the antibody of the invention is efficiently internalized in endosomes and lysosomes together with ETB. AS ETB is highly expressed in melanoma cells, the antibody of the invention could therefore be used forthe specific delivery of cytotoxic molecules into melanoma cells, increasing antibody-mediated cell killing.

Thus, the antibody of the invention has the unique properties to i) inhibit the ETB dependent G-protein signaling and migration of human melanoma cells, and ii) to be able to internalize cytotoxic drugs in human melanoma cells, therefore driving a high anti-tumoral efficacy. It is the first time that an antibody having such a high affinity for human ETB is shown to display these two important properties.

These particular properties make the antibody of the invention, namely rendomab-B4, a preferred tool for the development of immunological tools in the field of melanoma therapeutics.

The present invention therefore targets an antibody recognizing specifically the human endothelin receptor sub-type B (ETB) or antigen-binding fragments thereof, that can bind to the epitopes of SEQ ID NO:9 and SEQ ID NO:10 on human ETB expressed on melanoma cells, that can inhibit in said cells G-protein signalling dependent from this receptor, that can be internalized in said cells in the endo-lysosomal pathway, yet without competing with its natural ligand ET1 and/or ET3. This antibody or an antigen-binding fragment thereof can advantageously be used as a therapeutic anti-melanoma drug.

As used herein, the term “ETB” designates the endothelin receptor of sub-type B, that is also abbreviated as ET-BR or ET-B, or ETRB, or HSCR2. It is encoded by the EDNRB gene (also called HSCR2 gene). The protein P24530 (SEQ ID NO:11) is a G protein-coupled receptor which activates a phosphatidylinositol-calcium second messenger system. Its ligand, endothelin, consists of a family of three potent vasoactive peptides: ET1 , ET2, and ET3.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies, chimeric antibodies, and antigen-binding fragments. An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or an antigen-encoding nucleic acid.

A typical antibody is comprised of two identical light chains and two identical heavy chains that are joined by disulfide bonds. As used in the invention, the term “light chain” refers to mammalian immunoglobulin light chain, lambda (A) or kappa (K), having two successive domains: one constant domain and one variable domain. As used in the invention, the term “heavy chain” refers to chain of mammalian immunoglobulin denoted by: alpha (a), delta (6), epsilon (s), gamma (y), and mu (p). Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype. The variable region of each heavy chain is composed of a single Ig domain. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions" (“HVRs”), which are primarily responsible for binding an epitope of an antigen and are interspersed with regions that are more conserved, designated "Framework Regions" (FR). The CDRs thus direct the specificity of the binding of the antibody. They are usually referred to as CDR1 , CDR2, and CDR3, numbered sequentially from the N-terminus.

Each VH and VL is composed of three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid sequences to each domain is in accordance with well-known conventions (for example, the IMGT unique numbering convention as disclosed by Lefranc, M.- P.,et al., Dev. Comp. Immunol., 27, 55-77 (2003)). The functional ability of the antibody to bind a particular antigen depends on the variable regions of each light/heavy chain pair, and is largely determined by the CDRs. The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone (or hybridoma). By contrast, the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.

In one embodiment, the present application relates to polyclonal antibodies. A "polyclonal antibody" is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes producing non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

According to another embodiment, the antibody of the invention, or antigen-binding fragment thereof, is a monoclonal antibody, e.g., a murine monoclonal antibody, or an antigenbinding fragment thereof. As used herein, the term “monoclonal antibody” refers to an antibody arising from a nearly homogeneous antibody population. More particularly, the individual antibodies of a population are identical except for a few possible naturally-occurring mutations which can be found in minimal proportions. In other words, a monoclonal antibody consists of a homogeneous antibody population arising from the growth of a single cell clone (for example a hybridoma, a eukaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule coding for the homogeneous antibody, etc.) and is generally characterized by heavy chains of one and only one class and subclass, and light chains of only one type. Monoclonal antibodies are highly specific and are directed against a single antigen. An "antigen" is a predetermined molecule to which an antibody can selectively bind. The target antigen may be a polypeptide, a carbohydrate, a nucleic acid, a lipid, a hapten or any other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide.

In a preferred embodiment, the antibodies of the invention are monoclonal IgGs.

In a particular aspect, the antibody of the invention is a monoclonal lgG1 Kappa obtained from the hybridoma deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS CEDEX 15) on 14 March 2023 under the accession number CNCM I-5937.

In another particular aspect, the antibody and fragments of the invention comprise: a) a light chain comprising three CDRs of the sequences SEQ ID NO:1 , 2 or 3, or having a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequences SEQ ID NO:1 , 2 or 3 after optimal alignment and b) a heavy chain comprising three CDRs of the sequences SEQ ID NO: 4, 5 or 6, or having a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequences SEQ ID NO: 4, 5 or 6 after optimal alignment.

For ease of understanding, these CDR sequences of rendomab-B4 are listed in Table 1 :

Table 1 : amino acid sequences of SEQ ID NO:1-6

More precisely, the anti-ETs antibodies of the invention or antigen-binding fragments thereof comprise a light chain comprising the LCDR1 , LCDR2 and LCDR3 having respectively the amino acid sequences SEQ ID NO: 1 , 2 and 3; and a heavy chain comprising HCDR1 , HCDR2 and HCDR3 having respectively the amino acid sequences SEQ ID NO: 4, 5 and 6.

In one embodiment, the antibody of the invention, or antigen-binding fragment thereof, comprises: a) a light chain variable domain (VL) of sequence SEQ ID NO: 7, or an amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO: 7 after optimal alignment and b) a heavy chain variable domain (VH) of sequence SEQ ID NO: 8, or an amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO:8 after optimal alignment.

By “optimal alignment with a preferred sequence”, it is herein meant the two sequences have been aligned by means of a global alignment of the sequences in their entirety. This alignment is preferably performed by means of an algorithm that is well known by the skilled person, such as the one disclosed in Needleman and Wunsch (1970). Accordingly, sequence comparisons between two amino acid sequences or two nucleotide sequences can be performed for example by using any software known by the skilled person, such as the “needle” software using the “Gap open” parameter of 10, the “Gap extend” parameter of 0.5 and the “Blosum 62” matrix. Two sequences are “optimally aligned” when they are aligned so as to produce the maximum possible score for that pair of sequences, which might require the introduction of gaps in one or both of the sequences to achieve that maximum score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer- implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information website.

The invention also provides antibodies or fragments whose amino acid sequences are or contains sequences that are “similar” or “substantially similar” to SEQ ID NO:1 to SEQ ID NO:8. “Similarity” of two targeted amino acid sequences can be determined by calculating a similarity score for the two amino acid sequences. As used herein, the “similarity score” refers to the score generated for the two sequences using the BLOSUM62 amino acid substitution matrix, a gap existence penalty of 11 , and a gap extension penalty of 1 , when the two sequences are optimally aligned. Two amino acid sequences are substantially similar if their similarity score exceeds a certain threshold value. The threshold value can be any integer ranging from at least 1190 to the highest possible score for a particular reference sequence (e.g., SEQ ID NO:1-8). For example, the threshold similarity score can be 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, or higher. If in a particular embodiment of the invention, the threshold score is set at, for example, 1300, and the reference sequence is any of SEQ ID NO:1-8, then any amino acid sequence that can be optimally aligned with any of SEQ ID NO:1-8 to generate a similarity score of greaterthan 1300 is be held as “similar” to SEQ ID NO:1- 8. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978), and in Henikoff et al. (1992). To generate accurate similarity scores using NCBI BLAST, it is important to turn off any filtering, e.g., low complexity filtering, and to disable the use of composition based statistics. One should also confirm that the correct substitution matrix and gap penalties are used.

In a preferred embodiment, the antibody of the invention, or antigen-binding fragment thereof, comprises the light chain variable domain of SEQ ID NO:7 and the heavy chain variable domain of SEQ ID NO:8.

For ease of understanding, these CDR sequences are listed in Table 2:

Table 2: amino acid sequences of SEQ ID NO:7-8

An "epitope" is the site on the antigen to which an antibody specifically binds. It can be formed by contiguous residues or by non-contiguous residues brought into close proximity by the folding of an antigenic protein. Epitopes formed by contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by non- contiguous amino acids are typically lost under said exposure.

The present inventors have shown that the antibody of the invention binds preferentially on two sites of the N-terminal domain of ETB. The first epitope has the amino acid sequence ERGFPPDRATP (SEQ ID NO:9) and corresponds to the most N-terminal region of the mature receptor, once the signal peptide is cleaved. The second epitope has the amino acid sequence EVPKGDRT (SEQ ID NO:10). The absence of any similarity between the two sequences suggests that the antibody of the invention recognizes a conformational epitope formed by the juxtaposition of two distinct regions of the receptor. These two sequences are unique to human ETB and are not found in any other human proteins. Interestingly, the two sequences forming the epitope are not conserved between human and rodents, explaining why rendomab-B4 is unable to bind ETs-expressing rat cells.

In one embodiment, the invention provides antagonistic antibodies or antigen-binding fragments thereof capable of inhibiting the activation of ETB by its natural ligands ET1 and ET3 without impairing their binding to the receptor (figures 3A, 3B and figure 3C). Specifically, the antibodies of the invention or antigen-binding fragments thereof are capable of inhibiting the activating effect of ET1 and ET3 on protein G-dependent pathways (PLC activity and inositol phosphate production, figure 6A), but not the activating effect of ET1 and ET3 on ERK1/2 signaling (Figure 6B and C). In other words, the antibodies of the invention are capable of controlling differentially the various signalization pathways downstream of ETB. Rendomab-B4 is the first reported mAb to possess the ability to differentially affect ETs-coupled signaling pathways, behaving as a biased allosteric modulator inhibiting G protein-dependent processes but not p-arrestin-dependent ones.

Preferably, the antibodies of the present application have a high affinity for human ETB expressed by CHO and for human ETB naturally expressed by human melanoma cell lines (UACC-257, WM-266-4 and SLM8). More specifically, the antibodies of the invention or antigenbinding fragments thereof have a dissociation constant (KD) with human ETB of SEQ ID NO:11 expressed on melanoma cells such as UACC-257 about 0,15 nM as determined by flow cytometry (Guava Easycyte Plus, Millipore).

As used herein, the term "KD" refers to the dissociation constant of a particular antibody/antigen interaction. As used herein the term “binding affinity" or “affinity of binding” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

It must be understood here that the invention preferably does not relate to antibodies in natural form, i.e., they are not taken from their natural environment but are isolated or obtained by purification from natural sources or obtained by genetic recombination or chemical synthesis and thus they can carry “unnatural” amino acids as will be described below. They can also be multispecific, for example TandAb or Flexibody.

In another aspect, the invention relates to chimeric or humanized antibodies, or antigenbinding fragments, which can be obtained by genetic engineering or by chemical synthesis.

