Anti-AMHR2 antibodies and uses for the prevention and treatment of polycystic ovary syndrome

Abstract

In the present invention, inventors shows that exposure to high AMH levels during mini-puberty in mice causes PCOS-like reproductive and metabolic defects in both sexes. Inventors designed and developed several neutralizing antibodies against AMH receptor 2 (AMHR2) and discovered that administering the neutralizing antibody Ha13 during mini-puberty to a preclinical model of PCOS termed PAMH, which retains high circulating AMH levels, prevents the appearance of reproductive and metabolic abnormalities in adulthood. These findings suggest that exposure to elevated serum AMH levels during early postnatal life plays a causal role in the pathophysiology of PCOS. They also demonstrate the promise of AMHR2-targeting therapy in preventing both the onset and inheritance of PCOS-traits. Accordingly, the invention relates to new neutralizing antibodies that specifically binds with high affinity AMHR2, especially the epitope region involved in the interaction with the natural ligand AMH. Thus, these specific antibodies can be used for the prevention and / or therapy of polycystic ovary syndrome (PCOS).

Description

[0001] ANTI-AMHR2 ANTIBODIES AND USES FOR THE PREVENTION AND TREATMENT OF POLYCYSTIC OVARY SYNDROME

[0002] FIELD OF THE INVENTION:

[0003] The invention relates to new antibodies that specifically binds anti-Mullerian hormone receptor type 2 (AMHR2), especially the epitope region involved in the interaction with its natural ligand the anti-Mullerian hormone (AMH). These specific antibodies can be used for therapy of Polycystic Ovary Syndrome (PCOS).

[0004] BACKGROUND OF THE INVENTION:

[0005] Polycystic ovary syndrome (PCOS) is a highly prevalent endocrine condition, affecting up to 13% of women of reproductive age worldwide, and is characterized by a range of endocrine, reproductive, metabolic and psychological abnormalities1,2. The origin of PCOS is largely unknown, leaving treatment limited to symptom management without a cure or mechanism-based options3. Thus, there is an urgent need to design effective therapeutic strategies aimed at preventing, curing and / or alleviating the long-term health-related consequences of PCOS. The international PCOS guideline2endorses the Rotterdam criteria, requiring two of these features: hyperandrogenism, irregular cycles (oligo-anovulation), and polycystic ovarian morphology (PCOM) or elevated AMH. Hyperandrogenism is a common feature among women with PCOS, affecting nearly 80% of women diagnosed using the Rotterdam definition4. The aberrant PCOS hormonal milieu inhibits follicular maturation, causing excess of small antral follicles and ovulatory disturbance5. Pre-antral and small antral production of AMH by granulosa-cell is enhanced in the PCOS ovary, with serum concentrations of AMH two to three times above normal6,7. Indeed, circulating levels of AMH are elevated in > 90% of women with PCOS, reliably predicting PCOM8.

[0006] AMH, a Transforming-Growth Factor (TGF)-beta family member9, is produced by ovarian granulosa cells and released into circulation, suggesting hormonal functions in tissues with the AMHR2 receptor10'12.

[0007] PCOS has a strong heritable component13'17which might relate to interactions between genetic and epigenetic factors18, the latter induced by the aberrant maternal-fetal environment in PCOS. Among factors that have been proposed to predispose to PCOS adult manifestation is exposure during fetal life to excess of maternal androgens and / or AMH17,19. Consistently, women with PCOS retain higher circulating levels of both hormones during pregnancy than inpregnant women without PCOS5,20. To model these clinical findings, we previously developed a preclinical PCOS mouse model (PAMH) based on late-gestation AMH exposure21, replicating key reproductive, neuroendocrine, and metabolic traits of human PCOS19,21,22. However, the impact of AMH on early postnatal PCOS programming remains unknown. Interestingly, clinical studies and recent meta-analyses have shown that AMH levels are higher in daughters and sons bom to PCOS mothers from mini-puberty until the peripubertal period compared to daughters and sons born to non-PCOS women13,23'25. Mini-puberty refers to the transient gonadindependent activity of the hypothalamic-pituitary -gonadal (HPG) axis that occurs in both boys and girls shortly after birth26. According to preclinical data, mini-puberty could play an important role in the maturation of the HPG axis and reproductive function in both sexes27,28.

[0008] Herein, inventors developed an animal model mimicking PCOS-related reproductive and metabolic abnormalities. Excess AMH exposure during mini-puberty programmed females to develop PCOS-like phenotypes and males to exhibit metabolic features in adulthood. Blocking AMH signaling with an anti-AMHR2 antibody during this period prevented the manifestation of defects in adulthood and their transmission to future generations. Chronic anti-AMHR2 treatment of adult PCOS-like animals corrected neuroendocrine and reproductive alterations linked to PCOS, thus pointing out that this pharmacological agent is a promising candidate for treating PCOS.

[0009] SUMMARY OF THE INVENTION:

[0010] The present invention provides for an anti AMHR2 antibody, wherein said antibody binds to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1) of the AMHR2 protein.

[0011] In particular embodiment, said anti AMHR2 antibody is a neutralizing antibody.

[0012] The invention further relates to a therapeutic method of Polycystic ovary syndrome (PCOS) using the anti AMHR2 antibody according to the invention.

[0013] DETAILED DESCRIPTION OF THE INVENTION:

[0014] Polycystic Ovary Syndrome (PCOS), the most common endocrinopathy in women, causes significant reproductive and metabolic comorbidities, with no current cure. Gestational androgen and anti-Mullerian hormone (AMH) excess are linked to PCOS, and prenatal aberrant exposure to these hormones induces PCOS-like traits in animal models. However, whether the AMH effects on PCOS programming could extend to early postnatal life remains unknown. Clinical observations show higher AMH levels during mini-puberty in infants of mothers withPCOS, but whether this contributes to PCOS development is uncertain. Here inventors shows that exposure to high AMH levels during mini-puberty in mice causes PCOS-like reproductive and metabolic defects in both sexes. An example of a neutralizing antibody targeting AMH receptor 2 (AMHR2) prevented these defects when administered during mini-puberty and alleviated symptoms when given in adulthood. These findings highlight the causal role of elevated AMH in PCOS and suggest AMHR2-targeting therapy as a potential preventive or therapeutic approach

[0015] More precisely, inventors designed and developed several neutralizing antibodies against AMH receptor 2 (AMHR2) and discovered that administering the neutralizing antibody, named Hal3, during mini-puberty to a preclinical model of PCOS termed PAMH19,21,22, which retains high circulating AMH levels, prevents the appearance of reproductive and metabolic abnormalities in adulthood. These findings suggest that exposure to elevated serum AMH levels during early postnatal life plays a causal role in the pathophysiology of PCOS. They also demonstrate the promise of AMHR2-targeting therapy in preventing both the onset and inheritance of PCOS-traits.

[0016] Furthermore, and as demonstrated in Experimental section (Example 1 and 2), with a therapeutic goal in mind, inventors designed several anti-AMHR2-blocking antibodies that targets an epitope that, upon computational modelling, was found to interact with specified amino acids in the ligand-binding pocket of the AMHR2. The assumption was made that the binding of the antibody to that site would prevent entry of active AMH into this restricted pocket, and hence, reduce AMH action. Inventors selected and functionally validated a monoclonal antibody both in vitro, in an immortalized murine ovarian granulosa cell line, previously shown to be a suitable model to study AMH signalling as these cells retain AMHR2 expression and endogenous secretion of AMH40,41, and ex vivo, by electrophysiological recording of GnRH neurons in hypothalamic slice preparations of PAMH mice, which we previously showed to display persistent GnRH hyperactivity52. Proof-of-principle in vivo studies show that the inhibition of AMH action, via pharmacological blockade using the epitope-specific AMHR2 antibody Hal3, results in benefits to the reproductive, endocrine, and metabolic systems in a previously validated PCOS-like mouse model (PAMH)19,21’22. Indeed, this treatment, when applied during mini-puberty as well as during adulthood, was effective in preventing the manifestation of all reproductive and metabolic cardinal defects of PCOS in adulthood.

[0017] Importantly, the epitope-specific AMHR2 antibody was observed to accumulate in both the ovaries and GnRH neurons, indicating that its action is targeted to these key sites. Thus, thebeneficial effects of this antibody appear to operate through both peripheral and central mechanisms. Peripherally, the antibody likely exerts its effects on the ovaries, counteracting the aberrant AMH signaling. Indeed, ovarian over-secretion of AMH, together with elevated testosterone, is known to promote pre-antral follicle survival with AMH inhibition of FSH-induced antral follicle growth53, and to contribute to oligo-anovulation, polycystic ovarian morphology and type-2 diabetes in women with PCOS54In addition, in vitro cultures of rat preantral follicles showed that AMH inhibits FSH-induced aromatase expression and activity55, contributing to hyperandrogenism and, in several species, it has also been reported that AMH reduces aromatase (CYP19A1) gene expression56,57Altogether, these data point out a tenable mechanism underlying the beneficial effects of this pharmacological approach. Here, it is proposed a model in which the epitope-specific AMHR2 antibody protects against PCOS development by inhibiting ovarian androgen production and rescuing normal ovulation.

[0018] Finally, inventors of the Hal 3 epitope-specific AMHR2 monoclonal antibody, generated a Hal 3 scFv antibody (see example 3).

[0019] Antibodies according to the invention

[0020] Epitope

[0021] The present invention provides for an isolated anti-AMHR2 antibody, wherein said antibody binds to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO: 1) of the AMHR2 protein.

[0022] In particular embodiment said anti AMHR2 antibody is a neutralizing antibody (inhibit the interaction between AMHR2 and its ligand AMH).

[0023] In a particular embodiment said antibody binds to AMHR2 protein with a KD of with a KD of 50nM or less, 40nM or less, 30nM or less, 20nM or less, lOnM or less.

[0024] According to the present invention, “antibody” or “immunoglobulin” have the same meaning, and will be used equally in the present invention. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen, or single chains thereof. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies, antibody fragments, and fusion protein comprising an antigen-binding portion of an antibody.

[0025] In naturally occurring antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two typesof light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have each three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0026] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0027] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutant versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis.

[0028] The term "recombinant antibody", as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a immunoglobulin gene, sequences to other DNA sequences.

[0029] The term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.

[0030] According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains substantially the same CDRs of a non-human antibody.

[0031] As used herein the term “humanized” refers to a antibody of the invention wherein an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH and VL domain has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VH and VL sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a variable heavy chain (VH) from a conventional chain antibody from a human being. Methods for humanizing antibodies are well known in the art. Typically, the humanizing substitutions should be chosen such that the resulting humanized antibodies still retain the favourable properties of antibodies of the invention. The one skilled in the art is able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions. For example, the antibodies of the invention may be suitablyhumanized at any framework residue depicted in Table 1 provided that the antibodies remain soluble and do not significantly lose their affinity for AMHR2 .

[0032] The term "antibody fragment" refers to a fragment of an antibody which contains the variable domains comprising the CDRs of said antibody. The basic antibody fragments include Fab, Fab', F(ab')2 Fv, scFv, dsFv, diabodies, tribodies, or tetrabodies and the like. For example of antibody fragment see also for review, Holliger et al Nature Biotechnology 23, issue 9 1126 - 1136 (2005), which is included herein by reference.

[0033] The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.

[0034] The term “F(ab')2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

[0035] The term “Fab1” refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.

[0036] A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. “dsFv” is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

[0037] The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0038] By "purified" and "isolated" it is meant, when referring to an antibody according to the invention or to a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biologicalmacromolecules of the same type are present. An “isolated” nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0039] As used herein, "neutralizing antibody" according to the invention refers to an antibody, for example, a monoclonal antibody or a scFv antibody, capable of binding specifically to AMHR2 (anti-Mullerian hormone receptor type 2) to act as an antagonist in order to prevent and / or to compete the binding of the natural agonist Anti-Mullerian Hormone (AMH) to the AMHR2.

[0040] By "antagonist" or "receptor antagonist" is meant a natural or synthetic compound that has a biological effect opposite to that of an agonist. An antagonist binds the receptor and blocks the action of a receptor agonist by competing with the agonist for receptor. An antagonist is defined by its ability to block the actions of an agonist.

[0041] In one another embodiment, the anti-AMHR2 neutralizing antibody according to the invention, binds to the AMHR2 receptor and blocks the biological effect of AMH on AMHR2. Such a neutralizing antibody can act by occupying the ligand binding site or a portion thereof of the AMHR2 (corresponding to 28 amino acid peptide (GCESLHCDPSPRAHPSPGSTLFTCSCGT) (SEQ ID NO: 1), spanning the N-terminal region of AMHR2 involved in the interaction with AMH, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced. To identify an anti-AMHR2 neutralizing antibody able to block the biological effect between AMH on AMHR2, a test based on the effect of the neutralizing antibody candidate on the inhibition of autocrine stimulation of the specific AMH-AMHR2 signaling pathway (through phosphorylation of SMAD1 / 5 assay) as explained in the examples may be used, such specific signaling pathway leading to PCOS-like reproductive and metabolic defects.