Specifically, the anti-ETs antibodies of the invention are chimeric antibodies.

The term “chimeric antibody” as used herein refers to an antibody containing a natural variable region (light chain and heavy chain) derived from an antibody of a given species in combination with constant regions of the light chain and of the heavy chain of an antibody of a species heterologous to said given species. Thus, a “chimeric antibody”, as used herein, is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged, so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass. “Chimeric antibody” also refers to an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged, so that the constant region is linked to a variable region of a different species, or belonging to another antibody class or subclass. Such chimeric antibodies, or fragments of same, can be prepared by recombinant engineering. For example, the chimeric antibody could be produced by cloning recombinant DNA containing a promoter and a sequence coding for the variable region of a non-human monoclonal antibody of the invention, notably murine, and a sequence coding forthe human antibody constant region. A chimeric antibody according to the invention coded by one such recombinant gene could be, for example, a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from the murine DNA and its isotype determined by the constant region derived from human DNA.

In a preferred embodiment, the present invention relates to a chimeric antibody, or an antigen binding fragment thereof, comprising a light chain variable domain (VL) comprising CDR- L1 , CDR-L2 and CDR-L3 having respectively the amino acid sequence SEQ ID NO: 1 , 2 and 3; and a heavy chain variable domain (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 having respectively the amino acid sequence SEQ ID NO: 4, 5 and 6.

In another embodiment, the present invention relates to a chimeric antibody, or an antigen-binding fragment thereof, comprising a light chain variable domain (VL) comprising the amino acid sequence SEQ ID NO: 7 and a heavy chain variable domain (VH) comprising the amino acid sequence SEQ ID NO: 8.

In a specific embodiment, the present invention relates to a chimeric antibody, or an antigen-binding fragment thereof, comprising a light chain variable domain (VL) of sequence SEQ ID NO: 7 and a heavy chain variable domain (VH) of sequence SEQ ID NO: 8.

In another aspect, the present invention provides humanized antagonistic anti-ETs antibodies, or antigen-binding fragments thereof.

As used herein, the term "humanized antibody" refers to a chimeric antibody which contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR are typically no more than 6 in the Heavy (H) chain, and in the Light (L) chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

The goal of humanization is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted for nonrejection by other mammals, may be produced using several technologies such as resurfacing and CDR grafting. As used herein, the resurfacing technology uses a combination of molecular modeling, statistical analysis and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host.

Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed in US 5,639,641 , which is hereby incorporated in its entirety by reference. Briefly, in a preferred method, (1) position alignments of a pool of antibody heavy and light chain variable regions is generated to give a set of heavy and light chain variable region framework surface exposed positions wherein the alignment positions for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) a set of heavy and light chain variable region framework surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified; (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5 angstroms (A) of any atom of any residue of the complementarity- determining regions of the rodent antibody; and (5) the humanized rodent antibody having binding specificity is produced. Antibodies can be humanized using a variety of other techniques including CDR- grafting (EP0239400; WO91/09967; US5,530,101 ; and US5,585,089), veneering or resurfacing (EP0592106; EP0519 596), and chain shuffling (US5,565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods (US4,444,887, 4,716,1 11 , 5,545,806, and 5,814,318).

Another technique which may be employed either as an alternative, or in addition, to the methods described above for reducing immunogenicity, is the “deimmunisation”. Deimmunisation technology involves the identification and removal of T helper (Th) cell epitopes from antibody and other protein biological therapeutic agents. Th cell epitopes comprise short peptide sequences within proteins that have the capacity to bind to MHC class II molecules. The peptide- MHC class II complexes can be recognized by T cells and can trigger the activation and differentiation of Th cells, which is required to initiate and sustain immunogenicity through interaction with B cells, thus resulting in the secretion of antibodies that bind specifically to the administered biological therapeutic agent. For antibody deimmunisation, the Th-cell epitopes are identified within the antibody sequence, for example by a computer-based method for predicting the binding of peptides to human MHC class II molecules. To avoid recognition by T cells, the Th cell epitopes thus identified are eliminated from the protein sequence by amino acid substitutions. This may be achieved through the use of standard molecular biology techniques, such as for example site-directed mutagenesis to alterthe nucleic acid sequence encoding the Th cell epitope in the therapeutic protein. In this way, an antibody or antigen-binding fragment may be modified so that HAMA (Human anti mouse antigenic) and/or anti-idiotypic response(s) are reduced or avoided. Thus, in specific embodiments, the antibodies of the invention have been modified to remove any Th cell epitopes present in their sequence. Such binding molecules are referred to herein as deimmunised antibodies. The humanized antibodies of the invention arise from the murine antibody described above.

More particularly, the invention relates to a humanized antibody, or antigen-binding fragments thereof, comprising a light chain variable domain comprising CDR-L1 , CDR-L2 and CDR-L3 having respectively the amino acid sequence SEQ ID NO. 1 , 2 and 3; and a heavy chain variable domain comprising CDR-H1 , CDR-H2 and CDR-H3 having respectively the amino acid sequence SEQ ID NO: 4, 5 and 6.

Thus, in a specific embodiment, the present invention provides humanized antibodies or antigen-binding fragments thereof which specifically bind ETB and inhibit its protein-G dependent subsequent signaling.

In another specific embodiment, the antagonistic antibody or antigen-binding fragment of the invention is fully human. The term "fully human" as used herein relates to an antibody or antigen-binding fragment whose amino acid sequences are derived from (i.e. originate or may be found in) humans. Preferably, it is a full-human antibody comprising a light chain comprising the CDR-L1 , CDR-L2 and CDR-L3 having respectively the amino acid sequences SEQ ID NO: 1 , 2 and 3; and a heavy chain comprising CDR-H1 , CDR-H2 and CDR-H3 having respectively the amino acid sequences SEQ ID NO: 4, 5 and 6.

Antibody fragments of the invention

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641 ,870); single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of crosslinking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The term “Fv” as used herein refers to the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hyper variable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the n and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.

In a preferred embodiment, the antibody or fragment of the invention is multispecific, and in particular bispecific. As such, it can be chosen in the group consisting of: bispecific IgGs, IgG- SCFV2, (scFv)4-lgG, (Fab')2, (scFv)2, (dsFv)2, Fab-scFv fusion proteins, (Fab-scFv)2, (scFv)2-Fab, (SCFV-CH2-CH3-SCFV)2, bibody, tribody, bispecific diabody, disulfide-stabilized (ds) diabody, 'knob- into whole' diabody, single-chain diabody (scDb), tandem diabody (TandAb), flexibody, DiBi miniantibody, [(scFv) 2-Fc] 2, (scDb-Ch3)2, (scDb-Fc)2, Di-diabody, Tandemab., etc. “Functional fragments” of the antibodies of the invention comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

As used therein, the term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “cross-over” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161.

More particularly, the invention provides an anti-ETs functional fragment selected among the antibody fragments Fv, Fab, (Fab’)2, Fab’, scFv, scFv-Fc and diabodies, or any fragment whose half-life has been increased by chemical modification.

The chemical modification as cited above, may be such as the addition of polyalkylene glycol such as polyethylene glycol (PEGylation) (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG and Fab’-PEG), or by incorporation in a liposome, microspheres or Poly (D, L-lactic-co-glycolic acid) (PLGA), said fragments possessing at least six of CDRs of the invention which is notably capable of exerting in a general manner activity, even partial, of the antibody from which it arises.

Preferably, said antigen-binding fragment will comprise or include a partial sequence of the variable heavy or light chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same binding specificity as the antibody from which it arises and sufficient affinity, preferably at least equal to 1/100, more preferably at least 1/10 of that of the antibody from which it arises. Preferably, this antigen-binding fragment will be of the types Fv, scFv, Fab, F(ab’)2, F(ab’), scFv-Fc or diabodies, which generally have the same binding specificity as the antibody from which they result.

According to the present invention, antigen-binding fragments of the invention can be obtained from the antibodies described above by methods such as enzyme digestion, including pepsin or papain, and/or by cleavage of the disulfide bridges by chemical reduction. The antigens- binding fragments can be also obtained by recombinant genetics techniques also known to a person skilled in the art or by peptide synthesis by means, for example, of automatic peptide synthesizers such as those sold by Applied BioSystems, etc.

Antibody conjugates

An important characteristic of the antibodies of the invention is their ability to be internalized in endosomes and lysosomes of human melanoma cells such as UACC-257. They are therefore useful for targeting drugs into melanoma cells, in order to block their proliferation or kill them.

Assays for measuring the ability of an antibody to be internalized are known. For example, they rely on cytometry experiments or on microscopy analysis (see examples below).

In a preferred embodiment, the antibodies or fragments of the invention are coupled to a functional domain. This further functional domain may be a toxin molecule, a cytotoxic group, a label, or an effector group.

Said toxin molecule can be a ribosyl transferase, serine protease, guanyl cyclase activator, calmodulin- dependent adenyl cyclase, ribonuclease, DNA alkylating agent or mitosis inhibitor (e.g. doxorubicin) and the like, that may be used to target and kill melanoma cells.

Said cytotoxic group can be a group directly or indirectly toxic for the cells targeted by the antibody according to the present invention. By "directly cytotoxic", it is meant a group which is cytotoxic on its own. Direct cytotoxic group can be chosen in the group of the chemotherapeutic agents such as alkylating agents (mechlorethamine or chlorambucile; methotrexate; 5-fluoro- uracil; vinblastine; gemcitabine; fludarabine; nicotinamide; doxorubicin; mitomycin; L- asparaginase; cisplatin; taxol, etc.). It can also be a cytotoxic polypeptide group such as ricin, abrin, Pseudomonas exotoxin, TNFa or interleukin 2. They are poorly cytotoxic as such but are able to give, in particular after an enzymatic reaction or an irradiation, a cytotoxic substance (or “drug”). By "indirectly cytotoxic", it is meant a group which, although not cytotoxic on its own, can induce a cytotoxicity, for example by its action on another molecule or by a further action on itself. Indirectly cytotoxic agents (“prodrugs”) can be cytotoxic chemotherapeutic agents such as methotrexate-alanine; mitomycin phosphate, 5-fluorocytosine, photofrin and capecitabine or cytotoxic polypeptide such as carboxypeptidase, aminopeptidase, endopeptidase, phosphatase, sulphatase, amidase, kinase, glycosidase, deaminase, reductase, or oxidase. The cytotoxic group can also be a nucleic acid molecule which is directly or indirectly cytotoxic such as an antisense oligonucleotide or an aptamer.

Said label is preferably an "easily detectable group", i.e., a group that can be detected by implementing an advantageously non-invasive appropriate detection technique such as microscopy, scintigraphy, positon emission tomography (TEP) and magnetic resonance imaging (MRI).