[0042] As used herein, an antibody that binds to an “epitope” of AMHR2 protein, refers to an antibody that binds to said AMHR2 protein, in an in vitro assay for example in a Western blot or in vivo assay. In specific embodiments, the antibody of the invention binds to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1) spanning the N-terminal region of AMHR2 involved in the interaction with AMH and showing high homology among different mammalian species (human, mouse, and rats).Antibody binding to AMHR2 can be assayed by conventional methods known in the art. The mature form of AMHR2 protein, is preferably used for assaying antibody binding to the epitope of AMHR2 protein. Many different competitive binding assay format(s) can be used for determining epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such as radioimmunoassay, ELISA, “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York). For example, the BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed. An example of a suitable ELISA assay is also described in the Example below.

[0043] As used herein, the term "Affinity" refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, the stronger the affinity. Affinity can be determined by measuring KD. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka(i.e. Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A method for determining the KD of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore® system.

[0044] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

[0045] The percent identity between two amino acid sequences can be determined using the algorithm of E. Myers and W. Miller (Comput. Appl. Biosci. 4: 1 1-17, 1988) which has been incorporated into the ALIGN program. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:443- 453, 1970) algorithm which has been incorporated into the GAP program in the GCG softwarepackage. Yet another program to determine percent identity is CLUSTAL (M. Larkin et al., Bioinformatics 23:2947-2948, 2007; first described by D. Higgins and P. Sharp, Gene 73:237-244, 1988) which is available as stand-alone program or via web servers (see http : / / www. clustal . org / ) .

[0046] The percent identity between two nucleotide amino acid sequences may also be determined using for example algorithms such as the BLASTN program for nucleic acid sequences using as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0047] The terms "cross-block", "cross-blocked" and "cross-blocking" are used interchangeably herein to mean the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to AMHR2 in a standard competitive binding assay.

[0048] The ability or extent to which an antibody is able to interfere with the binding of another antibody or binding molecule to AMHR2, and therefore whether it can be said to cross-block according to the invention, can be determined using standard competition binding assays. One suitable assay involves the use of the Biacore technology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-blocking uses an ELISA-based approach as described in the Example (see Section 1 of the Example).

[0049] For example, an antibody is defined as cross-blocking in an ELISA assay, if the solution phase anti-AMHR2 antibody is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the AMHR2 detection signal (i.e. the amount of AMHR2 bound by the coated antibody) as compared to the AMHR2 detection signal obtained in the absence of the solution phase anti-AMHR2 antibody (i.e. the positive control wells).

[0050] The term “AMHR2 ”, also known as Mullerian Inhibiting Substance Type II Receptor, ( or AMHR, MISR2, MISRII, MRII, anti -Mullerian hormone receptor type 2) is a receptor for the anti -Mullerian hormone (AMH). Furthermore, anti-Mullerian hormone receptor type 2 is a protein in humans that is encoded by the AMHR2 gene (Gene ID: 269) AMHR2 belongs to TGF-P type II receptor family and adopts the characteristic three-finger toxin fold. However, it displays a unique extended finger 1 loop (see image) that is critical to effectively binding its ligand, AMH. The palm region and other fingers are also implicated in binding to AMH, but are relatively unremarkable when compared to other TGF-P type II receptors. As such, these interactions do not contribute significantly to the observed ligand specificity for AMH like thefinger 1 loop. The AMHR2 gene is present in both men and women. AMHR2 is a Type 2 receptor that binds AMH (Anti-Mullerian hormone). This hormone is responsible for Mullerian Duct regression in vertebrates once the SRY gene has been expressed. High circulating AMH continues on after testis development and is secreted from the Sertoli Cells. AMH binding to the AMHR2 in mammals causes regression of the oviducts, uterus, and upper 2 / 3 of the vagina. The protein sequence of human AMHR2 protein, and its isoforms, may be found in Uniprot database with the following access numbers: Q16671.

[0051] Neutralizing anti-AMHR2 antibodies of the invention

[0052] As described in experimental section, inventors reported the generation, structure, and function of epitope-specific AMHR2 -blocking antibodies.

[0053] Based on the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2, they identified one peptide sequence, GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1), as one of the most promising to produce antibodies with blocking-function activity. Indeed, this peptide presents 100% homology with the human and primate sequences and 92% and 89% homology, respectively, for the rat and mouse AMHR2 sequences (Figure 3B) The synthesized peptide was used to immunize mouse females and to generate 15 monoclonal anti-AMHR2 antibodies (2 different immunizing peptides were tested: see example 2).

[0054] An ELISA test with the unconjugated peptides was carried out on the serum of the mice to identify the animal with the higher antibody titer for each peptide and one peptide sequence, GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1) was selected. To further confirm the Clone 1 peptide 3 (producing anti-AMHR2 antibody) ability to inhibit AMH-AMHR2 interaction, the purified monoclonal antibodies were again tested, using the ovarian granulosa cell line (KK1) model, characterized by an autocrine stimulation of the specific AMH-AMHR2 signalling pathway. Each purified antibody was tested to evaluate its ability of inhibiting the phosphorylation (thus the activation) of SMAD proteins, one of the key protein family characterizing the AMH-AMHR2 downstream cascade

[0055] A specific monoclonal anti-AMHR2 antibody, named Hal3, was selected, with a KD of 37.1 nM, suggesting a relatively strong binding affinity between the ligand and its receptor). Finally, from monoclonal Hal 3 antibody a ScFv antibodies was produced.

[0056] The inventors have cloned and sequenced the variable domain (VL) of the light chain, and the variable domain (VH) of the heavy chain of the murine monoclonal antibody HA13. The location of the sequences encoding the complementarity determining regions (CDRs) of said antibody have been determined with reference to other antibody sequences (Kabat EA etal., 1991). This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.

[0057] These sequences derived from mAb Hal3 are described below in Table 1 (for the heavy chain and the light chain).

[0058] Table 1 Protein sequences of Hal3 variable domains. Complementarity-determining regions (CDRs) present in the variable domains are highlighted (CDR1, CDR2, and CDR3 in bold) (analysis with Kabat scheme) and nucleic acid sequence. VH: heavy chain variable domain; VL: light chain variable domain:

[0059]

[0060]

[0061] The CDRs sequences of Ha 13 are in bold

[0062] The present invention thus relates to an isolated antibody characterized in that it binds to the epitope of AMHR2 of the invention and in that it comprises:

[0063] a heavy chain having the VH-CDR1 as set forth in SEQ ID NO:4, VH-CDR2 as set forth in SEQ ID NO: 5 and VH-CDR3 as set forth in SEQ ID NO: 6 and a light chain having the VL-CDR1 as set forth in SEQ ID NO:7, VL-CDR2 as set forth in SEQ ID NO:8 and VL-CDR3 as set forth in SEQ ID NO:9.

[0064] Other antibodies of the invention include those having amino acids that have been mutated by amino acid deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95 or 100 percent identity in the CDR regions with the CDR regions depicted in the sequences described above. In some embodiments, the antibody of the invention is a mutant variant of any one of mAb Hal3, wherein said mutant variant antibody include mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated by amino acid deletion, insertion or substitution in the CDR regions when compared with the CDR regions depicted in the sequences described above.Accordingly, the invention also provides an antibody comprising a heavy chain and / or a light chain of antibody Hal 3 wherein:

[0065] - the VH domain having at least 70, 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VH domain of the antibody Hal3 (SEQ ID NO:2) and

[0066] - the VL domain having at least 70, 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VL domain of the antibody Hal3 (SEQ ID NO:3).

[0067] -that binds to AMHR2 with substantially the same affinity as an antibody having a variable light chain domain (VL) and / or a variable heavy chain domain (VH) of the antibody Hal 3.

[0068] In one embodiment, the antibody of the present invention comprises:

[0069] a heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO:2 and

[0070] a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 3

[0071] In another specific embodiment, the invention also provides an antibody comprising a heavy chain and a light chain wherein the variable domains comprise:

[0072] - a VH-CDR1 having at least 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VH-CDR1 of the VH chain of the antibody Hal 3 and / or,

[0073] - a VH-CDR2 having at least 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VH-CDR2 of the VH chain of the antibody Hal 3 and / or,

[0074] - a VH-CDR3 having at least 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VH-CDR3 of the VH chain of the antibody Hal 3 and / or,

[0075] - a VL-CDR1 having at least 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VL-CDR1 of the VL chain of the antibody Hal 3 and / or,

[0076] - a VL-CDR2 having at least 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VL-CDR2 of the VL chain of the antibody Hal 3 and / or,

[0077] - a VL-CDR3 having at least 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the VL-CDR3 of the VL chain of the antibody Hal3,

[0078] -that specifically binds to AMHR2 with substantially the same affinity as an antibody having a variable light chain domain (VL) comprising the VL-CDR1, VL-CDR2 and VL-CDR3 of the VL domain of the antibody Hal3and / or a variable heavy chain domain (VH) comprisingthe VH-CDR1, VH-CDR2 and VH-CDR3 of the VH domain of the antibody Hal3, and more preferably with substantially the same affinity as the antibody Hal3.

[0079] Yet other antibodies of the invention include antibodies selected from the group consisting of:

[0080] (i) an antibody comprising a heavy chain having the VH-CDR1 as set forth in SEQ ID NO:4, VH-CDR2 as set forth in SEQ ID NO:5 and VH-CDR3 as set forth in SEQ ID NO: 6 and a light chain having the VL-CDR1, CDR1 as set forth in SEQ ID NO: 7, VL-CDR2 as set forth in SEQ ID NO: 8 and VL-CDR3 as set forth in SEQ ID NO: 9;

[0081] (ii) antibody which comprises the VH domain of SEQ ID NO: 2 and the VL domain of SEQ ID NO:3;

[0082] (iii) a chimeric or humanized antibody obtained from the antibody i) or ii); and, (iv) the antigen-binding fragments of any of the antibodies i) to iii) above.

[0083] In another embodiment the antibody of the invention comprises the VL domain and the VH domain of the antibody Hal 3.

[0084] In another embodiment, the antibody of the invention is a chimeric antibody, which comprises the variable domains (VL and VH) of the antibody Hal 3.

[0085] In another embodiment, the antibody of the invention is a humanized antibody comprising the CDRs of the antibody Hal 3.

[0086] The present invention thus provides antibodies comprising functional variants of the VH and VL region including FRs and / or one or more CDRs of antibody Hal3. A functional variant of a VH (FR, or CDR) used in the context of an antibody of the present invention still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or the specificity / selectivity of the parent antibody (i.e. antibody Hal3) and in some cases such an antibody of the present invention may be associated with greater affinity, selectivity and / or specificity than the parent antibody. Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discusses these methods of affinity maturation. Such functional variants typically retain significant sequence identity to the parent antibody (or VHH). The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative substitutions; for instance at leastabout 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue replacements. The sequences of CDR variants may differ from the sequence of the CDRs of the parent antibody sequences through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:

[0087] Aliphatic residues I, L, V, and M

[0088] Cycloalkenyl-associated residues F, H, W, and Y

[0089] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y

[0090] Negatively charged residues D and E

[0091] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0092] Positively charged residues H, K, and R

[0093] Small residues A, C, D, G, N, P, S, T, and V

[0094] Very small residues A, G, and S

[0095] Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E, and R

[0096] More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathic / hydrophilic properties and residue weight / size also is substantially retained in a variant CDR as compared to a CDR of Hal 3. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap= 1 and Gapextension= 1). Suitable variants typically exhibit at least about 70% of identity to the parent protein. According to the present invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence. According to the present invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.

[0097] In some embodiments, the antibody of the present invention comprises :

[0098] (a) a heavy chain wherein the variable domain comprises

[0099] a VH-CDR1 having at least 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR1 of antibody Hal3 (SEQ ID NO:4), a VH-CDR2 having at least having at least 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR2 of antibody Hal3 (SEQ ID NO:5) and

[0100] a VH-CDR3 having at least 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR3 of antibody Hal3 (SEQ ID NO:6).

[0101] (b) a light chain wherein the variable domain comprises

[0102] a VL-CDR1 having at least 16, 15, 14, 13, 12, 1110, 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VL-CDR1 of antibody Hal 3 (SEQ ID NO: 7),

[0103] a VL-CDR2 having at least having at least, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR2 of antibody Hal3 (SEQ ID NO:8) and a VL-CDR3 having at least 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR3 of antibody Hal3 (SEQ ID NO:9).

[0104] As used herein, a “Hal 3 analogue” or “Hal 3 derivative” refers to an antibody exhibiting at least the same, or better, binding to AMHR2 protein and at least one of the biological activities of an antibody Ha 13 with a VH of SEQ ID NO: 2 and with a VL of SEQ ID NO: 3. The Hal3 analogue may for example be characterized in that it is capable of inhibiting the interaction between AMHR2 and its ligand AMH.

[0105] The biological activities of the antibody of the invention are, for example, to inhibit the interaction between AMHR2 and its ligand AMH and / or inhibit the stimulation of the specific AMH-AMHR2 signaling pathway as described above (through phosphorylation of SMAD1 / 5assay). The evaluation of the antagonist action on AMHR2 allows to determine the therapeutic properties of the antibody such as the correction of cognitive impairment observed in PCOS.

[0106] Said antibodies may be assayed for specific binding by any method known in the art. Many different competitive binding assay format(s) can be used for epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York). For example, the BIACORE® (GE Healthcare, Piscataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.

[0107] In the present invention, in addition of neutralizing antibody Hal3, inventors also generate several clones (Clone 2 / 3 ; Clone 3 / 3 ; Clone 4 / 3 ; Clone 5 / 3 ; Clone 6 / 3) . All these antibodies bind to the same epitope of the AMHR2 protein (GCESLHCDPSPRAHPSPGSTLFTCSCGT: SEQ ID NO:1) and said antibodies have at least the same binding affinity than Hal 3 (see Example 2 and table 3).

[0108] Cross-blocking antibodies

[0109] In yet another embodiment, antibodies that cross-block mAb Hal 3 and / or that bind to the same epitope as mAb Hal 3.

[0110] The antibody mAb Hal 3 has been shown to bind to an original epitope of AMHR2 (see SEQ ID NO: 1), enabling specific neutralization properties. Therefore, additional antibodies can therefore be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of AMH to AMHR2), in a statistically significant manner with other antibodies of the invention, for example mAb Hal3, in standard AMHR2 binding assays. Test antibody may first be screened for their binding affinity to AMHR2, for example from murine hybridomas, or human recombinant antibody libraries, using for example phage display technologies as described below. The ability of a test antibody to cross-compete with or inhibit the binding of antibodies of the present invention to AMHR2 demonstrates that the test antibody can compete with that antibody for binding to GP EBOV; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitopeon GP EBOV as the antibody with which it competes. Examples of Biacore or Elisa-based cross-blocking assays are described in detail in the Examples.

[0111] Accordingly, in one embodiment, the invention provides an isolated antibody which cross-blocks or is cross-blocked by at least mAb Hal 3 or an antibody having the same 6 CDRs of mAb Hal 3, from binding to the AMHR2 epitope of invention,

[0112] In a particular embodiment said antibody binds to the AMHR2 epitope of invention with a KD of 200nM or less, lOOnM or less, 50nM or less, 40nM or less, 10M or less.

[0113] In another embodiment, the invention provides antibodies that bind to the same epitope as does mAb Hal 3 as described herein.

[0114] Accordingly, the invention provides an isolated antibody which cross-blocks or is crossblocked by at least mAb Hal3 or an antibody having the same 6 CDRs of mAb Hal3, from binding to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO: 1) of the AMHR2 protein.

[0115] Optionally, in order to further confirm that the above-described cross-blocking antibodies have the same properties as mAb Hal3, it can be further screened for one or more of the following properties of mAb Hal 3 :

[0116] - inhibit the interaction between AMHR2 and its ligand AMH;

[0117] - inhibit the autocrine stimulation of the specific AMH-AMHR2 signaling pathway (through phosphorylation of SMAD1 / 5 assay).

[0118] In a preferred embodiment, the antibodies of the invention are able to neutralize the interaction between AMHR2 and its ligand AMH by reducing AMH-AMHR2 signaling pathway.

[0119] Nucleic acid molecules encoding antibodies of the invention

[0120] Another aspect of the invention pertains to nucleic acid molecules that encode the antibodies of the invention. Examples of variable light chain and variable heavy chain nucleotide sequences are those encoding the variable light chain amino acid sequences of mAb Hal3, the latter sequences being derived from the Table 1.

[0121] The invention also pertains to nucleic acid molecules that derive from the latter sequences having been optimized for protein expression in mammalian cells, for example, CHO cell lines, insect cells, fungal cells or bacterial cells or other available expression systems.

[0122] The nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDStreatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In an embodiment, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector.

[0123] Nucleic acids of the invention can be obtained using standard molecular biology techniques. Once DNA fragments encoding, for example, VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to an scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed under control of a desired promoter.

[0124] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.

[0125] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as to a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.

[0126] To create an scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al., 1988 Science 242:423-426; Huston et at., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature 348:552-554).

[0127] Antibody polypeptide and derivatives

[0128] • monospecificA further aspect of the invention refers to a polypeptide comprising at least one antibody of the invention.

[0129] Typically, the polypeptide of the invention comprises an antibody of the invention, which is fused at its N terminal end, at its C terminal end, or both at its N terminal end and at its C terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein. According to the invention the polypeptides that comprise a sole antibody are referred to herein as "monovalent" polypeptides. Polypeptides that comprise or essentially consist of two or more antibodies according to the invention are referred to herein as "multivalent" polypeptides.

[0130] In some embodiments, the two or more antibodies according to the invention (“multivalent" polypeptides) can be linked to each other directly (i.e. without use of a linker) or via a linker.

[0131] The linker is typically a linker peptide and will, according to the invention, be selected so as to allow binding of the two antibodies to the same epitopes of two distinct AMHR2 proteins. Suitable linkers inter alia depend on the epitopes and, specifically, the distance between the epitopes on two distinct AMHR2 proteins to which the antibodies bind to, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine experimentation. Also, when the two antibodies that bind to two distinct AMHR2 protein may also be linked to each other via a third antibody (in which the two antibodies may be linked directly to the third domain antibody or via suitable linkers). Such a third antibody may for example be an antibody that provides for an increased half-life. For example, the latter antibody may be an antibody that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin, as further described herein. In some embodiments, two or more antibodies that bind to distinct AMHR2 proteins are linked in series (either directly or via a suitable linker) and the third (single) antibody (which may provide for increased half-life, as described above) is connected directly or via a linker to one of these two or more aforementioned antibodies.

[0132] Suitable linkers are described herein in connection with specific polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutical purposes, the linker is preferably non-immunogenic in the subject to which the anti- AMHR2 protein polypeptide of the invention is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Other examples are poly-alanine linker sequences such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly / Ser linkers of different length including (gly4ser)3 , (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0133] According to a specific embodiment, the at least two antibodies according to the invention (“monospecific multivalent" polypeptides) are connected with linkers derived from the hinge region of heavy chain antibodies.

[0134] According to the invention, the antibodies and polypeptides of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The antibodies and polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The antibodies and polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

[0135] • Multispecific

[0136] In some embodiments, the polypeptide comprises at least one antibody of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is typically also an antibody. Such a polypeptide is referred to herein as "multispecific" polypeptide; in opposition to a polypeptide comprising the same antibodies (“monospecific” polypeptide). Thus, in some embodiments, the polypeptide of the invention may also provide at least one further binding site directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope. Said binding site is directed against tothe same protein, polypeptide, antigen, antigenic determinant or epitope for which the antibody of the invention is directed against, or may be directed against a different protein, polypeptide, antigen, antigenic determinant or epitope) from the antibody of the invention.

[0137] Typically, the one or more further binding site may comprise one or more parts, fragments or domains of conventional chain antibodies (and in particular human antibodies) and / or of heavy chain antibodies. For example, an antibody of the invention may be linked to a conventional (typically human) VH or VL optionally via a linker sequence.

[0138] A "bispecific" polypeptide of the invention is a polypeptide that comprises at least one antibody directed against a first antigen (i.e. AMHR2 protein) and at least one further binding site directed against a second antigen (i.e. different from AMHR2 protein), whereas a "trispecific" polypeptide of the invention is a polypeptide that comprises at least one antibody directed against a first antigen (i.e. AMHR2 protein), at least one further binding site directed against a second antigen (i.e. different from [antigen]) and at least one further binding site directed against a third antigen (i.e. different from both i.e. first and second antigen); etc.

[0139] In some embodiments, the polypeptide is as described in W02006064136. In particular the polypeptide may consist of i) a first fusion protein wherein the CL constant domain of an antibody is fused by its N-terminal end to the C-terminal end to an antibody according to the invention (i.e. a antibody directed against AMHR2 protein) and ii) a second fusion protein wherein the CHI constant domain of an antibody is fused by its N-terminal end to the C-terminal end of a antibody directed against an antigen different from AMHR2 protein. In another particular embodiment, the polypeptide consists of a first fusion protein wherein the CHI constant domain of an antibody is fused by its N-terminal end to the C-terminal end of an antibody directed against an antigen different from AMHR2 protein and a second fusion protein wherein the CL constant domain of an antibody is fused by its N-terminal end to the C-terminal end to a antibody of the invention (i.e. AMHR2 protein).

[0140] In some embodiments, the polypeptide is a biparatopic polypeptide. As used herein, the term "biparatopic" polypeptide means a polypeptide comprising an antibody and a second antibody as herein defined, wherein these two antibodies are capable of binding to two different epitopes of one antigen (e.g. AMHR2 protein), which epitopes are not normally bound at the same time by one monospecific immunoglobulin, such as e.g. a conventional antibody or one antibody. The biparatopic polypeptides according to the invention are composed of antibodies which have different epitope specificities, and do not contain mutually complementary variable domain pairs which bind to the same epitope. They do therefore not compete with each other for binding to AMHR2 protein.In some embodiments, the two antibodies of the biparatopic polypeptide of the present invention can be linked to each other directly (i.e. without use of a linker) or via a linker.

[0141] The linker is typically a linker peptide and will, according to the invention, be selected so as to allow binding of the two antibodies to each of their at least two different epitopes of AMHR2 protein. Suitable linkers inter alia depend on the epitopes and, specifically, the distance between the epitopes on AMHR2 protein to which the antibodies bind, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine experimentation. Also, when the two antibodies that bind to AMHR2 protein may also be linked to each other via a third antibody (in which the two antibodies may be linked directly to the third domain antibody or via suitable linkers). Such a third antibody may for example be an antibody that provides for an increased half-life. For example, the latter antibody may be an antibody that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin, as further described herein. In some embodiments, two or more antibodies that bind to AMHR2 protein are linked in series (either directly or via a suitable linker) and the third (single) antibody (which may provide for increased half-life, as decribed above) is connected directly or via a linker to one of these two or more aforementioned antibodies. Suitable linkers are described herein in connection with specific polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutical purposes, the linker is preferably non-immunogenic in the subject to which the anti-AMHR2 protein polypeptide of the invention is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Other examples are poly-alanine linker sequences such as Ala-Ala-Ala. Further preferred examples of linker sequences are Gly / Ser linkers of different length including (gly4ser)3 , (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0142] Methods of producing antibodies of the invention

[0143] Methods for obtaining such antibodies are well known in the art.

[0144] Accordingly, a further object of the invention relates to a nucleic acid sequence encoding an antibody according to the invention.In a particular embodiment, the invention relates to a nucleic acid sequence encoding the VH and VL domain of the antibody of the invention (e.g. antibody Hal 3)..

[0145] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0146] The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

[0147] So, a further object of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like. Examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.

[0148] A further object of the present invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention and expressing an antibody according to the invention.

[0149] Accordingly, such recombinant host cells can be used for the production of antibodies of the invention.

[0150] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

[0151] The nucleic acids of the invention may be used to produce an antibody of the invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E.coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cell"), and the like.

[0152] The present invention also relates to a method of producing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained (iii), recovering the expressed antibody.

[0153] Pharmaceutical Composition and therapeutic methods of the invention:

[0154] A further object of the invention relates to a pharmaceutical composition comprising an antibody or a nucleic acid sequence of the invention or a vector of the invention.

[0155] A further object of the invention relates to a pharmaceutical composition comprising an antibody of the invention or a nucleic acid sequence of the invention or a vector of the invention for use in therapy.

[0156] A further object of the invention relates to a pharmaceutical composition comprising an antibody of the invention or a nucleic acid sequence of the invention or a vector of the invention for use in the treatment of PCOS.

[0157] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of developing a disease or suspected to have a disease as well as subjects who have been diagnosed as suffering from a disease or clinical condition. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimenmay include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). In its broadest meaning, the term "treating" or "treatment" refers to reversing, alleviating, inhibiting the progress of Polycystic Ovary Syndrome (PCOS). In particular, "prevention" or "prophylactic treatment" of Polycystic Ovary Syndrome (PCOS) may refer to the administration of the compounds of the present invention that prevent the symptoms of Polycystic Ovary Syndrome (PCOS).

[0158] A further object of the invention relates to a method for preventing or treating PCOS comprising administering a subject in need thereof with a therapeutically effective amount of an antibody of the invention or the nucleic acid sequence of the invention or the vector of the invention.