A compound according to the invention comprising such an easily detectable group is particularly suitable for the field of imaging and diagnosis. It enables in particular sites at which the ETB is over-expressed to be identified and localized because of the ETB binding specificity of the antibody of the invention. This label can be an enzyme or a molecule capable of generating a detectable and possibly quantifiable signal under particular conditions (such as when putting into contact with an adapted substrate). By way of illustrating and non-limiting examples, biotin, digoxigenin, 5-bromodeoxiuridin, an alkaline phosphatase, a peroxidase, an acetylcholine esterase (AChE), a glucose amylase and a lysozyme can be mentioned. The label can also be can be a fluorescent, chimiofluorescent or bioluminescent label such as fluorescein and derivatives thereof, rhodamine and derivatives thereof, GFP (Green Fluorescent Protein) and derivatives thereof, umbelliferone; luminol; luciferase or luciferin. The label can also be a radioactive label or isotope such as iodine-123, iodine-125, iodine-126, iodine-133, indium- 111 , indium-113m, bromine-77, gallium-67, gallium-68, ruthenium-95, ruthenium-97, technetium-99m, fluorine-19, fluorine-18, carbon-13, nitrogen-15, oxygen-17, scandium- 47, tellurium-122m, thulium-165 and yttrium-199. It should be observed that some radioactive atoms used as easily detectable groups can also be cytotoxic groups because of the radioactivity quantity they can deliver.

Said effector group can be a group capable of specifically recognizing a melanoma marker, or which makes it possible to recruit (i) an effector cell of the immune system i.e. NK cells, cytotoxic T cells, macrophages or (ii) the complement system. By "group capable of specifically recognizing a melanoma marker", it is meant, within the scope of the present invention, a ligand of a melanoma marker; an antibody (identical to or different from the antibody according to the present invention); a protein; a peptide; or a nucleic molecule such as a DNA, an RNA, an RNAi, an aptamer, a PNA or an LNA. By " melanoma label", both an ETB and another membrane marker are contemplated. The effector group can recognize a melanoma marker such as S100, HMB-45 and Melan-A which is expressed at the surface of melanoma cells, thus ensuring better recognition specificity and thus increased targeting of melanoma cells. The effector group can alternatively exhibit a recognition specificity for a marker specifically present at the surface of effector cells of the immune system, i.e. NK cells, macrophages or cytotoxic T cells. Such a recruitment ensures targeted lysis of the melanoma cells recognized by the antibody of the present invention. The effector group can also have a recognition specificity for the complement system and, in particular, for protein C1 or its truncated form C1q, which initiates the cascade of molecular events which result in the death of the targeted melanoma cell. The effector group can also exhibit a recognition specificity for the complement system and, in particular, for protein C3 or its truncated form C3b, thus ensuring recruitment of effector cells of the immune system, which cells induce the death of the targeted melanoma cell.

In a particular embodiment, it is possible to conjugate the antibody of the invention with a ERK1/2 inhibitor known in the art, such as TCS ERK 11 e (Asundi J. et al, 2014) or with the BRAF inhibitors vemurafenib, dabrafenib and encorafenib that are used in the treatment of patients with BRAF-mutant melanoma (Proietti I. et al, 2020).

Those skilled in the art know different techniques enabling such groups to be conjugated with an antibody according to the present invention once the latter is obtained or produced. These techniques allow a covalent coupling between an antibody according to the invention and a cytotoxic group by taking advantage of particular chemical groups carried by the antibody according to the invention and by the cytotoxic group. Among these particular chemical groups, a thiol group, an ester group, an amino group, an acid group and any chemical element likely to be implemented in "click-chemistry" can be mentioned. Alternatively and in particular when the cytotoxic group is a group of peptidic nature, this conjugation can consist in producing the compound according to the invention as a fusion compound by genetic recombination techniques, wherein a polynucleotide comprises respective regions coding the antibody according to the present invention and the cytotoxic group, which are adjacent to each other, juxtaposed or separated by a region coding a peptide linker which does not destroy the desired properties of the final hybrid compound. In the case where the label is a radioactive label, it can be introduced into the peptide sequence of the antibody according to the invention. This introduction can take place during the synthesis of the antibody by using one or more labelled amino acids. Alternatively, this introduction can take place following this synthesis by binding the radioactive label on residues of the peptide sequence of the synthesized antibody. For example, yttrium-90 can be bound via a lysine residue. Further alternatively, the radioactive label can be indirectly bound to the antibody by known means. For example, EDTA or another chelating agent can be bound to the antibody according to the invention and used to bind indium-111 .

Irrespective of the technique used to conjugate an antibody according to the present invention with a cytotoxic group, the only requirement to meet within the scope of this conjugation is that the conjugated antibody preserves its ETB binding specificity and its antagonist property. Preferred mutations in the Fc domain

It is also possible to increase the half-life of the antibodies of the invention by modifying the antibody amino acid sequence itself.

For example, the half-life of the antibodies or fragments of the invention can be increased by introducing the following amino acid mutations:

• M252Y/S254T/T256E (“YTE”): this mutation increases the binding of the IgG or Fc domains to the IgG recycling receptor, FcRn, leading to a prolonged half-life (Dall'Acqua WF et al. 2002).

• M428L/N434S (“LS”): this mutation increases the binding of the IgG or Fc domains to the IgG recycling receptor, FcRn, leading to a prolonged half-life (Zalevsky J et al, 2010).

• L309D/Q311 H/N434S (“DHS”): this mutation increases the binding of the IgG or Fc domains to the IgG recycling receptor, FcRn, leading to a prolonged half-life (Lee CH et al, 2019)

• T307A/E380A/N434A: this mutation increases the binding of the IgG or Fc domains to the IgG recycling receptor, FcRn, leading to a prolonged half-life (Shields RL, et al. 2001).

Moreover, as the antibodies and fragments of the invention are intended to be used in the treatment and/or therapy in humans, their potential immunogenicity and deleterious effects should be minimized by any means.

It is therefore recommended to modify the Fc regions of these antibodies in order to abolish their effector functions, as already proposed in the art. In particular, it is better to mutate the Fc region of the antibodies in order to avoid the activation not only of the receptors FcyR (FcyRI, FcyRII, FcyRIII, FcyRIIIA, FcyRIIIB, Fcyn) but also of the C1q component of the complement, which plays important roles in opsonization, lysis of cell pathogens, and inflammatory responses. As used herein, the term "Fc region" is used to define a C-terminal region of an IgG heavy chain. Although the boundaries may vary slightly, the human IgG heavy chain Fc region is defined to stretch from Cys226 to the carboxy terminus. The Fc region of an IgG comprises two constant domains, CH2 and CH3. The CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341 . The CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447. The CH2 domain of a human IgG Fc region usually extends from amino acid 231-340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG.

As used herein, an Fc region that "lacks effector function" does not bind the Fc receptor and/or does not bind the C1q component of complement nor trigger the biological responses characteristic of such binding.

It is possible to impair the effector function of antibodies by generating Fc regions that are not glycosylated (or “aglycosylated”) at its usual glycosylation sites.

The term "glycosylation site" refers to an amino acid residue that is recognized by a mammalian cell as a location for the attachment of sugar residues. Amino acid residues to which carbohydrates, such as oligosaccharides, are attached are usually asparagine (N-linkage), serine (O-linkage), and threonine (O-linkage) residues. The specific sites of attachment usually have a characteristic sequence of amino acids, referred to as a "glycosylation site sequence." The glycosylation site sequence for N-linked glycosylation is : -Asn-X-Ser- or -Asn-X-Thr-, where X can be any of the conventional amino acids, other than proline. The Fc region of human IgG has two glycosylation sites, one in each of the CH2 domains. The glycosylation that occurs at the glycosylation site in the CH2 domain of human IgG is N-linked glycosylation at the asparagine at position 297 (Asn 297).

All the mutations proposed for the 3G8 antibody in WO 2007/009065 are herewith encompassed (cf., in particular, [0101] to [0110] and [0116] to [0122]). In particular, it is possible to modify the Fc regions of the antibodies of the invention by mutating them with any of the following mutations:

• N297A: this mutation replaces the asparagine able to receive N-glycosylation. This N-glycosylation is necessary for the interaction between the Fc region of IgG and human low-affinity FcyR (CD32A, CD32B, CD32C, CD16A, CD16B). It does not affect the interaction of IgG with the high-affinity FcyR, FcyRI/CD64.

• N297D: similar mutation to N297A with same consequences on FcyR binding.

• L234A, L235A (LALA): this double mutation abolishes the interaction between the Fc region of IgG and human low-affinity FcyR (CD32A, CD32B, CD32C, CD16A, CD16B). It does not affect the interaction of IgG with the high-affinity FcyR, FcyRI/CD64.

• L234A, L235A, P329G (LALAPG): this triple mutation abolishes the interaction between the Fc region of IgG and all human FcyR, whether low-affinity FcyR (CD32A, CD32B, CD32C, CD16A, CD16B) or high-affinity FcyR (CD64).

Any of this mutation can be used to generate an efficient therapeutic antibody that can be safely administered to human beings.

Moreover, all the mutations known in the art to enhance the efficiency and reduce the adverse side effects of therapeutic antibodies are herewith encompassed.

Hybridoma, antibody production and polynucleotides

In another aspect, the present invention targets an isolated polynucleotide that encodes the antibody of the invention, or complementary sequences thereof, or fragments thereof. In a particular embodiment, the present invention targets : i) a polynucleotide encoding an antibody as previously defined; ii) a polynucleotide complementary to the polynucleotide as defined in (i); or iii) a polynucleotide of at least 18 nucleotides, capable of hybridising under high stringency conditions with the polynucleotides as defined in (i) and (ii).

By "polynucleotide", it is meant, within the scope of the present invention, a nucleic acid, a nucleic sequence, a nucleic acid sequence, an oligonucleotide, a polynucleotide sequence, a nucleotide sequence, a single strand DNA, a double strand DNA or an RNA. A polynucleotide according to the present invention can comprise natural nucleotides and non-natural nucleotides.

As an example, the following polynucleotides are encompassed by the present invention:

SEQ ID NO:12, corresponding to one polynucleotide encoding the CDR1 of the light chain of Rendomab-B4

SEQ ID NO:13 (ctggtgtct), corresponding to one polynucleotide encoding the CDR2 of the light chain of Rendomab-B4

SEQ ID NO:14, corresponding to one polynucleotide encoding the CDR3 of the light chain of Rendomab-B4

SEQ ID NO:15, corresponding to one polynucleotide encoding the variable light chain of Rendomab-B4

SEQ ID NO:16, corresponding to one polynucleotide encoding the CDR1 of the heavy chain of Rendomab-B4

SEQ ID NO:17, corresponding to one polynucleotide encoding the CDR2 of the heavy chain of Rendomab-B4

SEQ ID NO:18, corresponding to one polynucleotide encoding the CDR3 of the heavy chain of Rendomab-B4

SEQ ID NO:19, corresponding to one polynucleotide encoding the variable heavy chain of Rendomab-B4

The polynucleotide according to the invention does not correspond to a nucleotide sequence in its natural state i.e., in its natural chromosomal environment. On the contrary, the polynucleotide according to the invention has been isolated and possibly purified, its environment has consequently been modified. The polynucleotide according to the invention can also be obtained by genetic recombination or chemical synthesis.