[0159] A further object of the invention relates to a method for preventing or treating a PCOS patient by administering an anti AMHR2 antibody of the invention comprising means for binding human AMHR2 protein or means for binding an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1) of the AMHR2 protein.

[0160] A further object of the invention relates to a method for preventing or treating PCOS comprising administering a subject in need thereof with a therapeutically effective amount of an isolated antibody, which cross-blocks or is cross-blocked by at least one antibody of the present invention from binding to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1) of the AMHR2 protein.

[0161] As used herein, the term “PCOS” or “Polycystic Ovary Syndrome” refers to a highly prevalent endocrine condition, and characterized by a wide range of endocrine, reproductive,metabolic and psychological abnormalities1,2. Polycystic Ovary Syndrome (PCOS) is the main cause of female infertility, affecting up to 13% of women of reproductive age worldwide (Dumesic et al., 2015; March et al., 2010). It is characterized by a wide range of clinical symptoms including hyperandrogenism, oligo-anovulation and, in many cases, metabolic disorders (type 2 diabetes, hypertension and cardiovascular disease) (Boyle and Teede, 2016; Dokras et al., 2017).

[0162] PCOS has a strong heritable component (Crisosto et al., 2007; Gorsic et al., 2019; Gorsic et al., 2017), as witnessed by the fact that -60-70% of daughters bom to women with PCOS will eventually manifest the disease (Crisosto et al., 2019; Risal et al., 2019). In line with that, a recent study showed that daughters of mothers with PCOS have a fivefold-increased risk of being diagnosed with PCOS later in life (Risal et al., 2019). It has been suggested that environmental factors, such as excessive androgen (Abbott et al., 2002; Franks and Berga, 2012; Padmanabhan and Veiga-Lopez, 2013; Risal et al., 2019; Walters et al., 2018b), or elevated levels of anti -Mullerian hormone (AMH) exposure (Tata et al., 2018), may be in part responsible for the development of PCOS.

[0163] In particular embodiments, the subject of the present invention suffers from PCOS and / or have been previously diagnosed (or one parent) with PCOS.

[0164] In particular embodiments, the subject diagnosed with PCOS (or born to a mother diagnosed with PCOS) to be treated with an antibody according to the invention, is a girl during the mini-puberty (between 1 and 6 month old) or a pre-adolescent girl (between 8 and 10 years old) or an adolescent girl (between 11 and 17 years old) or an adult woman (18 and more than 18 years old).

[0165] By a "therapeutically effective amount" of the antibody of the invention is meant a sufficient amount of the antibody to treat said PCOS, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibody employed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0166] For administration, the antibody of the invention or the fragment thereof is formulated as a pharmaceutical composition. A pharmaceutical composition comprising an antibody of the invention or a fragment thereof can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic molecule is combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc. The pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular. To prepare pharmaceutical compositions, an effective amount of the antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The pharmaceutical forms include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. An antibody of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0167] The antibodies of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

[0168] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0169] Combination and uses of the invention

[0170] Inventors previously developed another therapeutic approach in order to treat woman affected PCOS. This previous study relates to a gonadotropin-releasing hormone (GnRH) antagonist for its use in women affected with polycystic ovary syndrome (PCOS) for preventing the occurrence of PCOS in the offspring of the said woman. This study also relates to a gonadotropin-releasing hormone (GnRH) antagonist for its use to rescue ovulation and fertility in post-puberal PCOS affected individuals (see Tata B, et al P (2018) Elevated prenatal anti-Miillerian hormone reprograms the fetus and induces polycystic ovary syndrome in adulthood. Nature Medicine. WO2018177746 (A GnRH antagonist for use in the treatment of a women affected with polycystic ovary syndrome)

[0171] Accordingly aspect of the invention relates to a combination of (i) an anti-AMHR2 antibody according to the invention, and (ii) an GnRH antagonist, for the simultaneous or sequential use in the treatment of PCOS.

[0172] An anti-AMHR2 antibody according to the invention is previously defined above. Any GnRH antagonist described in WO201817774 are included by reference.

[0173] As used herein, a “GnRH antagonist” encompasses a compound which combines with a GnRH receptor on a cell and, at least partially, prevents a physiological response in that cell. GnRH antagonists also encompass compounds that reduce or block the GnRH-encoding gene expression, as well as compounds that reduce or block the GnRH protein production or secretion. GnRH antagonists also encompass nitric oxide as well as nitric oxide donor compounds, and molecules providing the nitric oxide effector cyclic guanosine monophosphate (cGMP) or prolonging its half life that are known in the art to modulate GnRH neuronal activity(Clasadonte et al., 2008, Endocrinology, Vol. 149(2) : 587-596). GnRH antagonists also encompass phosphodiesterase inhibitors.

[0174] For practicing the present invention, any GnRH antagonist may be used. The one skilled in the art may notably refer to the various GnRH antagonists that are known to date. The one skilled in the art may notably refer to the review article of Schultze-Mosgau et al. (2005, Expert Opinion on Investigational drugs, Vol. 14 (n°9) : 1085-1097).

[0175] In some embodiments, a GnRH antagonist consists of an antagonistic peptide analogue of GnRH resulting from multiple amino acid substitutions, mainly at positions 1, 2, 3, 6, 8 and 10, in the GnRH decapeptide (Schultze-Mosgau et al., 2005, Expert Opinion on Investigational drugs, Vol. 14 (n°9) : 1085-1097).

[0176] In some other embodiments, a GnRH antagonist is a non-peptide antagonist compound, such as those described by Shu et al. (2004, Expert Opinion on Therapeutic Patents, Vol. 14 : 187-189) or by Armer et al. (2004, Curr Med Chem, Vol. 1 : 3017-3028).

[0177] Illustratively, a GnRH antagonist may be selected in a group comprising degarelix, ganirelix, cetrorelix abarelix, elagolix, relugolix.and Linzagolix choline

[0178] Degarelix is a GnRH antagonist having the CAS reference n° 214766-78-6 and having the IUPAC condensed formula Ac-D-2Nal-D-Phe(4-Cl)-D-3Pal-Ser-Phe(4-S-dihydroorotamido)-D-Phe(4-ureido)-Leu-Lys(iPr)-Pro-D-Ala-NH2. Illustratively, degarelix is available as the pharmaceutical specialty marketed under the brand name Firmagon®.

[0179] Ganirelix is a GnRH antagonist having the CAS reference n° 129311-55-3 and having the IUPAC condensed formula Ac-D-2Nal-D-Phe(4-Cl)-D-3Pal-Ser-Tyr-D-Lys(Unk)-Leu-Lys(Unk)-Pro-D-Ala-NH2. Illustratively, ganirelix is available as the pharmaceutical specialty marketed under the brand name Orgalutran® or Antagon®.

[0180] Cetrorelix is a GnRH antagonist having the CAS reference n° 120287-85-6 and having the IUPAC condensed formula Ac-D-2Nal-D-Phe(4-Cl)-D-3Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2. Illustratively, cetrorelix is available as the pharmaceutical specialty marketed under the brand name Cetrotide®.

[0181] Abarelix is a GnRH antagonist having the CAS reference n° 183552-38-7 and having the IUPAC condensed formula Ac-D-2Nal-D-Phe(4-Cl)-D-3Pal-Ser-N(Me)Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH2. Illustratively, abarelix is available as the pharmaceutical specialty marketed under the brand name Plenaxis®.

[0182] Elagolix is a (oral) GnRH antagonist having the CAS reference n° 834153-87-6 and having the IUPAC condensed formula 4-[[(lR)-2-[5-(2-fluoro-3-methoxyphenyl)-3-[[2-fluoro-6-(trifluoromethyl)phenyl]methyl]-4-methyl-2,6-dioxopyrimidin- 1 -y 1 ] - 1 -phenylethyl]amino]butanoic acid. Illustratively, Elagolix is available as the pharmaceutical specialty marketed under the brand name Orilissa®.

[0183] Relugolix is a (oral) GnRH antagonist having the CAS reference n° 737789-87-6 and having the IUPAC condensed formula l-[4-[l-[(2,6-difhiorophenyl)methyl]-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxothieno[2,3-d]pyrimi din-6-yl]phenyl]-3-methoxyurea. Illustratively, Relugolix is available as the pharmaceutical specialty marketed under the brand name Orgovyx®.

[0184] Linzagolix Choline is a (oral) GnRH antagonist having the CAS reference n° 1321816-57-2 and having the IUPAC condensed formula 3-[5-[(2,3-difluoro-6-methoxyphenyl)methoxy]-2-fluoro-4-methoxyphenyl]-2,4-dioxo-lH-thieno[3,4-d]pyrimidine-5-carboxylate;2-hydroxyethyl(trimethyl)azanium. Illustratively, Linzagolix Choline is available as the pharmaceutical specialty marketed under the brand name Linzagolix®.

[0185] The one skilled in the art may also refer to the GnRH antagonists that are disclosed in the patent or patent application documents US 2009 / 0197813, US 2012 / 0238494, US 4,409,208, US 4,547,370, US 4,565,804, US 4,569,927 and US 4,619,914, US 7,419,983, US 8,735,401, US 10,016,433.

[0186] The above-described GnRH antagonists may be administered in the form of pharmaceutically acceptable non-toxic salts or complexes, depending of the GnRH antagonist which is considered.

[0187] In other embodiments, a GnRH antagonist may consist of a relevant micro-RNA molecule (miR) such as the miR155 or miR200, as disclosed in the European patent application filed under n° EP 16305459.6 on April 20, 2016 in the name of Inserm. Micro-RNA miR155 is described notably by O’Connell et al. (2007, Proc Natl Acad Sci U S A, Vol. 104:1604-1609). Micro-RNA miR200 is described notably by Korpal et al. (2008, J Biol Chem, Vol. 283 (n°22) : 14910-14914). GnRH antagonists also encompass compounds, and especially antagomir nucleic acids, that block the binding of miR155 to a Cebpb-encoding nucleic acid sequence or that block the binding of miR200 to a Zeb 1 -encoding nucleic acid sequence.

[0188] Generally, the GnRH antagonists specified above are available as a powder for injectable formulation and a solvent for reconstitution of the powder (with the exception of Elagolix, Relugolix and Linzagolix choline, which are orally active GnRH antagonist) . The powder for injectable formulation is a lyophilisate containing the said GnRH antagonist and mannitol, and the solvent consists of water for injection provided in appropriately sized vials.Another GnRH antagonists are developed in several clinical phases. Accordingly a GnRH antagonist may be selected in a group comprising, Merigolix (CAS N° 1454272-94-6), SHR-7280 (Yi XU et al Front Pharmacol. 2022 Nov 23; 13: 1027648) CMS-D002.

[0189] In preferred embodiments, a GnRH antagonist, or alternatively a combination of two or more GnRH antagonists, is the sole active ingredient or combination of active ingredients that is administered for the purpose of preventing or treating a female individual against PCOS, i.e. either (i) for the purpose of preventing or treating PCOS in a female individual affected, or susceptible to be affected, with this pathology or (ii) for the purpose of preventing the occurrence of PCOS in the offspring of a pregnant woman affected with PCOS.

[0190] FIGURES:

[0191] Figure 1. Exposure to high AMH during mini-puberty induces PCOS-like reproductive features in female mice. (A) Illustration representing the mini-puberty AMH model. (B) Representative estrous cyclicity of mCNTR and mAMH mice during 15 consecutive days. D: Diestrus, P: Proestrus, E: Estrus. (C) Analysis of the percentage of the time spent in each estrous cycle stage over 15 days. The estrous cycle stage’s name is displayed on the x-axis. (mCNTR, n = T, mAMH, n = T, age: P60-P80 for both groups). (D) Number of complete estrous cycles during 15 days (mCNTR, n = T, mAMH, n = T, age: P60-P80 for both groups).

[0192] (E) Number of pups per litter over 2 months in mCNTR, n = 11 and mAMH, n = 11; age: P60-P120. (F) Mean number of corpora lutea (CL) in mCNTR, n = 5 and mAMH, n = 6 (age: P180 for both groups). (G) Mean circulating levels of gonadal sex steroids in diestrous female mice measured by mass spectrometry coupled with gas chromatography (GC-MS). Testosterone, androstenedione (delta4-Dione), estradiol (E2) and progesterone (P4) were measured in P90 mCNTR and mAMH females n = 6 for each animal group).