The high stringency conditions correspond to temperature and ionic strength conditions which enable a hybridization to be maintained between two complementary nucleotide sequences. Those skilled in the art will be able to determine the most suitable high stringency conditions in particular depending on the size of the nucleotide sequences by referring to the teaching of Sambrook et al, 1989 (Molecular cloning, Noland C. ed., New York: Cold Spring Harbor Laboratory Press).

Also, the present invention relates to a cloning and/or expression vector containing at least one polynucleotide according to the present invention. Such a vector is in particular useful to transform a host organism and express in the latter an antibody according to the present invention.

The vector according to the present invention further comprises one (or more) element(s) which enable(s) the polynucleotide according to the present invention to be expressed and/or the product resulting from the translation of the polynucleotide according to the present invention to be secreted. Among these elements, a constituent or inducible promoter, a transcription initiation signal or a transcription termination signal, a translation initiation sequence or a translation end signal can be mentioned.

Advantageously, the vector according to the present invention comprises a promoter, a polynucleotide of the invention and a terminator element which are operationally linked to each other. By "operationally linked to each other", according to the invention, it is meant elements linked to each other such that the functioning of one of the elements is affected by that of another one. By way of example, a promoter is operationally linked to a coding sequence when it is capable of affecting the expression of the same. The peptide transcription, translation and maturation regulating elements that the vector can comprise are known to those skilled in the art who are able to choose them depending on the host organism in which the expression or cloning should be made.

The vector according to the present invention is advantageously chosen from a plasmid, a cosmid, a bacteriophage and a virus such as a baculovirus. In particular, the vector of the invention is an autonomously replicating vector including elements enabling it to be maintained and replicated in the host organism as a replication origin. Further, the vector can include elements enabling it to be selected in the host organism as, for example, an antibiotic resistant gene or selection gene which ensures complementation with the respective gene deleted in the genome of the host organism. Such cloning and/or expression vectors are well known to those skilled in the art and widely described in the literature.

The invention also relates to a host organism transformed by or comprising a polynucleotide according to the present invention or a vector according to the present invention.

By "host organism", it is meant any isolated, single or multi-cell, lower or higher organism, in which a polynucleotide of the invention is introduced for producing an antibody according to the present invention.

Those skilled in the art know different methods for efficiently introducing a polynucleotide into a host organism in order to produce the antibody coded by said polynucleotide in the host organism. By way of example and in a non-exhaustive way, this method can be an electroporation, lipofection, biological transformation of a plant using Agrobacterium tumefasciens, a heat shock or a chemical process.

Advantageously, the host organism is a microorganism such as a yeast, bacterium or fungus. The transformation of such microorganisms enables the antibody of the invention to be produced at a semi-industrial or industrial scale. Alternatively, the host organism can be an animal cell such as mammal cell, plant cell, insect cell, animal except for a human, or a plant. Mammalian cells are commonly used for the expression of a recombinant therapeutic immunoglobulins, especially for the expression of whole recombinant antibodies. For example, mammalian cells such as HEK293 or CHO cells, in conjunction with a vector containing the major intermediate early gene promoter element from human cytomegalovirus, are an effective system for expressing the IgG antibody of the invention.

It is preferred to choose a host cell which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing of protein products may be important for the function of the protein. Appropriate cell lines or host systems are preferably chosen to ensure the correct modification and processing of the expressed antibody of interest. Hence, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, COS, HEK293, NS/0, BHK, Y2/0, 3T3 or myeloma cells (all these cell lines are available from public depositories such as the Collection Nationale des Cultures de Microorganismes, Paris, France, or the American Type Culture Collection, Manassas, VA, U.S.A.).

For long-term, high-yield production of recombinant proteins, stable expression is preferred. In one embodiment of the invention, cell lines which stably express the antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells are transformed with DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences known to the person skilled in art, and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for one to two days in an enriched media, and then are moved to a selective media. The selectable marker on the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and be expanded into a cell line. Other methods for constructing stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, the transformed DNA underthe control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences is integrated in the host cell genome at a specific target site which has previously been cleaved (US 5,792,632; US 5,830,729; US 6,238,924; WO 2009/054985; WO 03/025183; WO 2004/067753).

A number of selection systems may be used according to the invention, including but not limited to the Herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, glutamate synthase selection in the presence of methionine sulfoximide and adenine phosphoribosyltransferase genes in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside, G-418; and hygro, which confers resistance to hygromycin. Methods known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993). The expression levels of an antibody can be increased by vector amplification. When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in the culture will increase the number of copies of the marker gene. Since the amplified region is associated with the gene encoding the IgG antibody of the invention, production of said antibody will also increase. Alternative methods of expressing the gene of the invention exist and are known to the person of skills in the art. For example, a modified zinc finger protein can be engineered that is capable of binding the expression regulatory elements upstream of the gene of the invention; expression of the said engineered zinc finger protein (ZFN) in the host cell of the invention leads to increases in protein production. Moreover, ZFN can stimulate the integration of a DNA into a predetermined genomic location, resulting in high-efficiency site-specific gene addition.

The antibody of the invention may be prepared by growing a culture of the transformed host cells under culture conditions necessary to express the desired antibody. The resulting expressed antibody may then be purified from the culture medium or cell extracts. Soluble forms of the antibody of the invention can be recovered from the culture supernatant. It may then be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by Protein A affinity for Fc, and so on), centrifugation, differential solubility or by any other standard technique for the purification of proteins. Suitable methods of purification will be apparent to a person of ordinary skills in the art.

Another aspect of the invention relates to a method for the production of an antibody according to the invention, or antigen-binding fragments thereof, characterized in that said method comprises the following steps: a) culturing a host organism according to the present invention and in particular a singlecell host organism in a culture medium and under appropriate conditions; and b) recovering said antibody from the culture medium of said cultured host organism or from said cultured host organism.

In another aspect, the present invention targets the hybridoma deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS CEDEX 15) on 14 March 2023 under the accession number CNCM I-5937.

Another aspect of the invention relates to a method for the production of an antibody according to the invention, or antigen-binding fragments thereof, characterized in that said method comprises the following steps: a) culturing the hybridoma deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS CEDEX 15) on 14 March 2023 under the accession number CNCM I-5937 in a culture medium (e.g., RPMI 20%, SVF, L-glutamine 4mM, sodium pyruvate 1 mM, antibiotics) and under appropriate conditions; and b) recovering said antibody from the culture medium of said hybridoma. Pharmaceutical composition and treatment methods

An important characteristic of the antibodies of the invention is their ability to inhibit the migration of melanoma cells induced by ET. They can therefore be used, either alone, or coupled to a drug, to prevent the formation of metastasis and the worsening of melanoma in patients in need thereof. Assays for measuring the ability of an invention to inhibit cell migration are known. For example, they rely on Boyden chambers or microscopy analysis (see examples below).

In another aspect, the present invention therefore relates on a pharmaceutical composition comprising the antibody or fragment of the invention, and a pharmaceutically-acceptable carrier. Preferably, the pharmaceutical composition of the invention contains, in addition to the antibody of the invention, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, salt solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of media and agents for pharmaceutically active substances is well known in the art. A typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of the combination. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), and the 18th and 19th editions thereof, which are incorporated herein by reference.

The antibody in the composition preferably is formulated in an effective amount. An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result, such as treatment of melanoma. A “therapeutically effective amount” means an amount sufficient to influence the therapeutic course of a particular disease state. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.

The invention also relates to the antibody or fragment of the invention, or the pharmaceutical composition of the invention, for use as a medicament, in particular for the treatment of melanoma.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating the symptoms of a disorder (e.g., a melanoma) and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein "treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that the extent of the disease is decreased or prevented. For example, treating results in the reduction of at least one sign or symptom of the disease or condition. Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, and may be performed either prophylactically, or subsequent to the initiation of a pathologic event. Treatment can require administration of an agent and/ or treatment more than once.

In another embodiment, the present invention also relates to the use of an antibody or fragment or of a pharmaceutical composition according to the invention for the preparation of a drug and/or a medicament for the treatment of melanoma in a human being.

In a related aspect, the invention provides methods of treating melanoma by administering to a human being a therapeutically effective amount of a pharmaceutical composition comprising the antibody or fragment described herein. The antibodies and fragments of the invention can be administered in combination with other treatments directed to treat melanoma as well. In particular, since rendomab-B4 does not inhibit ERK1/2 pathway and this pathway is crucial for melanoma growth, it would be interesting to co-treat the patient with a ERK1/2 pathway inhibitor, or by directly coupling the antibody to such inhibitor.

The dosage of the compositions of the invention administered to a patient is typically about 0.1 mg/kg to about 10 mg/kg of the patient's body weight, e.g., about 0.1 , about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1 , about 1 .5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, and about 10 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between about 1 mg/kg and about 9 mg/kg of the patient's body weight. In other embodiments the dosage of the compositions of the invention is about 0.1 , about 0.3, about 1 .0 or about 3.0 mg/kg of the patient's body weight.

The antibodies of the invention can be administered according to the judgment of the treating physician, e.g., daily, weekly, biweekly or at any other suitable interval, depending upon such factors, for example, as the nature of the ailment, the condition of the patient and half-life of the antibody. In a preferred example, a subject is treated with the antibody or fragment of the invention in the range of between about 0.1 to about 10 mg/kg body weight, one time per week for between about 1 to about 10 weeks, preferably between about 2 to about 8 weeks, more preferably between about 3 to about 7 weeks, and even more preferably for about 4, about 5, or about 6 weeks. In other embodiments, the pharmaceutical compositions of the invention are administered once a day, twice a day, or three times a day. In other embodiments, the pharmaceutical compositions are administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once per year. It will also be appreciated that the effective dosage of the antibodies used for treatment may increase or decrease over the course of a particular treatment. In a most preferred embodiment, the composition of the invention is administered intravenously over about 30 minutes. In other embodiments, the composition of the invention is administered intravenously over at least about 1 hour, at least about 30 minutes, or at least about 15 minutes.

More generally, for therapeutic applications, the antibody of the invention can be administered to the subject as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.

Sequences SEQ ID NO: 2 and 13 do not have the minimum length required by the ST26 norm. They are therefore indicated as omitted sequences in the XML sequence listing. Consequently, the corresponding sequences themselves are not entered in the sequence list; instead of the sequence, the indication "000" appears.

The examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.

FIGURE LEGENDS

Figure 1 shows the binding properties of anti-human ETB specific monoclonal antibodies. To evaluate maximal binding (A) and apparent affinities (B, C), CHO and UACC-257 cells expressing hETs were incubated for 24 h at 4°C with increasing concentrations of ETB directed antibodies. Binding of anti-ETs antibodies were revealed using R-PE-labeled anti-mouse antibody and quantified by flow cytometry. Each antibody’s maximal binding (Bmax) and apparent affinity (1 /apparent Kd) were determined using GraphPad Prism software.

Figure 2 shows that rendomab-B4 specifically recognizes with high affinity human ETB overexpressed in CHO cell line. To check human ETB expression on CHO-ETB surface, cells were incubated with increasing concentrations of ET-1-FAM (A) and ET-3-FAM (B) for 24 h at 4°C in the dark. To determine rendomab-B4 affinity, increasing concentrations of the monoclonal antibody were incubated with CHO-ETB cells for 24 h at 4°C (C). Rendomab-B4 binding was revealed using R-PE-labeled anti-mouse antibody. The binding of labeled-peptides and monoclonal antibody were measured on a GUAVA flow cytometer and resulting curves (A, Band C), which correspond to mean fluorescence intensity (MFI) as a function of ligand concentrations, were plotted and fitted using GraphPad Prism software. Kd or apparent Kd (Kd*) corresponding to half maximal effective concentration values correspond to the mean § s.d. of three (A and B) or 5 (C) independent experiments. (D) CHO and CHO-ETB cells were fixed and incubated with 100nM of rendomab-B4, followed by Cy3-conjugated anti-mouse IgG antibody incubation.

Figure 3 shows that rendomab-B4 does not compete with ET-1 and ET-3 for binding CHO-ETB. CHO-ETB cells were incubated with 10 nM of ET-1-FAM (A) or ET-3-FAM (B) and simultaneously with increasing concentrations of isotype control antibody, rendomab-B4, selective receptor antagonists (BQ123 or BQ788) and the corresponding unlabelled endothelin (ET-1 or ET-3). (C) Cells were incubated simultaneously with 100 nM of rendomab-B4 and with increasing concentrations of endothelin (ET-1 or ET-3) or control peptide. Rendomab-B4 binding on cell surface was detected with a R-PE-labeled anti-mouse antibody. The fluorescence (A, B and C) was quantified by flow cytometry. Inhibition binding curves, corresponding to MFI relative to competitor concentration, were plotted and fitted with GraphPad Prism software. Two independent experiments were performed.

Figure 4 shows that rendomab-B4 specifically recognizes ETB in melanoma: Binding properties. (A) Human melanoma cell lines (UACC-257, WM-266-4 and SLM8), HEK293T and HUVEC were incubated with increasing concentrations of rendomab-B4. Rendomab-B4 binding was evaluated using R-PE-labeled anti-mouse antibody followed by flow cytometry analysis. The results are representative of 3 independent experiments. (B) To control human ETB expression at the surface of UACC-257 cells, the cells were incubated with varying concentrations of ET-3-FAM and fluorescence was measured by flow cytometry. Three independent experiments were performed. (C) UACC-257 melanoma cells were simultaneously incubated with 10 nM of ET-3-FAM and increasing concentrations of isotype control antibody, rendomab-B4, selective antagonists of ETA (BQ123) or ETB(BQ788) or unlabelled ligand ET-3. (D) Differential rendomab mAbs binding. The rendomab B1 was incubated at different concentrations on UACC-257, A-375 and WM-266-4 human melanoma cells. Rendomab B1 did not show binding curves on these melanoma cells except UACC-257. The fluorescence was quantified by flow cytometry. All these curves were plotted and fitted using GraphPad Prism software.

Figure 5 shows that rendomab-B4 is internalized in UACC-257 cells. Flow cytometry experiments were performed to study the internalization of ETB(A) and of rendomab-B4 itself (B). (A) Living, adherent UACC-257 cells were incubated at 4°C (ET-3 4°C curve) or 37°C (ET-3 37°C curve) in medium in the presence of 50 nM ET-3. After washing, cells were detached and incubated for 2 hours at 4°C with rendomab-B4 in order to evaluate ETB amount at the surface of the cells. Cells were then incubated with a fluorescent secondary antibody and the fluorescence was measured using a FACScalibur cytometer. Black curve corresponds to basal fluorescence. (B) UACC-257 living cells were incubated for 3hours at 4°C (B4 4°C curve) with 100nM rendomab-B4. Cells were incubated for 3 hours at 37° C with 100nM rendomab-B4 prior to 1 additional hour incubation at 37°C with 50 nM ET-3 (B4+ET-3 37°C curve) or not (B4 37°C curve). Cells were then detached and remaining antibodies at cell surface were detected using secondary antibody. The fluorescence was quantified by flow cytometry. (C and D) Living UACC-257 cells were incubated for 2 h at 4°C (C) or 37°C (D) with rendomab-B4, fixed and incubated in Cy3-anti-mouse antibody. Labeling in (E) corresponds to early endosomal antigen 1 (EEA1) staining. Merged image is represented in (F).

Figure 6 shows that rendomab-B4 inhibits PLC but not ERK response due to ETB receptor activation in UACC. (A) [³H]inositol-labeled UACC-257 cells were treated or not for 2 h with 150 nM of control isotype antibody or Rendomab-B4 before stimulation for 30 min in the presence or the absence of 50 nM ET-1 or ET-3. Total [³H]lnsPs amount was determined as described in materials and methods. Results are expressed as percent of InsPs production induced by ET-1 in the absence of antibody and are means ± SEM of 6 independent experiments, each performed in duplicate. (B) Cells were treated for 2 h with or without 150 nM of control isotype antibody or rendomab-B4 before the addition of 10 nM ET-1 or ET-3. After a 10 min incubation, cells were lysed and total proteins were analyzed by 10%SDS/ PAGE followed by immunoblotting with antiactive phosphorylated ERK1/2 (pERK) antibody and anti-total ERK2 (ERK). pERK and ERK signals were quantified (C), and the levels of phosphorylated ERK1/2 were normalized with respect to total ERK2 amount in the corresponding sample. Results were expressed as percent of ET-1 stimulation without antibody treatment (100%). Values are the means ± SEM of 3 independent experiments performed in duplicate.

Figure 7 shows that rendomab-B4 inhibits melanoma cell migration induced by endothelin. UACC-257 (150 nM) seeded in culture inserts were treated for 2 h with or without 150nM of rendomab-B4 or control isotype antibody before the addition of 10 nM ET-1. After 20 h of incubation, cells were stained with crystal violet and counted from 3 randomly selected images per insert. (A) Images of UACC-257 cells on the lower side of the insert membranes. (B) Quantification of the number of cells present on the lower side of the insert membranes. Values are the means ± SEM of 3 independent experiments.

EXAMPLES

1. Material and methods

Animals

Six-week-old female C57BL/6 mice (Elevage Janvier) were kept in a specific pathogen-free animal facility. All animal experiments complied with French animal experimentation regulations.

Plasmids The cDNA clone ofthe human ETswas subcloned in pcDNA3.1 vector (Life Technologies). The integrity ofthe con- struct was confirmed by sequencing.

Cell culture and transfection

CHO-K1 cells (ECACC) were cultured in Ham-F12 medium, HEK293T (EACC) and human umbilical vascular endothelial cells (HUVEC, ATCC) in DMEM medium. The melanoma cell line UACC-257 (NCI-60 cell collection) was cultured in RPMI-1640. Melanoma cell lines WM-266-4 (ECACC) and SLM8 were cultured in DMEM: Ham-F12 medium (1 :1). All media were supplemented with 10% fetal calf serum, 1 mM pyruvate, 1% nonessential amino acids, 2 mM glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin. Eker rat uterine leiomyoma cells (ELT-3) were cultured in DF8 medium supplemented with 10% fetal calf serum. Cells were maintained at 37 C in a humidified atmosphere of 5% CO2. All media and cell culture supplements were from Life Technologies. The ETB expression vector was transfected and stably overexpressed in CHO and HEK cells using FUGENE HD reagent (Roche Diagnostics). Stably transfected cells expressing human ETB or ETA were termed CHO-ETB, CHO-ETA or HEK-ETs, and they were always cultured in the presence of 1 mg/mL G1418.

DNA immunization protocol and production of monoclonal antibodies

MAb production, by DNA immunizations, was performed as previously described in Allard B. et al, 2011 ; collected splenocytes of the 2 best- responding 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. After subcloning by limiting dilutions, antibodies were isotyped using a mouse immunoglobulin isotyping kit according to the manufacturer (Pierce) instructions and purified by affinity chromatography on Protein A- Sepharose (Millipore). Flow cytometry analysis

Affinity determination

For affinity measurements, saturation binding experiments were performed with stably transfected CHO-ETB, melanoma cell lines (UACC-257, WM266-4 and SLM8), HEK293T and HUVEC. Collected cells were seeded (100,000 cells/well) in V-shaped 96-well plates. Plates were centrifuged, supernatant was discarded and cells were incubated overnight at 4°C with 100 |j_ of D-phosphate-buffered saline (PBS) supplemented with 0.1% BSA and 5% normal goat serum (NGS, Life Technologies) and containing increasing concentrations of rendomab- B4. After two washes with 150 pL of ice-cold D-PBS - 0.1% BSA - 1% NGS, cells were incubated for 2 h at 4° C in the dark with R-phycoerythrin (R-PE)-conjugated AffiniPure goat anti-mouse IgG (H⁺L) (Jackson ImmunoResearch). After two washes, cells were re-suspended in 100 L of D-PBS and their fluorescence was measured using a GUAVA flow cytometer (Guava Easycyte Plus, Millipore). Mean fluorescence intensity (MFI) of samples was then calculated. To control ETB expression at the cell surface, increasing concentrations of fluorescein-labeled ET-1 or ET-3 (ET-1-FAM or ET-3-FAM, Phoenix Pharmaceuticals) were used.

Competition experiments

For competition tests, CHO-ETB or melanoma cells (UACC- 257) were treated according to 2 different protocols: (i) cells were co-incubated with 10 nM ET-1-FAM or ET-3-FAM and varying concentrations of different competitors (rendomab-B4, control isotype antibody, ET-1 , ET-3, selective ETA receptor antagonist (BQ123) or selective ETB receptor antagonist (BQ788); and (ii) cells were co-incubated with fixed rendomab- B4 concentration (10 nM) and varying concentrations of competitor peptides ET-1 , ET-3. To reach equilibrium, cells were incubated overnight at 4° C. For type (i) experiments, cells were washed twice before measuring the fluorescence. For type (ii) experiments, after 2 washes, cells were incubated for 2 h with (R-PE)- conjugated secondary antibody. The MFI of the cells was measured using a GUAVA cytometer.