[0193] In all bar graphs, data are presented as mean ± s.d. *P < 0.05, **P < 0.01, ***p < 0.005, **** < 0.0001 and ns, not significant, using a two-way ANOVA followed by Holm-Sidak’s multiple comparisons post hoc test (C), a two-sided paired Mann-Whitney U test (D), a paired Student’s Ltest (E,) or an unpaired Student’s Ltest (F,G)

[0194] Figure 2 Exposure to high AMH during mini-puberty drives PCOS-like metabolic defects in female mice. (A) Body weight of mCNTR (n = 10; 6 months old) and mAMH adult diestrous females (n = 10; 6 months old). (B-D) Body composition of mCNTR (n = 7; 6 months old) and mAMH adult females (n = 7; 6 months old). (E) Curve representing glycaemia (glucose levels) during a glucose tolerance test (GTT) in mCNTR (n = 6) and mAMH (n = 4) 6 months old females. (F) Serum insulin concentrations during the first 30 min of a glucose tolerance testin in mCNTR (n = 6) and mAMH (n = 4) 6 months old females. (G) Homeostasis model assessment of insulin resistance (HOMA-IR) index in mCNTR (n = 6) and mAMH (n = 4) 6 months old females. (H) Relative expression of Esrl, Pome, Agrpl, Kissi, Pgr and Ar mRNAs in the MBH of mCNTR and mAMH adult females n = 4 animals per group, 6 months old). (I) Mean adipocyte area in mCNTR and mAMH adult females (n = 8 animals per group, 6 months old). (J) Visceral fat mass (in mg) in mCNTR (n = 8 females, 6 months old) and mAMH adult diestrous females (n = 4 females, 6 months old).

[0195] In all bar graphs, data are presented as mean ± s.d. *P < 0.05, **P < 0.01, ***p < 0.005, ****P < 0.0001 and ns, not significant, using a paired Student’s / -test (A-D), a two-way ANOVA followed by Holm-Sidak’s multiple comparisons post hoc test (E,F), an unpaired Student’s / -test (AUC in E and F, G, I, J) or a two-sided paired Mann-Whitney U test (H).

[0196] Figure 3. Generation and functional validation of an anti-AMHR2 neutralizing antibody. (A) Schematic of the AMH-AMHR2 signaling pathway and anti-AMHR2 antibody targets. (B) Sequence of the human AMHR2 N-terminal immunizing peptide with % homology across species. Conserved residues are green; mismatches are red. (C) Schematic of the experimental workflow for generating the anti-AMHR2 antibody (Hal 3). (D) Relative Amhr2 mRNA expression in indicated tissues. The graph represents the mean expression of Amhr2 ± s.e.m. (n = 3 female mice, P90). (E) Western blot experiments for Hal3 and actin were performed in ovaries (positive control), liver and pancreas (negative controls) dissected from three adult diestrous females (P90). (F) KK1 mouse granulosa cell line was treated with or without IgM antibodies and Hal3 antibody concentrated at 50 and 100 pg / ml. Western-blot experiments were performed using anti-phospho-Smad-1-5 and P-actin antibodies. (G) Quantification of the ratio P-Smad-1-5 over P-actin. Values are represented as the mean ± s.d. Statistics were calculated by one-way ANOVA with Dunnett’s multiple comparisons test, **P < 0.005; ****P <0.0001. (H) Illustration representing the whole-cell current-clamp recording set-up. (I) Whole-cell current-clamp recording showing the spontaneous burst firing of a GnRHPOAneuron recorded from a PAMH;GnRH-GFP female mouse (P60) before, during and after bath application of Hal 3 antibody. Bottom traces show expansions of the recording at the indicated time points 1 (min 1), 2 (min 11) and 3 (min 22). (J) Firing rate (Hz) of GnRHPOAneurons (n = 8-11 cells recorded cells from n = 3-5 mice per experimental group; P60. Values are represented as mean values for the groups: PAMH, PAMH+Hal3 treatment and PAMH+washes, and as mean ± s.e.m. for the control basal groups. Statistics were calculated by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test *P < 0.05.Figure 4. Hal3 treatment during mini-puberty prevents PCOS-like reproductive and neuroendocrine defects. (A) Schematic illustrating the experimental treatment groups and the phenotypic characterization as a function of age. (B) Representative estrous cycle profiles from the 4 groups of animals (P60 from n = 3 biological replicates). (C) Number of complete estrous cycles during 15 days in the 4 groups of animals (CNTR+veh, n = 11; PAMH+vehicle, n = 11; PAMH+IgM, n = 11; PAMH+Hal3, n = 8; age: P60 for both groups). (D) Analysis of the percentage of the time spent in each estrous cycle stage in the 4 groups of mice over 15 days (CNTR+veh, n = 11; PAMH+vehicle, n = 11; PAMH+IgM, n = 11; PAMH+Hal3, n = 8; age: P60 for both groups).

[0197] In all bar graphs, data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***p < 0.0005, ****P < 0.0001 and ns, not significant, using a two-way ANOVA with Tukey’s post hoc test (C, D).

[0198] Figure 5. Hal3 treatment during mini-puberty prevents PCOS-like metabolic defects. (A) Body weight and body composition in different groups (CNTR, PAMH+vehicle, PAMH+IgM, PAMH+Hal3; P180 females). (B) Glucose tolerance test (GTT) curve with corresponding area under the curve (AUC) analysis (CNTR, n = 6; PAMH + vehicle, n = 5; PAMH+IgM, n = 8; PAMH+Hal3, n = 6). (C) Insulin tolerance test (ITT) curve after 4-hour fasting with corresponding AUC analysis. CNTR, n = 5; PAMH + vehicle, n = 6; PAMH+IgM, n = 5; PAMH+Hal3, n = 5. (D) Mean lipid droplets area in the BAT (CNTR, n = 6; PAMH, n = 7; PAMH+IgM, n = 8; PAMH+Hal3, n = 6; 6 months old females). (E) Mean rectal temperature, expressed in C°, in the indicated animal groups (CNTR, n = 11; PAMH, n = 4; PAMH+IgM, n = 4; PAMH+Hal3, n = 10; 6 months old females). (F) Mean visceral fat weight (in mg) in 6 months-old females CNTR (n = 8), PAMH (n = 7), PAMH+IgM (n = 7), PAMH+Hal3 (n = T). (G) Mean adipocyte area in the visceral WAT (vWAT) of 6 months old females CNTR (n = 8), PAMH (n = 8), PAMH+IgM (n = 8), PAMH+Hal3 (n = 8). (H) Relative densitometric bar graphs of protein levels in the WAT of 6 months old females CNTR (n = 5; grey column), PAMH (n = 8; red column), PAMH+Hal3 (n = 5; blue column). GAPDH was used to normalize protein levels. Scale bar in E: 50 pm.

[0199] In all bar graphs, data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***p < 0.0005, ****P < 0.0001 and ns, not significant, using a one-way ANOVA with Tukey’s test (A, in AUC of B and C, and in D,-G, H), or a two-way ANOVA with Dunnett’s test (B, C). In B, letters define statistical differences (a: CNTR vs. PAMH, b: CNTR vs. PAMH+IgM, c: CNTR vs. all groups). In c, letters define statistical differences (a: CNTR vs. PAMH, b: CNTR vs. PAMH+Hal3).Figure 6. Hal3 treatment during mini-puberty prevents intergenerational transmission of PCOS-like traits to female offspring. (A) Schematic illustrating the experimental design. (B) Mean circulating levels of testosterone and androstenedione (delta4-Dione) measured by GC-MS in P60 females (CNTR F2, n = 7; PAMH F2, n = 7; PAMH F2+Hal3 (Fl), n = 10; age: P60 for all groups). (C) Percentage of time spent in each estrous cycle stage over 15 days in the three groups (CNTR F2, n = 6; PAMH F2, n = 6; PAMH F2+Hal3 (Fl), n = 6; age: P60 for all groups). (D) Number of complete estrous cycles during 15 days (CNTR F2, n = 6; PAMH F2, n = 6; PAMH F2+Hal3 (Fl), n = 6; age: P60 for all groups). (E) Number of LH pulses per hour in P60 diestrous females (CNTR F2, n = 11 PAMH F2, n = 17; PAMH F2+Hal3 (Fl), n = 19). (F) Body weight at Pl 50 (CNTR F2, n = 6; PAMH F2, n = 9; PAMH F2+Hal3 (Fl), n = 9). (G) Insulin tolerance test (ITT) after 4-hour fasting at P150 (CNTR F2, n = 6; PAMH F2, n = 7; PAMH F2+Hal3 (Fl), n = 7).

[0200] In all bar graphs, data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***p < 0.0005, ****p < 0.0001 and ns, not significant, using a one-way ANOVA with Tukey’s test (b, d-f), or a two-way ANOVA with Tukey’s test (c, g). In g, Statistical differences: a: CNTR F2 vs. PAMH F2, P < 0.01; b: PAMH F2 vs. PAMH F2+Hal3, P < 0.05.

[0201] Figure 7. Hal3 chronic treatment during adulthood corrects PCOS-like neuroendocrine and reproductive defects. (A) Schematic illustrating the experimental design. (B) Analysis of the percentage of the time spent in each estrous cycle stage in the 4 groups of mice over 15 days. Statistics by two-way ANOVA followed by Tuckey’s post hoc test, **P < 0.005, * P < 0.05, ns: not statistically significant (CNTR, n = 9; PAMH, n = 10; PAMH+IgM, n = 10; PAMH+Hal3, n = 8; age: P120 for all groups). (C) Number of complete estrous cycles during 15 days in the 4 groups of animals. Statistics by one-way ANOVA followed by Tuckey’s multiple comparisons test ****P < 0.0001 (CNTR, n = 9; PAMH, n = 10; PAMH+IgM, n = 10; PAMH+Hal3, n = 8; age: P120 for all groups). (D) Mean circulating levels of testosterone and androstenedione (delta4-Dione) measured by mass spectrometry coupled with gas chromatography (GC-MS). Testosterone and delta4-Dione were measured in P120 females. Values are mean ± s.e.m; statistics by one-way ANOVA followed by Tuckey’s post hoc test, **P < 0.005, * P < 0.05 (CNTR, n = 9; PAMH, n = 10; PAMH+IgM, n = 10; PAMH+Hal3, n = 8; age: P120 for all groups)..

[0202] Figure 8. Densitometric analysis of Western Blot anti-P-SMAD / anti-GAPDH. In order to test the ability of the anti-AMHR2 antibodies to inhibit AMH-AMHR2 interaction, all the positive supernatants (derived from the ELISA screening) were tested, using a cell model, characterized by an autocrine stimulation of the specific AMH-AMHR2 signalling pathway.Each supernatant was tested to evaluate its ability of inhibiting the phosphorylation (thus the activation) of SMAD, one of the key protein characterizing the AMH-AMHR2 downstream cascade. Experiments were performed focusing at short lead time of inhibition (< 3h), after evaluating the time course of SMAD phosphorilation in the experimental model. Data of 3 independent experiments were analyzed using ImageJ, and graphs were plotted by GraphPad Prism are reported the; all the samples are significantly different from control (CONT). P = peptide number.

[0203] Figure 9. Anti AMHR2 scFv production

[0204] Panel A) WB of scFv concentrations (2,5 pg / ml-50 pg / ml) and relative controls. pSMAD has a molecular weight of about 55 kDa and Tubuline of about 50 kDa. Ctr = cells incubated with culture medium (RPMI + 10% FCS).

[0205] Panel B). Densitometric values relative to scFv concentrations (2,5 pg / ml-50 pg / ml) compared to Ctr. Ctr = cells incubated with culture medium (RPMI + 10% FCS). Statistical analysis was carried out using Student t-test. 12 pg / ml, 25 pg / ml and 50 pg / ml concentrations are statistically significant with a p-value of 0,0035; 0,0042 and 0,0001 respectively. Data are means ± SEM of three separate experiments.

[0206] Panel C). Estimation plots of 50 pg / ml scFv concentration which present the magnitude of the effects, along with a visual representation of its precision.

[0207] The X axis shows the sample of 50 pg / ml and the control (Ctr).

[0208] The left Y axis shows data with a scatter graph showing individual points. The right Y axis is scaled to show the effect of the scFv concentration.

[0209] EXAMPLE 1:

[0210] Materials and Methods

[0211] Animals

[0212] C57BL / 6J mice (Charles River, USA) were housed at 21-22°C with a 12 h light / dark cycle and provided ad libitum access to water and standard laboratory chow (9.5 mm Pelleted RM3, Special Diets Services; Competence Centre for Lab Animal Science of SAFE®; France). Mice were randomly assigned to groups of five animals per cage at the time of purchase or weaning with litters within the same treatment allocation group mixed (pseudo-random mixing based on weight) to minimize any potential bias. No data were excluded from analyses.

[0213] Postnatal anti-Miillerian hormone mouse model (mini-puberty: mAMH) C57BL / 6J mice (Charles River, USA) were injected daily intraperitoneally (i.p.) from postnatal day 12 to 14 with either 100 L of 0.01 M phosphate-buffered saline (PBS) (controlgroup) or 0.26 pg / mouse / day human recombinant AMHc (R&D Systems, rhMIS, catalogue n°1737-MS-10) diluted in PBS (mAMH group).

[0214] Prenatal anti-Miillerian hormone (PAMH) mouse model

[0215] PAMH mice were generated according to previous studies35. Pregnant adult (3-4 months) female mice were injected daily intraperitoneally (i.p.) at embryonic day (E) 16.5, 17.5, and 18.5 with either 200 pL of 0.01 M phosphate-buffered saline (PBS) only (control group) or 0.12 mg / Kg / day human recombinant AMHc (R&D Systems, rhMIS 1737-MS-10) diluted in PBS (PAMH group). Mouse pregnancy was timed considering the detection of vaginal plugs as GD 0.5. Six PAMH female mice were bred with Gnrh-Gjp1homozygous mice to generate PAMH;GnRH-GFP+ / ' mice that were used for electrophysiology experiments.

[0216] Assessment of reproductive status and blood sampling

[0217] Reproductive cycles were followed and characterized according to well-described guidelines of the mouse estrous cycle according to well-established guidelines36. Briefly, vaginal smears were collected using 10 pL of sterile saline, transferred to glass slides, and evaluated during the morning period at 8:00-9:00h (room lights were on at 7:00h). Vaginal cytology was performed with freshly collected samples using an inverted microscope with maximum microscope condenser distance allowing a better contrast for proper visualization. Estrous cyclicity was followed for 15-17 consecutive days. Blood sampling for LH and testosterone levels was conducted when mice were in diestrus. For LH-level profile studies, mice were trained to interact with the investigator and allow restraint of the tail for at least one week before the commencement of each set of experiments. A 4 pL of the whole blood sample from the tail tip was taken every 5 min (LH profile) for 2-3h. Samples were collected into Eppendorf tubes pre-loaded with 50 pL of 0.1M phosphate-buffered saline (PBS)-0.05% Tween (pH = 7.4), thoroughly mixed, and snap-frozen with dry ice and stored at -80 °C. Plasma testosterone samples were collected from the cheek or tail tip at a single point for each treatment. Samples were kept in ice for one hour followed by centrifugation at 6,8 ref for 15 minutes at 4 °C. Plasma supernatant was transferred to sterile 200-pL Eppendorf tubes and stored at -80 °C.

[0218] Hormone measurements and analysis

[0219] For this study, we used an ultrasensitive ELISA method to measure circulating LH levels in the whole blood in mice as validated previously to assess pulsatile LH release in female mice over the estrous cycle37,38. Briefly, this ELISA uses a 96-well high-affinity binding microplate (Corning) coated with 50 pL of primary capture antibody (bovine LHP subunit, 518B7; L. Sibley; University of California, UC Davis) at a dilution of 1:1000. The mouse LH-RP reference used for this assay was provided by Dr. Albert F. Parlow (National Hormone and Pituitary Program, Torrance, California, USA) and used to generate a standard curve with a twofold serial dilution from a start standard of 4 ng / mL to 0.0019 ng / mL LH-RP reference diluted in 0.2% bovine serum albumin (BSA)-O.IM PBS-0.05% Tween solution. Whole blood samples were transferred to the coated as singlets for each point of sampling and incubated for 2 h under agitation at room temperature. A rabbit LH antiserum primary antibody (AFP240580Rb; NIDDK- NHPP) was used at 1:10,000 dilution and a secondary horseradish peroxidase-conjugated antibody (goat anti-rabbit; Vector Laboratories, PI- 1000) was used at 1:10000 dilution. We used 100 pL of 1-Step Ultra TMB-Elisa Substrate Solution (ThermoFisher Scientific, catalogue n° 34028) for the final revelation step followed by a stop solution with 50 pL of 3M HC1 per well. The assay sensitivity of this LH ELISA was 0.04 ng / ml and intra-assay coefficient of variation was 3.9% and the inter-assay coefficient of variation was 8.3%. LH pulse analysis was analysed based on previous studies38 40. Briefly, mean LH levels were considered as the average of all measured values within the 2-h blood sampling protocol. LH pulse peaks were determined as a single point with a value more than 20% from 1 of the 2 previous points followed by a decrease by >10% in 1 of the next 2 subsequent points74. Next, LH pulse frequency was determined by counting the number of identified pulses per hour. The average time distance (in min) between each LH pulse peak was used to calculate the LH pulse interval. The LH pulse amplitude was calculated considering the peak of each LH pulse minus its preceding zenith values followed by a final average calculation of all amplitudes within the 2-h blood sampling protocol. Basal LH level analysis considered the average of all LH nadir and non-peak values. The area under the curve (AUC) analysis is considered the total area summed over the 2-h blood sampling protocol.

[0220] Plasma AMH levels were measured using a commercial rat and mouse ELISA kit following the manufacturer’s instructions (AnshLabs®, catalogue n° AL- 113). The assay sensitivity of this ELISA was 0.011 ng / mL as reported by the manufacturer, and the intra-assay coefficient of variation was 5.2%. Quality controls were also provided by the manufacturer.

[0221] Measurement of mouse steroid levels in serum by GC / MS

[0222] Hormonal parameters

[0223] Testosterone (T), Dihydrotestosterone (DHT), Estradiol (E2), A4-androstenedione (4-dione), and Progesterone (Prog) were measured by GC / MS. Twice charcoal dextran-stripped aged female rabbit serum was used as matrix for calibrators and quality control (QC) standards.

[0224] Sample extraction and purificationBriefly, each sample (200 pl of serum per mouse, calibration standards, quality controls, and blank matrix) was collected in a 4 ml borosilicate tube. A spiking solution of deuterated steroid internal standards (IS) (10 pl containing 0.5 ng of T-d5, 0.5 ng of DHT-d3, 50 pg of E2-d4, 2 ng of 4-dione-d7, and 3 ng of Prog-d9, except for blank matrix) (CDN Isotopes, Inc., Point-Claire, Canada), 200 pl of saline water, and 2.8 ml of 1 -chlorobutane were added to each sample. After a 2 min vortex-step followed by rapid centrifugation, the upper organic phase was collected on a conditioned Hypersep SI 500 mg SPE mini-column (Thermo Scientific). The column and adsorbed material were then washed with ethyl acetate / hexane (6 ml; 1 / 9). The second fraction containing the steroids of interest was eluted with ethyl acetate / hexane (4 ml; 1 / 1), then evaporated at 60 °C to dryness.

[0225] Derivatization reactions and determination of steroid levels in the supernatant T, DHT, and E2 were derivatized as previously described75with pentafluorobenzoyl chloride (PFBC) (Sigma-Aldrich, catalogue n° 103772-1G). Final extracts were reconstituted in 20 pl of isooctane, then transferred into conical vials for GC injection.

[0226] 4-dione, and Progesterone were derivatized with 50 pl of heptafluorobutyric anhydride (Sigma-Aldrich) and anhydrous acetone (1 / 1) mixture. Final extracts were reconstituted in 20 pl of anhydrous n-hexane, then transferred in conical vials for injection into the GC system (GC-2010 Plus, Shimadzu) using a 50 % phenylmethylpolysiloxane VF-17MS capillary column (Agilent Technologies). A TQ8050 triple quadrupole mass spectrometer (Shimadzu) equipped with a chemical ionization source (NCI), and operating in Q3 single ion monitoring (SIM) mode, was used for the detection of DHT, T, and E2, and with an electron impact source (El) operating in multiple reaction monitoring (MRM) mode for the detection of 4-dione, and Progesterone. For NCI detection, the reagent gas was methane, and the GC was performed in pulsed splitless mode with a 1 min pulsed splitless-time. The oven temperature was initially 150°C for 0.50 min, further increased to 305°C at 20°C / min and held at 305°C for 3.60 min, and then to 335°C at 30°C / min and held at 335°C for 1.7 min. The injection port and transfer line temperatures were respectively 290 and 280°C. The flow rate of helium (carrier gas) was maintained constant at 0.96 ml / min. The mass spectrometer CI source temperature was 220°C. For El detection, GC was performed in the splitless mode with a 1-min splitless-time. The temperature in the oven was initially 70°C for 1 min, further increased to 238°C at 25°C / min, and then to 261 °C at 5°C / min. The injection port and transfer line temperatures were respectively 290 and 280°C. The flow rate of carrier gas was maintained constant at 0.70 ml / min. The mass spectrometer El source was 230°C.The linearity of steroid measurement was confirmed by plotting the ratio of the steroid peak response / internal standard (IS) peak response to the concentration of steroid for each calibration standard.

[0227] Ovarian histology

[0228] Ovaries were collected from 3- to 4-month-old diestrus mice, immersion-fixed in 4% PFA solution and stored at 4 °C. Paraffin-embedded ovaries were sectioned at a thickness of 5 pm (histology facility, University of Lille 2, France) and stained with hematoxylin-eosin (Sigma Aldrich, catalogue n° GHS132, HT1103128). Sections were examined throughout the ovary. Corpora lutea (CL) were classified and quantified as previously reported76. To avoid repetitive counting, CL were counted every 100 pm by comparing the section with the preceding and following sections. CL were characterized by a central cavity filled with blood and follicular fluid remnants or by prominent polyhedral to round luteal cells.

[0229] Physiological measurements

[0230] Body composition

[0231] Body composition was measured weekly over several experiments using a Minispec LF Series (Bruker Corporation). Fat and lean mass data are expressed as percentage body weight.

[0232] Analysis of basal metabolism

[0233] Mice were analyzed for total energy expenditure, oxygen consumption, CO2 production, food intake and ambulatory movements (total beam breaks h-1) using calorimetric cages (TSE Systems) and standard procedures. Mice were individually housed and acclimatized to cages for 48 h before measurements. Pair-fed animals received the mean weight of food eaten by the control group during the previous 24 h. RER and energy expenditure (EE) were calculated as reported previously77, and FA oxidation as reported by Brass and colleagues78: FA oxidation (kcal h ') = EEx(1 - RER / 0.3).

[0234] Glucose tolerance

[0235] After overnight fasting (12 h), tail blood samples for insulin measurement were taken using glass capillaries before and 15 and 30 min after glucose administration. Blood samples were kept on ice before centrifugation (4 °C, 6.8 ref, 15 min) and plasma was frozen at -80 °C until an ultrasensitive mouse insulin ELISA (Mercodia, catalogue n° 10-1249-01) was performed on 12.5 pL of plasma per time point. Basal blood glucose levels were measured before i.p. glucose administration (2 g glucose kg-1 body weight) and 15, 30, 45, 60 and 120 min after glucose administration using a Accu-Chek Guide glucometer.

[0236] Insulin response measurementBasal blood glucose levels were measured after a 6-h fast for the ITT before i.p. insulin administration (0.5 UI kg-1 body weight, Eli Lilly and Company, catalogue n° HI0210) and 15, 30, 45, 60 and 120 min after glucose administration using a Accu-Chek Guide glucometer.

[0237] Immunoblot

[0238] Tissues, all dissected during the diestrous stage, were homogenized using a TissueLyser II (Qiagen) in cold RIPA buffer (200 mM Tris / HCl pH 7.4, 130 mM NaCl, 10% (v / v) glycerol, 0.1% (v / v) SDS, 1% (v / v) Triton X-100, 10 mM MgC12) with anti-proteases and antiphosphatases (Sigma-Aldrich), and lysates were centrifuged for 30 min at 18,000g and 4 °C. Liver and white adipose tissue (intra-abdominal adipose tissue) total protein lysates were separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to polyvinylidene difluoride membranes and probed successively with the following antibodies: hormone-sensitive lipase / HSL (1:1,000; Abeam, catalogue n° ab45422); phospho-HSL (Ser660) (1:1000; Cell Signalling Technology, catalogue no. 4126); ACC (1:1000; Millipore, catalogue n° 04-322); phospho-Acc (Ser79) (1:1000; Cell Signalling Technology, catalogue n° 3661); lipoprotein lipase (1:1000; H-53, Santa Cruz Biotechnology, catalogue n° sc-32885); FAS antibody (1:5000; Abeam, catalogue n° abl28870); and P-actin (1:5000; Sigma-Aldrich, catalogue n° A2228), after incubation of membranes with 5% BSA blocking buffer. Proteins were detected using horseradish peroxidase-conjugated secondary antibodies (Dako, catalogue n° PI-2000). Specific immunolabelling was visualized using chemiluminescence following the manufacturer s instructions (Pierce ECL Western Blotting Substrate, ThermoScientific), and values expressed relative to P-actin.

[0239] Adipose tissue histomorphology

[0240] Visceral (intra-abdominal) white adipose tissue and intrascapular brown adipose tissue were collected during the diestrous stage, fixed in 10% formalin for 24 h, dehydrated and paraffin-embedded. Sections of 5-pm thickness were cut on a microtome, stained using standard alcoholic hematoxylin and eosin (BioOptica), and observed and photographed using a Provis AX70 microscope (Olympus). Two 20x pictures per sample / mouse / treatment were analysed.