Receptor and rendomab-B4 internalization To study receptor and antibody internalization in UACC-257 cell line, 300,000 cells were used per assay. The internalization of the receptor due to ET was evaluated by incubating living adherent cells for 1 h at 4°C or 37° C with 50 nM ET-3. After detachment, cells were incubated for 2 h at 4° C in D-PBS - 0.1% BSA - 5%NGS with 100 nM rendomab-B4, washed twice and were incubated for 2 h at 4° C with (R-PE)-conjugated AffiniPure goat anti-mouse IgG (H⁺L). Cells were washed twice and the fluorescence was analyzed. For antibody internalization experiments, living adherent cells were incubated for 3 h at 4° C or 37° C with either 100 nM rendomab-B4 or control isotype antibody. Cells were incubated for one additional hour with or without 50 nM ET-3. Then, cells were detached and incubated for 2 h at 4° C with (R-PE)- conjugated AffiniPure goat anti-mouse IgG (H⁺L) and were washed twice again. For these 2 kinds of experiments, cell fluorescence was finally assayed using a FACSCalibur flow cytometer.

Microscopy analysis

Wide-field microscopy analysis

Wide-field microscopy analysis was performed on CHO and CHO-ETB cell lines seeded on glass coverslips at the density of 15,000 cells per cm2. After 48 h, cells were washed in PBS and fixed for 15 min in 4% paraformaldehyde (PFA) at room temperature. Cells were then incubated for 10 min in 50 mM NF CI and washed once with PBS. Cells were then incubated at room temperature for 1 hour in the presence of 100 nM of rendomab-B4 in PBS-1 % BSA. This incubation is performed in the absence of detergent in order to only detect membrane receptors at the cell surface. After three successive washes with PBS, cells were incubated for 1 h with PBS - 1 % BSA containing Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch). After three washes, cells were mounted on slides with Fluoroshield containing DAPI (Sigma-Aldrich). Images were taken using a fluorescence microscope (Zeiss Axiophot II), at 40x magnification.

Confocal microscopy analysis Confocal microscopy analysis was performed on UACC-257 cells seeded on glass coverslips at a density of 30,000 cells per cm2. For internalization experiments, living cells were incubated at 4 or 37°C for 1 h with 100 nM of rendomab-B4 before fixation with 4% PFA. To stain the endosomal compartment, cells were permeabilized by treatment for 15 min with PBS -1 % BSA - 0.2% Triton X-100 before incubation with an anti-EEA1 antibody (Millipore, France). After three washes, cells were incubated in PBS - 1 % BSA - 0.2% Triton X-100 containing Cy3-conjugated anti-mouse IgG antibody and FITC-conjugated anti-rabbit antibody. Images were taken using a confocal microscope (Zeiss LSM510), at 63x magnification.

Peptide synthesis and ETs-binding epitope mapping

The entire extracellular amino acid sequence of the ETB receptor was synthesized on a cellulose membrane using the previously described SPOT technique (Laune D. et al, 2002), laying down overlapping 12-mer peptides, frameshifted by one residue. Rendomab-B4 epitope mapping was performed according to a protocol previously described (Allard B. et al, 2011), 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). Imaged software was used to quantify the signal obtained for each spot. Peptides were considered as antigenically relevant if they were part of a consecutive series of reactive spots, presenting at least one signal peak times higher than the background (Laune D. et al, 2002).

Cloning of GFP-tagged rat and human ETB

Rat ETB CDNA was cloned by RT-PCR technique from rat ELT3 cells, which express high level of ETB (Raymond MN et al, 2009). Total RNA from these cells was prepared using NucleoSpin ® RNAII (Macherey-Nagel) according to the manufacture’s protocol.

The reverse transcription reaction was performed with 5 pg of total RNA using 200 units of Moloney Murine Leukemia Virus (M-MLV)-reverse transcriptase, 200 pM deoxynucleoside triphosphates (dNTPs), and 10 pM random hexamer primers. PCR primers for amplification of rat EBT 0"ETB) were based on the GenBank sequence (NM_017333). The primers span the start codon and introduced a modified codon in place of the stop codon and contain Hindlll and BamHI sites (italics):

50-GCAAGC7TATGCAATCGTCCGCAAGCC-30 (Forward primer, SEQ ID NO:20) and 50-GCGGATCCACAGATGAGCTGTATTTATTGCTGG -30 (Reverse primer, SEQ ID NO:21). For hETs cloning, hETs cDNA was amplified by PCR from pCDNA3.1-ETs plasmid (described above) with PCR primers based on the GenBank sequence (NM_000115.3). As for rETs cloning, the primers span the start codon and introduced a modified codon in place of the stop codon and contain Hindlll and BamHI sites (italics):

50-GCAAGC7TATGCAGCCG CCTCCAAGTC-30 (Forward primer, SEQ ID NQ:20) and 50-GCGGATCCACAGATGAGCTGT ATTTATTACTGG-30 (Reverse primer, SEQ ID NO:21). The PCR reactions were performed, in a thermal cycler (icycler; Bio-Rad) with 3 ml of the RT reaction (rETs) or 10 ng pCDNA-ETs plasmid (hETs) using the Phusion® high fidelity DNA Polymerase according to the manufacturer’s protocol.

The amplified condition was 98°C for 30 s followed by 30 cycles of 98°C for 10 s, 70°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min. The PCR products were digested by BamHI and Hindlll restriction enzymes and ligated into pEGFP-N1 (Clonetech), using T4 DNA ligase according to the manufacturer’s indication. Resulting plasmids were named phETs-GFP and prETs-GFP, and their sequences were verified by sequencing (Eurofins MWG Operon).

Immunoprecipitation of ETB

CHO cells seeded in 24-well plates were transfected with phETs-GFP or prETs-GFP plasmids using Lipofectamine LTX (Life Technologies) according to the manufacturer’s instructions. 24 h after transfection, cells were washed with PBS and lysed in 200 pL of ice cold lysis buffer containing 10 mM Tris/ Cl pH 7.5; 150 mM NaCI; 0.5 mM EDTA; 1 % Triton X-100. Lysates were centrifuged for 5 min at 15,000 x g and the supernatants diluted to 1 mL with lysis buffer without Triton X-100.

Antibodies, 4 pg of rendomab-B4 or 4 pL of GFP-Trap beads (Chromotek), were added and the samples were incubated on a wheel for 4 h at 4 C. When rendomab-B4 was used, 20 pL G-protein conjugated beads (Santa Cruz) were added after the 2 first hours of incubation. At the end of the incubations, samples were washed 4 times with 1 mL ice cold lysis buffer without detergent. Immunoprecipitated samples were then analyzed by protein gel blot using an anti-GFP antibody (Cell Signaling Technologies) and a secondary anti-rabbit IgG antibody conjugated to HRP (Cell Signaling Technologies).

Measurement of PLC activity

Confluent UACC-257 cells seeded in 12-well plates were serum starved for one night in MEM in the presence of 10 pCi/mL Myo-[2-³H]inositol (16 Ci/mmol; PerkinElmer). The cells were washed 3 times with MEM and then incubated at 37°C for 2 h in 800 pl of MEM in the presence of 150 nM rendomab-B4 or control isotype antibody. Cells were then exposed to the agonists ET-1 or ET-3 (50 nM) in the presence of 10 mM LiCI (added 20 min before the agonists). Total inositol phosphates (InsPs) produced by PLC were quantified as previously described in Raymond MN et al, 2009. Results were expressed as the mean

± SEM of at least 3 independent experiments performed in duplicate.

Western blot analysisof phosphorylated ERK1/2

Confluent UACC-257 cells seeded in 12-well plates were serum starved for one night in RPMI and then incubated at 37 C for h in 800 pl of MEM in the presence of 150 nM rendomab-B4 or control isotype antibody. Cells were then exposed to the agonists ET-1 or ET-3 (50 nM) for 5 min and then lysed in 50 ml lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCI, 100 mM NaF, 10% glycerol, 10 mM Na4P2O?, 200 mM Na3VO4, 10 mM EDTA, 1 % Triton X-100, 10 mg/mL aprotinin and leupeptin). Detergent-extracted proteins were analyzed by western blot technique using mouse monoclonal anti-active phosphorylated ERK1/2 (Cell Signaling Technology) and rabbit polyclonal anti-ERK2 (Santa Cruz Biotechnology) antibodies (1 :5000 each). The membranes were then incubated with antirabbit IgG antibody conjugated to IRDye 800CW (LICOR Biosciences) and antimouse IgG antibody conjugated to AlexaFluor 680 (Life technologies) for 1 h at 37°C. Protein bands were detected and quantified on a 2color Odyssey Infrared Imaging System (LICOR Biosciences).

Migration assay

UACC-257 cells were seeded within cell culture inserts (Falcon PET membrane with 8 mm porosity) placed in a 24-well plate (30,000 cells/insert). After 8 h of culture, media were removed and the cells were incubated for 2 h in RPMI medium without FCS, in presence or absence of 150 nM rendomab-B4 or 150 nM antibody control. Cells were then stimulated or not by addition of 50 nM ET-1 in the 2 compartments. After 20 h, inserts were removed and the cells were stained with crystal violet (0.1 % in 20% ethanol). After scrapping off non-migrated cells, the inserts were observed on an Axiophot II Zeiss microscope and images were taken at low magnification. The mean number of cells present on each insert was determined from 3 randomly selected images.

2. Results

Characterization of anti-human ETB specific monoclonal antibodies

As described previously, rendomab-B1 was initially selected from 24 antibodies obtained through genetic immunization based on its unique property to behave as a remarkably potent antagonist of human ETB (Allard B. et al, 2013). However this mAb displayed a very low affi ity for ETB expressed on tumor cells, which prompted us to look for the potential presence, among the 23 other anti-ETs mAbs that we produced, of antibodies that could recognize the tumor-associated form of human ETB. Toward that end, the binding properties of all the mAbs on Chinese hamster ovary (CHO) cells stably transfected with human ETB (CHO-ETB) and different melanoma cell lines were investigated. As expected, all mAbs were able to bind CHO-ETB, although with different intensities, since this cell line was used for the screening of anti-ETs antibodies- expressing hybridomas. This is illustrated in Fig. 1 A for 9 mAbs selected here for their high Bmax value on CHO- ETB (rendomab B4 and 8 other rendomabs, BS1 to BS8); 3 mAbs belonged to the lgG2B subclass, 3 to the lgG1 and 3 to the lgG3 subclass. The affinities of these 9 mAbs for CHO- ETB are shown in Fig. 1 B, and, as can be observed, no correlation was found between the isotypic class of the mAbs, Bmax, and affinity.

Most interestingly, all 9 selected mAbs appeared to be able to bind ETB expressed at the surface of the 3 melanoma cell lines tested: UACC-257, WM-266-4 and SLM8. Fig. 1 C shows their apparent affinities for UACC-257, and a detailed analysis of rendomab-B4 binding on the 3 different melanoma cells is shown below. In addition, among the 23 new antibodies that we tested, some other mAbs were totally unable to bind ETB expressed on any tumoral cell line, as was observed for the rendomab B1 that was already described (Allard B. et al, 2013) whereas others recognized ETB expressed only on a given tumoral cell line (results not shown). Since, as illustrated in Fig. 1 B and C, rendomab B4 (Rmb B4) exhibited the highest apparent affinity values not only for CHO-ETB, but above all for the tumoral UACC-257 cell line, it was selected for further characterization of its functional properties.