[0241] 8-bit images of adipose tissue sections were processed with Fiji (https: / / doi.org / 10.1038 / nmeth.2019) as follows. Automatic thresholding was first performed using the Triangle method. To correct for damaged and open membranes, two rounds of erosion were applied to the resulting binary image, before using the particle analysis tool to segment adipocytes and extract their pixel area. To exclude potential artifacts, partial adipocytes on the edges of the images, as well as particles of small size (< 2300 px) were excluded from the detectionStructural modelling of AMH-AMHR2 complex

[0242] The 3D fold of the peptide sequence GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO: 1) was predicted by using Alphafold2. Out of the 5 models generated, the best one was used for further analysis. The overall pLDDT was 67.2 with the regions of the peptide predicted to form b-strands with a pLDDT > 70 (high confidence of prediction).

[0243] The structural models of murine AMH and AMHR, generated by Alphafold2, are available on Uniprot database. In particular, the domains involved in the complex formation have a high or very high model confidence with pLDDT >70 and, for some regions, >90. Structural superimposition was generated using UCSF Chimera and the crystal structure of the human complex (PDB ID 7L0J) as a template. From the superimposition, a structural alignment was generated by UCSF Chimera and the structural conservation of the amino acids involved in the AMH- AMHR interaction in the human and murine proteins were analyzed.

[0244] Immunization and generation of hybridomas

[0245] A 28 amino acid peptide (GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO: 1)), spanning a terminal region of AMHR2 involved in the interaction with AMH and showing high homology among different mammalian species (human, mouse, and rats), was synthesized and conjugated to KLH carrier molecule (Primm Sri) by Di.V.A.L. Toscana S.r.l. (Italy). Hybridomas were produced according to Kohler and Milstein's standard protocol79. Briefly, six-week-old female Balb / c mice, purchased from Envigo (Envigo-Harlan Laboratories, Italy) were immunized by intraperitoneal injection of an emulsion of 100 pg KLH-conjugated peptide in PBS and Complete Freund’s Adjuvant (Sigma- Aldrich) by Di.V.A.L. Toscana S.r.l. (Italy). Three more intraperitoneal injections of 50 pg KLH-conjugated peptide emulsioned with Incomplete Freund’s Adjuvant (Sigma-Aldrich) were performed every 2-3 weeks and a final immunization with 100 pg peptide was administered intravenously. Three days after the last immunization, spleen cells from immunized mice were fused with the aminopterin-sensitive myeloma cells (NS0) by the polyethylene glycol (PEG; Sigma-Aldrich) method80. Hybridomas were cultured in DMEM with 20% Fetal Clone I serum medium supplemented with hypoxanthine-aminopterin-thymidine (HAT; Sigma-Aldrich) in humidified air at 37° C with 5% CO2 and supernatants were analyzed by ELISA assay.

[0246] Monoclonal anti-AMHR2 neutralizing antibody production

[0247] The best hybridoma population underwent clonal isolation by Di.V.A.L. Toscana S.r.l. (Italy) using the soft agar cloning method. Different numbers of hybridoma cells (10000, 5000, 2500, 1250 cells) were plated in soft agar 60mm dishes composed of a Base Layer (0.5% low melting point agarose in DMEM added 20% Fetal Clone I serum and lx HAT) and a Top Layer(cells in 0.25% low melting point agarose in DMEM added with 20% Fetal Clone I serum and lx HAT). About 15 days after seeding, clones were picked up, amplified and gradually adapted to grow in DMEM with Fetal Clone I serum 5%. The supernatants of hybridomas were added to 96-well plates pre-coated with 10 pg / ml of the uncoupled form of the peptide used for immunization followed by blocking with 3% BSA (Sigma-Aldrich). After a two-hour supernatants' incubation, 10 pg / ml anti-mouse secondary antibody, conjugated with horseradish peroxidase (Sigma-Aldrich) was added to each well for 1 hour. Finally, TMB (3, 3', 5,5'-Tetramethylbenzidine) substrate (Sigma-Aldrich) was added and optical density was determined by absorbance at 405 nm.

[0248] Monoclonal anti-AMHR2 neutralizing antibody purification

[0249] Anti-AMHR2 monoclonal antibody was purified by Affinity Chromatography, using AKTA Pure Protein Purification System (Cytiva). Clone supernatant was exchanged with Phosphate buffer 20mM pH 8,0 by HiPrep 26 / 10 Desalting column (Cytiva). The purification was hence performed with HiTrap Protein A HP column (Cytiva). The monoclonal antibody was eluted with a 0, IM citric acid buffer pH 4,0 immediately neutralized with IM Tris-HCl pH 9,0. The fractions were collected and dialyzed in PBS.

[0250] Determination of kinetics and affinity constants of anti-AMHR2

[0251] The equilibrium dissociation and affinity constant (KD and Ka) between the Hal 3 mAb and rhAMHR2 (Human MIS RII (AMHR2) Protein, His Tag; Acrobiosystems #MII-H52H6, batch 5726-21CQF1-ZL) was determined using a T200 Biacore instrument (GE Healthcare Bio-Sciences, Piscataway, NJ). Capture antibodies (anti-His) were immobilized on a CM5 sensor chip surface and the kinetics and affinity constants were determined by performing a single cycle kinetics (SCK) of anti-AMHR2 antibodies at 2.5 nM, 7.4 nM, 22.2 nM, 66.7 Mn and 200 nM over captured anti-His antibodies. For each determination, a first cycle was performed with injection of buffer instead of antibody (blank cycle). The response curve of this blank cycle was subtracted from one of the cycles with the antibody. The kinetics and affinity constants were calculated using a Langmuir 1 : 1 fitting model.

[0252] In vitro studies using anti-AMHR2 neutralizing antibody

[0253] Cell lines

[0254] The mouse granulosa cell line KK-181has previously been shown to be a suitable model to study AMH signalling40. Cells were cultured in DMEM / F12 (Life Technologies) containing ImM Pyruvate, 2mM L-Glutamine (Life Technologies), 100 pg / ml streptomycin, 100 U / ml penicillin and 9 mg / ml glucose (MP Biomedicals, Santa Ana CA, USA), supplemented with10% v / v fetal bovine serum (FBS, Life Technologies) in a 10% CO2 humidified atmosphere at 37 °C.

[0255] Western blot analysis

[0256] KK-1 cells were plated at 106cells / well in 6-well plates and treated with either IgM or Hal3 neutralizing antibody at 50 and 100 pg / ml, respectively for 2 hours. Cells were then scraped into ice-cold PBS, centrifuged at 4°C for 30 s at 16000g, and cell pellets re-suspended in RIPA buffer with protease and phosphatase inhibitors (lOmM Tris-HCl pH 7.5, 500 mM NaCl, 0,1% SDS, 1% NP40, 1% sodium deoxycholate, 2 mM EDTA, 2 mM Na2VO4, 2 mM Na4P2O7, 2mM NaF) and stored at -80°C. Protein content was assayed by the BCA protein Assay Kit (Pierce, Rockford, IL 61101 USA). Total proteins were fractionated by SDS electrophoresis on NuPage 4-12% Bis-Tris gel (Life Technologies) and transferred onto nitrocellulose membranes (Hybond-C super, Amersham Biosciences, Bucks HP979NA, UK). Western blotting was performed using equal amounts of protein from each treatment group with the relevant antibodies against pSMAD proteins 1 / 5 (Rb polyclonal, Cell Signalling, catalogue n° 4 ID 10, 1 : 1000). Actin (anti- P-actin, 1 : 5000; Sigma-Aldrich, catalogue n° A2228) was used as the reference protein and loading control. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Gt anti-rabbit HRP, DAKO, catalogue n° P0448) diluted 1 :2000 in 5% non-fat milk TBST for 1 hr at RT.

[0257] Electrophysiology

[0258] Electrophysiological recordings were performed on living brain slices from 8 to 12-week-old PAMH;Gnrh-GFP+ / ' and CNTR;Gnrh-GFP+ / ' mice. Mice were put under isoflurane anesthesia and killed by decapitation. The brain was dissected and rapidly placed in ice-cold aCSF containing: 120 mM NaCl, 3.2 mM KC1, 1 mM NaH2PO4, 26 mM NaHCCh, 1 mM MgCh, 2 mM CaCh, 10 mM glucose (300 mOsm, pH 7.4) and bubbled with 95% O2 to 5% CO2. 200 pm coronal slices containing the rostral preoptic area were cut using a VT1200 vibratome (Leica). Slices were incubated at 34°C in oxygenated aCSF for a recovery period of 1 hour and then placed at room temperature until patch-clamp recording. Individual brain slices were placed in a submerged recording chamber (Warner Instruments) and continuously perfused at a rate of 3mL / min with oxygenated aCSF maintained at 32.8°C by a heater controller (TC-344C-Wamer Instrument). GnRH neurons were visualized under xlO and x40 magnification using an upright fluorescence microscope with infrared differential interference contrast (DM-LFSA, Leica) and an ORCA-Frash4.0 digital CMOS camera (Hamamatsu). Recording pipettes were pulled from borosilicate glass capillaries (1.5 mm outer diameter, 1.12 mm inner diameter; World Precision Instruments) using a P1000 Flaming Brown puller (SutterInstruments) and had a resistance of 7-9 MQ when filled with an internal solution containing 140 mM K-gluconate, 10 mM KC1, 1 mM EGTA, 2 mM Na2-ATP, and 10 mM HEPES, pH 7.3, with KOH. Whole-cell patch-clamp recordings were performed in current-clamp mode using a Multi clamp700B Amplifier, digitized with the Digidata 1322A interface, and acquired with pClamp 10.2 software (Molecular Devices). For antibody functional characterization, 25 pg / mL of Hal 3 (anti-AMHR2 IgM) was added to the aCSF bathing medium by the use of the perfusion system after stable baseline recording. Recordings were analyzed using Clampfit 10.2 pClamp software (Molecular Devices). For each recording, the membrane potential and mean firing rate were determined before and during the bath application of Hal3. Only cells that showed less than 20% change in access resistance throughout the recording were included in this study. The junction potential was corrected in the data analysis.

[0259] In vivo studies using anti-AMHR2 neutralizing antibody

[0260] For in vivo Hal3 functional validation, we administered daily intraperitoneal injections of either Hal 3 or mouse IgM (4 mg / Kg / inj ection) to infantile PAMH female mice from P10 to P15. This dose of antibody was chosen based on previous works using another anti-AMHR2 neutralizing antibody to suppress AMH proliferative effect on ovarian adenocarcinoma in mice82. We next characterized the effects of the Ha 13 treatment applied during mini-puberty on the neuroendocrine / reproductive and metabolic outcome of the adult animals, respectively at 3 months and 6 months.

[0261] For the intergenerational study, PAMH female offspring (Fl) were administered daily with intraperitoneal injections of either Hal 3 (4 mg / Kg / inj ection) or PBS from P10 to Pl 5 and then mated at P60 with CNTR males (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5 as described above) to generate PAMH F2 offspring. CNTR F2 female offspring were generated by breeding CNTR females (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5 and treated at mini-puberty with PBS) with CNTR unrelated males (generated by prenatally treating gestating mice with PBS from El 6.5 to E18.5). F2 female offspring were subjected to phenotypic testing as described above. The exact number of mice used for each procedure and their sex and age are given in the figure legends.

[0262] RNA extraction and RT-qPCR

[0263] Ovaries, perigonadal fat, liver and pancreas were harvested from diestrous female mice and snap-frozen in liquid nitrogen. Frozen tissues were homogenized using 1 ml of Trizol (ThermoFisher Scientific, catalogue n° 15596026) with a tissue homogenizer and total RNA was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen; catalogue n° 74804) following the manufacturer’s instructions. For gene expression analyses, cDNA was synthesized from 1000ng of total RNA using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, catalogue n° 4387406) using the manufacturer’s recommended cycling conditions. Real-time PCR was carried out on Applied Biosystems QuantStudio3 7900HT Fast Real-Time PCR system using exon-boundary-specific TaqMan Gene Expression Assays (TermoFisher Scientific). Data were analyzed by using the 2-AACTmethod83and normalized to housekeeping gene Rnl8S (Thermofisher #Mm03928990) Beta-actin (AciB) levels. The other primers were directed against Esrl (Thermo-fisher #Mm00433149_ml); Pome (Thermo-fisher #Mm00435874_ml); Agrp (Thermo-fisher #Mm00475829_gl); Kissi (Thermo-fisher #Mm03058560_ml); Pgr (Thermofisher #Mm00435628_ml); Ar (Thermo-fisher #Mm00442688_ml): Amhr2 (Thermo-fisher #Mm00513847_ml). Values are expressed relative to control values, as appropriate, set at 1.