Rendomab-B4 specifically recognizes with high affinity human ETB overexpressed in CHO cell line

It is well established that the 3 ETs bind ETB with comparable affinities. First, we checked the binding properties of ETs on CHO-ETB by using fluorescent ET-1 and ET-3 (ET-1-FAM and ET- 3-FAM, respectively). Flow cytometry experiments showed that the 2 labeled ETs bound to CHO- ETB with the same Kd value ~ 1 nM, and the maximal binding was obtained at 100 nM for the 2 ligands (Fig. 2A and B). Data presented in Fig. 2C show that rendomab-B4 binding was dosedependent, saturable, and reached a plateau for a 50 nM concentration, half maximal binding was about 0.15±0.03nM (mean ± s.d), reflecting the high affinity of rendomab-B4 for hETs. Additional experiments show that rendo- mab-B4 bound neither to untransfected CHO cells that do not express ETB, nor to a rat leiomyoma cell line (ELT-3) that endogenously expresses ETB at high level (Raymond MN et al, 2009), and did not cross-react with ETA expressed in CHO cells (data not shown). Altogether, these data indicated that rendomab-B4 was specific to human ETB. This specific binding of rendomab-B4 to ETB was visualized by immunofluorescence experiments showing that the mAb displayed a marked membrane labeling in CHO-ETB cells, but not on control untransfected cells (Fig. 2D).

Rendomab-B4 does not compete with ET-1 and ET-3 for binding on CHO-ETB

Competition experiments were performed to characterize the interaction of rendomab-B4 with ETB expressed in CHO in the presence of ET-1-FAM or ET-3-FAM. Data depicted in Fig. 3 show that ET-1 and ET-3 inhibited ET-1 -FAM (Fig. 3A) and ET-3-FAM (Fig. 3B), respectively, with IC50 estimated at 0.17 ± 0.07 and 0.31 ±0.01 nM, respectively. The specific ETA antagonist BQ123 failed to reduce ET-1-FAM and ET-3-FAM binding in CHO-ETB. In contrast, BQ788, the specific antagonist for ETB, inhibited ET-1-FAM and ET-3-FAM binding in a dose-dependent manner, with IC50 values around 25.2 ± 2.8 and 8.9 ± 4.0 nM, respectively. Rendomab-B4, and its corresponding isotype control mAb, failed to impair the binding of ET-1 -FAM (Fig. 3A) and ET-3- FAM (Fig. 3B). Conversely, rendomab-B4 binding was not inhibited by ET-1 , ET-3 or random control peptide (Fig. 3C). These data demonstrated that rendomab-B4 and ETs are not competing for receptor binding, suggesting that their binding sites on ETB are distinct.

Rendomab-B4 recognizes a discontinuous epitope on hETs N-terminal domain

To further characterize the binding site of rendomab-B4 on hETs, epitope mapping experiments were performed. Pepscan membranes spotted with overlapping dodecapeptides, frame- shifted by one residue, and corresponding to the N-terminal part and extracellular loops (E1 , E2, E3) of the hETs were used. The immunization protocol that was carried out was expected to lead to the production of antibodies directed against the extracellular parts of the ETB.

The membrane incubated with rendomab-B4 displayed 2 immunoreactive regions, one from A21 to B8 and another from C17 to C24 spots, corresponding to ETB residues 28 to 38 and 70 to 77, respectively. The two series of immunoreactive peptides are part of the N-terminal domain. The deduced minimal sequences were ERGFPPDRATP (SEQ ID NO:9) and EVPKGDRT (SEQ ID NO:10). The first sequence corresponds to the most N-terminal region of the mature receptor, once signal peptide is cleaved. The absence of any similarity between the 2 sequences suggested that rendomab- B4 recognized a conformational epitope formed by the juxtaposition of 2 regions of the receptor. These two sequences are unique to human ETB and are not found in any other human proteins. Interestingly, the 2 sequences forming the epi- tope are not conserved between human and rodents, and each peptide differs by 3 amino acids. This may explain why rendomab-B4 is unable to bind ETs-expressing rat cells, and suggest that these residues are crucial for rendomab-B4 binding.

To confirm this, human and rat ETB were expressed in CHO cells and tested for their ability to bind rendomab-B4 in an in vitro immunoprecipitation assay. Because no anti-ETs antibody that works well in western blot was available, human and rat ETB were expressed as GFP fusion proteins and detected with an anti-GFP antibody. Human (hETs) and rat (rETs) GFP-fused ETB, immunoprecipitated with an anti-GFP nanobody followed by protein gel blot analysis with another anti GFP antibody, appear as several immunoreactive bands. For each receptor, the band of higher apparent molecular mass is likely to represent the full-length GFP-tagged receptor as its mass is consistent with the calculated theoretical mass of the protein (about 75 kDa). The other bands of lower molecular mass may represent N-terminally truncated forms of the receptor since the receptors were immunoprecipitated through their C-termi- nal GFP fusion.

When hETs-GFP was subjected to immunoprecipitation with rendomab-B4 followed by western blot analysis with an anti-GFP antibody, only one immunoreactive band was detected. This band corresponds to the higher mass band visible in the lower panel. This result shows that rendomab- B4 is able to interact and precipitate the full length ETB, but not its N-terminally truncated forms. This result is consistent with the finding of the epitope sequence in its N-terminal domain. When the same experiment was per- formed on rETs-GFP instead of hETs-GFP, no immunoreactive band was detected, confirming that rendomab-B4 does not cross-react with rETs and strengthening the hypothesis that the amino acids that differ between human and rat ETB are important for rendomab-B4 binding.

Rendomab-B4 specifically recognizes ETB in UACC-257 melanoma cell line: binding properties Considering that rendomab-B4 was able to bind human ETB in CHO cells with high affinity and specificity, its binding on human melanoma cell lines and on non-malignant cell lines expressing ETB was further investigated. Results in Fig. 4A show that rendomab-B4 clearly displays saturable binding curves on UACC-257 cells. Binding curves obtained with WM- 266-4 and SLM8 also tend to saturate and could be easily fitted to sigmoid curves. Analysis of the binding curves gave half- maximal binding concentration values of 0.14 ± 0.03 nM, 1 .5 ± 0.32 nM and ~10 nM for UACC- 257, WM-266-4 and SLM8, respectively. In contrast, binding curves obtained with HEK293T and HUVEC clearly did not tend to saturate, and these curves could not be fitted to sigmoid curves, indicating that no specific binding of rendomab-B4 was observed in these non-tumoral, yet ETB- expressing cells (Fig. 4A). This result further supports our previous assumption of the occurrence of tumor-specific conformations of ETB.25

Since its apparent affinity was also higher in UACC-257 than in the 2 other melanoma cell lines and, in addition, among the 9 selected mAbs, rendomab-B4 displayed the best apparent affinity for UACC-257 (Fig. 1 C), this cell line was chosen for further characterization of the fine binding properties and pharmacological effects of the antibody on tumor cells. Fig. 4B shows the saturable binding on UACC-257 cells of labeled ET- 3, which is a specific agonist of ETB, known to play a leading role in melanoma development. This binding is characterized by an EC50 value of 3.8 ± 0.1 nM, which is in the same order of magnitude as that obtained in CHO-ETB cells (Fig. 2B). Competition experiments revealed that, as expected, ET-3 and BQ788 were able to fully inhibit ET-3-FAM binding, while control antibody and BQ123 were without any effect (Fig. 4C).

Moreover, as already observed in CHO-ETB cells, rendomab- B4 was not able to reduce ET-3- FAM in UACC-257 cells, confirming that rendomab-B4 is not a competitor of ET-3 binding on hET_B.

Rendomab-B4 is internalized in UACC-257 cells

Given that ETs-targeting-rendomab-B4 binds UACC-257 cells with high affinity, and that ETB is known to have high internalization and turnover rates (Oksche A. et al, 2000; Bremnes T. et al, 2000), the internalization of the rendomab-B4 was studied. Internalization of a mAb is a crucial property because it allows uptake of a cytotoxic drug coupled to the mAb into cancer cells expressing the target.17,29 Cytometry experiments were carried out to study the internalization of ETB in UACC-257 cells (Fig. 5A). Because no competition was observed between ET-3 and rendomab-B4 binding on UACC-257 (Fig. 4C), rendomab-B4 was used to quantify receptors present at the surface of the cells after incubation of living adherent cells with ET-3 in different conditions. The maximal binding was determined by incubating the cells with ET-3 for h at 4°C, a condition where internalization is blocked. Incubation of the cells with ET-3 for 1 h at 37°C, a temperature for which internalization can occur, led to a decrease of the fluorescent signal by about 70%. This signal decrease reflects a massive ET-3-mediated internalization of ETB.

We also examined the internalization of the rendomab-B4 itself, and the results are shown in Fig. 5B. To avoid internalization of the receptor and to obtain the maximal binding, adherent UACC- 257 cells were first incubated for 3 hours at 4°C with the rendomab-B4. To allow the internalization, the cells were incubated for 3 hours at 37°C in the presence of the antibody. This resulted in a signal decrease by about 40%. Moreover, when ET-3 was added to the cells and incubated for one additional hour at 37°C, still in the presence of rendomab-B4, the signal was further reduced by about 80%. In control experiments, no binding was observed with an isotypic control anti- body (data not shown). Thus, rendomab-B4 is internalized by UACC-257 cells, and this effect is increased in the presence of the agonist ET-3, reaching a percentage of internalization higher than that obtained with ET-3 alone, suggesting that rendomab-B4 by itself is able to promote ETB endocytosis to some extent. Immunofluorescence analysis of rendomab- B4 binding on UACC-257 living cells at 4°C is shown in Fig. 5C.

The result clearly shows that rendomab-B4 bound to the UACC-257 cell surface. After 2 hours of incubation at 37°C, the labeling disappeared from the cell membrane (Fig. 5D) and appeared as a punctuate labeling in the cytoplasm, reflecting the internalization of the mAb. As expected, this labeling obtained with rendomab-B4 mainly colocalized with Early Endosome Antigen 1 (EEA1) staining (Fig. 5E and F), indicating that after 2 h ET-3 treatment, a large proportion of rendomab- B4 is located in early endosomes.

Rendomab-B4 inhibits PLC but not ERK response due to ETB receptor activation in UACC Although rendomab-B4 does not compete with ET binding on hETs, we cannot exclude the possibility that it might exert allosteric modulation on receptor activity or might reduce the number of receptors at the cell surface, as suggested by the internalization results described above. Therefore, the effect of rendomab-B4 on signaling pathways coupled to hETs in UACC-257 cells was investigated. It is well established that ETB is coupled to Gq and/or Gi families of G proteins, leading to the activation of phospholipase C (PLC) and ERK1/2 MAP kinases pathways. Data shown in Fig. 6A showed that incubation of UACC-257 cells with ET-1 , but also with ET-3, stimulated the PLC activity by about 5 times, as reflected by the increased production of inositol phosphates (InsPs). Incubation of the cells for 2 hours in the presence of control isotype antibody had no effect. In contrast, a similar treatment with the rendomab-B4 strongly reduced (by about 75%) the production of InsPs stimulated both by ET-1 and ET-3. These data indicate that rendomab-B4 is able to reduce the PLC transduction path- way without interfering with the binding of endogenous receptor ligands.

The MAP kinases pathway is known to be involved in tumor progression in melanoma. Therefore, we also investigated the potential effect of rendomab-B4 on this pathway. Our data confirmed that in UACC-257, both ET-1 and ET-3 increased ERK1/2 phosphorylation and activation, as previously shown (Asundi J. et al, 2011).

Indeed, in UACC-257, the 2 ETs induced a rapid increase of ERK1/2 phosphorylation, with a maximal response obtained at 5 to 10 min, followed by a progressive decrease (data not shown). The effect of rendomab-B4 was then tested on the activation of ERK1/2 by ET-1 and ET-3 (Fig. 6B and C). Data depicted in Fig. 6C clearly indicate that the activation of ERK1/2 was unaffected when UACC-257 were treated in the presence of rendomab-B4, as it was observed using control mAb. These results show that rendomab-B4 exhibits differential inhibitory effects on PLC and ERK pathways activated by ETs.

Rendomab-B4 inhibits melanoma cell migration induced by endothelin

It is now well established that the ET axis plays an important role in cancer progression, triggering cellular events involved in cancer cells invasion. Given this crucial role, we next studied the effect of rendomab-B4 on ET-1 -induced migration of UACC-257. Data shown in Fig. 7 demonstrated that the migration of the melanoma cell line was increased by 3-fold in the presence of ET-1. Incubation of the cells with control antibody failed to modify the cell migration due to ET-1. By contrast, rendomab-B4 completely abolished UACC-257 cells migration induced by ET-1. We verified that under similar conditions, the viability and the number of cells were not affected by

ET-1 and rendomab-B4 (data not shown). Altogether, these results show that, although rendomab-B4 does not prevent ET binding on ETB, it is able to inhibit several of its effects on melanoma cells.

BIBLIOGRAPHIC REFERENCES

Allard B, Priam F, Deshayes F, Ducancel F, Boquet D, Wijkhuisen A, Couraud JY. Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor. DNA Cell Biol 2011 ; 30:727-37.

Allard B, Wijkhuisen A, Borrull A, Deshayes F, Priam F, Lamourette P, Ducancel F, Boquet D, Couraud JY. Generation and characterization of rendomab-B1 , a monoclonal antibody displaying potent and specific antagonism of the human endothelin B receptor. MAbs 2013; 5:56-69.

Asundi J, Lacap JA, Clark S, Nannini M, Roth L, Polakis P. MAPK pathway inhibition enhances the efficacy of an anti-endothelin B receptor drug conjugate by inducing target expression in melanoma. Mol Cancer Ther 2014; 13:1599-610.

Asundi J, Reed C, Area J, McCutcheon K, Fernando R, Clark S, Luis E, Tien J, Firestein R, Polakis P. An antibody-drug conjugate targeting the endothelin B receptor for the treatment of melanoma. Clin Cancer Res 2011 ; 17:965-75.

Berger Y, Bernasconi CC, Juillerat-Jeanneret L. Targeting the endothelin axis in human melanoma: combination of endothelin receptor antagonism and alkylating agents. Exp Biol Med (Maywood) 2006; 231 :1 111-9.

Bremnes T, Paasche JD, Mehlum A, Sandberg C, Bremnes B, Attramadal H. Regulation and intracellular trafficking pathways of the endothelin receptors. J Biol Chem 2000; 275:17596-604.

Kefford RF, Clingan PR, Brady B, Ballmer A, Morganti A, Hersey P. A randomized, double-blind, placebo-controlled study of high-dose bosentan in patients with stage IV metastatic melanoma receiving first-line dacarbazine chemotherapy. Mol Cancer 2010; 9:69.

Kondoh et al, 1990 ("Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization", BBRC, vol. 172, pages 503-510

Lahav R. Endothelin receptor B is required for the expansion of melanocyte precursors and malignant melanoma. Int J Dev Biol 2005; 49:173-80.

Laune D, Molina F, Ferrieres G, Villard S, Bes C, Rieunier F, Chardes T, Granier C. Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contribute to antigen binding. J Immunol Methods 2002; 267:53-70.

Maguire JJ, Davenport AP. Endothelin receptors and their antagonists. Semin Nephrol 2015; 35:125-36.

Oksche A, Boese G, Horstmeyer A, Papsdorf G, Furkert J, Beyermann M, Bienert M, Rosenthal W. Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin- 1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein. J Cardiovasc Pharmacol 2000; 36:S44-7.

Proietti I. et al. BRAF Inhibitors: Molecular Targeting and Immunomodulatory Actions. Cancers 2020, 72(7), 1823.

Raymond MN, Robin P, De Zen F, Vilain G, Tanfin Z. Differential endothelin receptor expression and function in rat myometrial cells and leiomyoma ELT3 cells. Endocrinology 2009; 150:4766- 76. Recondo G, Diaz Canton E, de la Vega M, Greco M, Valsecchi ME. Therapeutic options for HER- 2 positive breast cancer: Perspectives and future directions. World J Clin Oncol 2014; 5:440-54.

Rosand, L., Spinella, F., Bagnato, A., 2013. Endothelin 1 in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 13, 637-651 .

Russo A, Ficili B, Candido S, Pezzino FM, Guarneri C, Biondi A, Travali S, McCubrey JA, Spandidos DA, Libra M. Emerging targeted therapies for melanoma treatment (review). Int J Oncol 2014; 45:516-24.

Shihoya, W., Nishizawa, T., Okuta, A., Tani, K., Dohmae, N., Fujiyoshi, Y., Nureki, O., Doi, T., 2016. Activation mechanism of endothelin ETB receptor by endothelin-1 . Nature 537, 363.

Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, Brahmer JR, Lawrence DP, Atkins MB, Powderly JD, et al. Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol 2014; 32:1020-30.

Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014; 515:568-71.

Weathers, S.-P., Rood-Breithaupt, J., de Groot, J., Thomas, G., Manfrini, M., Penas-Prado, M., Puduvalli, V.K., Zwingelstein, C., Yung, W.K.A., 2021. Results of a phase I trial to assess the safety of macitentan in combination with temozolomide for the treatment of recurrent glioblastoma. Neurooncol Adv 3, vdab141 .

Wouters, J., Hunger, R.E., Garrod, T., Dubuis, B., Hunziker, T., Oord, J.J. van den, Coutre, R.L., 2015. First-in-Human Proof-of-Concept Study: Intralesional Administration of BQ788, an Endothelin Receptor B Antagonist, to Melanoma Skin Metastases. The Oncologist 20, 1121 — 1122.

Yamaguchi et al, 2004 ("Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells", Biotechnology Letters, vol. 26, pages 293-299)

Claims

1. A specific antibody against the human endothelin receptor sub-type B (ETB) expressed by human melanoma cells, said antibody comprising: a) a light chain comprising three CDRs of the sequences SEQ ID NO:1 , 2 or 3, or having a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequences SEQ ID NO:1 , 2 or 3 and b) a heavy chain comprising three CDRs of the sequences SEQ ID NO: 4, 5 or 6, or having a sequence of at least 80%, preferably 85%, 90%, 95% and 98% identity with sequences SEQ ID NO: 4, 5 or 6, or an antigen-binding fragment thereof.

2. The antibody or fragment of claim 1 , comprising: a) a light chain variable domain (VL) of sequence SEQ ID NO: 7, or an amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO: 7 and b) a heavy chain variable domain (VH) of sequence SEQ ID NO: 8, or an amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO:8.

3. The antibody or fragment according to any of claims 1 to 2, wherein the dissociation constant (KD) of said antibody or fragment with human ETB is subnanomolar.

4. The antibody or fragment according to any of claims 1 to 3, said antibody being a chimeric antibody comprising a light chain variable domain (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 having respectively the amino acid sequence SEQ ID NO: 1 , 2 and 3, and a heavy chain variable domain (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 having respectively the amino acid sequence SEQ ID NO: 4, 5 and 6, preferably comprising a light chain variable domain (VL) having the amino acid sequence SEQ ID NO:7 and a heavy chain variable domain (VH) having the amino acid sequence SEQ ID NO:8.

5. The antibody or fragment according to any of claims 1 to 4, said antibody being a humanized antibody comprising a light chain variable domain (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 having respectively the amino acid sequence SEQ ID NO: 1 , 2 and 3, and a heavy chain variable domain (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 having respectively the amino acid sequence SEQ ID NO: 4, 5 and 6.

6. The antibody or fragment according to any one of claims 1 to 5, wherein it is a full-human antibody comprising a light chain comprising the CDR-L1 , CDR-L2 and CDR-L3 having respectively the amino acid sequences SEQ ID NO: 1 , 2 and 3; and a heavy chain comprising CDR-H1 , CDR-H2 and CDR-H3 having respectively the amino acid sequences SEQ ID NO: 4, 5 and 6.

7. The antibody or fragment according to any one of claims 1 to 6, wherein it is an lgG1/kappa type immunoglobulin.

8. The antibody or fragment according to any one of claims 1 to 7, wherein it is a monoclonal antibody.

9. The antibody or fragment according to any one of claims 1 to 8, wherein it is produced by the hybridoma deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS CEDEX 15) on 14 march 2023 under the accession number CNCM I-5937.

10. The antibody or fragment according to any one of claims 1 to 9, wherein it is coupled with a toxin molecule, a cytotoxic group, a label, or an effector group.

11. A pharmaceutical composition comprising the antibody or fragment according to any one of claims 1 to 10, and a pharmaceutically-acceptable carrier.

12. The antibody or fragment according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11 , for use as a medicament.

13. The antibody or fragment according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 12, for use in the treatment of melanoma.

14. The hybridoma deposited at the CNCM (Collection Nationale de Cultures de

Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS CEDEX 15) on 14 March 2023 under the accession number CNCM I-5937.

15. A method for producing the antibody according to claim 1-10, or antigen-binding fragments thereof, said method comprising the following steps: a) culturing the hybridoma deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, F-75724 PARIS CEDEX 15) on 14 March 2023 under the accession number CNCM I-5937 in a culture medium and under appropriate conditions; and b) recovering said antibody from the culture medium of said hybridoma.