[0264] In vivo biodistribution of Hal3

[0265] To image the biodistribution of Hal3 in vivo, anesthetized P16 female mice were intravenously injected (orbital venous sinus) with 30 pl of anti-AMHR2-BF antibody (Hal 3, 4mg / Kg) conjugated to a fluorophore (ATTO-565). Animals were sacrificed 5 min or 30 mins later. The brain, ovaries, visceral adipose tissue and pancreas were dissected at diestrus and immersion-fixed in 4% PFA solution overnight at 4°C, cryoprotected overnight in PBS / sucrose 30% at 4°C, embedded in OCT embedding medium (Tissue-Tek), and frozen and stored at -80 °C until cryosectioning. Tissues were cut at 35-pm-thick sections and processed as free-floating sections for immunofluorescence. At the end of the immunofluorescence experiments, sections were coverslipped with Fluoromount-GTM with DAPI (Invitrogen, Cat: 00-4959-52) and stored at 4°C.

[0266] RNAscope fluorescence in situ hybridization

[0267] Fluorescence in situ hybridization (FISH) was performed on fresh-frozen brain sections of the ME of adult female mice using the RNAscope Multiplex Fluorescent Kit v.2, according to the manufacturer’s instructions (Advanced Cell Diagnostics). Specific probes were used to detect Pome (Advanced Cell Diagnostics, catalogue n° 314081), Agrpl (Advanced Cell Diagnostics, catalogue n° 400711), and Esrl (Advanced Cell Diagnostics, catalogue n° 478201) mRNAs. Hybridization with a probe against the Bacillus subtilis dapB gene (Advanced Cell Diagnostics, catalogue n° 320871) was used as a negative control. Image acquisition was performed using an inverted confocal microscope (LSM 710, Zeiss).

[0268] Ethics statement

[0269] Animal studies were approved by the Institutional Ethics Committees of Care and Use of Experimental Animals of the Universities of Lille 2 (France). All experiments were performed in accordance with the guidelines for animal use specified by the European UnionCouncil Directive of September 22, 2010 (2010 / 63ZEU) and the approved protocol (APAFIS# 29172-2020121811279767 v5) by the Ethical Committee of the French Ministry of Education and Research. All efforts were made to minimize animal suffering and animal care was supervised by veterinarians and animal technicians skilled in rodent healthcare and housing.

[0270] Statistics and data analysis

[0271] All analyses were performed using 10.0.2 GraphPad Prism software (GraphPad; San Diego, CA, USA). The normality of each group was determined by Shapiro-Wilk test. We did not assume equal variances. Statistically significant P values were considered when P < 0.05. No statistical methods were used to pre-determine sample sizes but our sample sizes were similar to those reported in previous publications22’27’84. Animals were randomized to experimental groups and investigators were blinded to group allocation during data collection and analyses. For each experiment, replicates are described in the figure legends. No samples were excluded from the analyses. For the normal distribution, data were compared using unpaired two-sided Student’s / -test and one-way and two-way ANOVA followed by Tukey’s or Holm-Sidak’s multiple comparisons test tests. For non-normally distributed values, the Mann-Whitney U test was used. The numbers of biologically independent experiments, sample size, P values, and ages and sexes of the animals together with the details of the statistical tests used for the analyses are all indicated in the main text or figure legends.

[0272] Results

[0273] AMH treatment during mini-puberty in mice triggers the neuroendocrine and reproductive disturbances of PCOS

[0274] Clinical studies have shown that infants born to mothers with PCOS have twice the levels of AMH during mini-puberty compared to those born to non-PCOS women13,23’24. Based on this, we assessed whether our recently established PCOS-like mouse model, created by prenatal treatment with high AMH (PAMH model)19’21, which display the major cardinal neuroendocrine12,21, reproductive21and metabolic PCOS defects19, could also mimic the elevated AMH levels observed in daughters of women with PCOS during mini-puberty. In female mice and rats, mini-puberty starts the second week of postnatal life, concluding before the emergence of the first external signs of sexual maturation (around P30)28. We thus measured AMH in female PAMH mice and controls at P12, which corresponds to the peak of minipuberty in female mice27'29. Our findings showed that PAMH mice had elevated AMH levels compared to controls during this critical early developmental stage (Data not Shown),replicating the human scenario. We also assessed plasma LH levels at P12 and we did not observe any differences between the two groups (Data not Shown).

[0275] To determine whether elevated AMH levels during infancy in offspring of women with PCOS are a bystander condition or a driving force behind the condition, we generated another animal model by exposing female mice to exogenous AMH during mini-puberty and by assessing whether this exposure could lead to PCOS defects later in life. We administered phosphate-buffered saline (PBS: mini-puberty-control mice: mCNTR) or human recombinant AMHc (mini-puberty-AMH mice: mAMH) to infantile mice via intraperitoneal daily injection (i.p.) from P12 to P14 and we examined the neuroendocrine reproductive and metabolic characteristics of these animals during adulthood (Figure 1A).

[0276] At P21, we collected the blood from mCNTR and mAMH animals and we used mass spectrometry coupled with gas chromatography (GC-MS) to measure gonadal steroids in these animals. At this postnatal stage, circulating androgens (testosterone and androstenedione [delta4-Dione]), estradiol (E2) and progesterone (P4) levels did not display any difference between the two groups of animals (Data not Shown). Since AMH levels during mini-puberty have been documented to be higher also in boys born to PCOS mothers as compared to boys bom to non-PCOS women25, we also assessed gonadal steroid levels in males treated with AMH or PBS during mini-puberty. We found that mAMH P21 males did not display any difference in circulating testosterone, dihydrotestosterone (DHT) or delta4-Dione as compared to control males (Data not Shown). However, at this stage, mAMH males had significantly higher P4 levels than in mCNTR males (Data not Shown).

[0277] We monitored whether the age of puberty onset could be affected by aberrant exposure to AMH at mini-puberty. Vaginal opening is initiated by mini-puberty-induced gonadal estrogen production, and first estrus correlates with the first ovulation at puberty in rodents. Vaginal opening was significantly delayed by ~ 5 d in mAMH female mice compared with control females (Data not Shown). Similarly, the first estrus was significantly delayed in mAMH versus mCNTR mice (Data not Shown). Males mAMH mice showed normal puberty timing, as revealed by the analysis of the balano-preputial separation (bps), a marker of pubertal onset in males (Data not Shown). Interestingly, mAMH mice of both sexes displayed a significant increase in body weight as compared to control animals from P21 to adulthood (P120; Data not Shown).

[0278] Next, we characterized the neuroendocrine and reproductive features of adult female mice exposed either to PBS or AMH during mini-puberty. Similar to the clinical condition, mAMH mice exhibited signs of oligo-anovulation (Figure IB, 1C), displaying prolonged timein diestrus and significantly less time in proestrus as compared to mCNTR females (Figure 1C), together with a significant reduction in the number of completed ovulatory cycles observed for 15 days as compared to controls (Figure ID). In addition, mAMH female mice also showed impaired fertility, as indicated by a significantly reduced litter size (Figure IE). Ovarian histology of mAMH animals showed abnormalities consistent with their oligo-anovulatory phenotype, with the presence of fewer post-ovulation corpora lutea as compared to control animals (Figure IF). Circulating androgens (testosterone and delta4-Dione), measured at diestrus, were significantly elevated in mAMH mice compared to controls, whereas estradiol (E2) and progesterone (P4) levels did not display any difference between the two groups (Figure 1G). We then evaluated LH pulsatility by serial blood sampling in diestrous mice (Data not Shown) and found that mAMH animals had a significantly higher mean LH levels (Data not Shown) and LH pulse frequency (Data not Shown), followed by a shortened pulse interval time (Data not Shown) as compared to controls.

[0279] We did not observe any difference in testicular morphology (Data not Shown), relative testis weight (normalized by body weight; Data not Shown), nor circulating testosterone levels (Data not Shown) between control and mAMH males, suggesting that exposure to high AMH levels over mini-puberty does not significantly impact the neuroendocrine system in male mice.

[0280] AMH treatment during mini-puberty triggers PCOS-like metabolic disturbances We have previously shown that prenatal exposure to high AMH leads to PCOS-like metabolic dysfunctions in female offspring, which progressively manifest with age (~5-6 months of postnatal life)19. Herein, we investigated whether AMH exposure during minipuberty also promotes metabolic alterations in adulthood both in females and males. Six-month-old mAMH females displayed higher body weight and fat mass (Figure 2A, 2B), and lower lean mass (Figure 2C) compared with controls. The percentage of free body fluids was comparable between all groups (Figure 2D). In addition, glucose tolerance was lower in mAMH females than in controls following i.p. glucose administration (Figure 2E).

[0281] Consistently, this was followed by exacerbated insulin release after glucose injection in mAMH animals (Figure 2F) and increased HOMA-IR compared with control mice (Figure 2G). Using an indirect calorimetric system to characterize the energy metabolism of these animals, we found that mAMH females displayed modest modifications in their feeding patterns, with a significant increase in food-intake observed only during the first hours of the nocturnal active phase (Data not Shown). In addition, no difference was observed in respiratory exchange ratio, locomotor activity, energy expenditure or in the Z rearing between the two groups of animals (Data not Shown). In males, we found that food intake was comparable in mAMH and mCNTRanimals (Data not Shown). However, we found that weight gain observed in male mAMH mice (Data not Shown) was associated with an increased respiratory exchange ratio (RER) during the second part of the dark phase (Data not Shown) and decreased energy expenditure (EE) (Data not Shown). Locomotor activity and Z rearing were comparable between mAMH and control males (Data not Shown). Given the increased RER in mAMH male mice, indicative of increased consumption of carbohydrates over lipids to meet energy requirements30, we next examined lipid metabolism in these animals and we found that the changes in RER and food-intake-independent body-weight gain in mAMH males appeared to be due to decreased fatty acid (FA) oxidation (Data not Shown). Comparable alterations as those detected in females, in terms of glucose tolerance, insulin release and insulin resistance were also detected in mAMH males (Data not Shown).

[0282] Because anorexigenic proopiomelanocortin (POMC) and orexigenic agouti -related peptide (AgRP) neurons located in the hypothalamic arcuate nucleus (ARC) are key neuronal regulators of food intake and energy homeostasis31, we then evaluated the transcript expression of Pome and Argpl in the ARC / mediobasal hypothalamus (MBH) of control and mAMH females. We did not observe any difference between the groups of animals in Pome and Argpl gene expression in the ARC / MBH as revealed by RNAscope analyses (Data not Shown) and by qRT-PCR experiments (Figure 2H).

[0283] Hyperandrogenemia, acting through the androgen receptor (AR), has been shown to reduce both estrogen receptor alpha (ERa) and progesterone receptor (PR) expression in the arcuate nucleus of the MBH32,33. This contributes to diminished estradiol- and progesterone-mediated hypothalamic negative feedback, perpetuating LH hypersecretion and ovarian hyperandrogenism in PCOS34'37. Additionally, decreased estradiol-mediated ERa activity in the MBH has been linked to obesity, type 2 diabetes, and hyperlipidemia38. We thus investigated the expression of Esrl mRNA in the ARC / MBH in these animals, together with other genes whose expression might be altered in PCOS, namely Kisspeptin (Kissi), progesterone receptor (Pgr), and Ar. Our results showed that mAMH females have significantly lower levels of Esrl expression in the ARC / MBH as compared to mCNTR females (Data not Shown and Figure 2H). In contrast, Kissi, Pgr and Ar transcript expression was similar between the two animal groups (Figure 2H).

[0284] High androgen levels in PCOS patients often leads to obesity, characterized by increased subcutaneous and visceral adipose tissue1, we next focused on the morphology of the white adipose tissue (WAT) of these animals. Similar to the clinical condition, mAMH females presented larger white adipocytes in the visceral fat (Figure 21), and increased visceral fat mass,compared to controls (Figure 2J), suggesting that mAMH mice have lipid accumulation in white adipocytes. Like mAMH females, also mAMH males presented increased visceral fat mass (Data not Shown) and larger white adipocytes in the visceral fat (Data not Shown) compared to controls.

[0285] Development and characterization of an AMHR2 blocking-function antibody Current pharmacologic interventions for PCOS mainly address symptomatic management. Considering the data presented, along with previous preclinical12and clinical studies showing that abnormal serum AMH levels are a hallmark of PCOS6'8, it is biologically and clinically logical to explore AMH inhibition as a potential preventive or therapeutic strategy for PCOS. Here, we report the generation, structure, and function of an epitope-specific AMHR2 -blocking antibody (Figure 3A).

[0286] Initially, a research investigation was undertaken to identify the amino acid sequences within the N-terminal region of AMHR2, exhibiting antigenic properties and significant homology across various mammalian species. Based on the X-ray crystal structure of AMH bound to the extracellular domain of AMHR239, we identified one peptide sequence, GCESLHCDPSPRAHPSPGSTLFTCSCGT, as one of the most promising to produce antibodies with blocking-function activity. Indeed, this peptide presents 100% homology with the human and primate sequences and 92% and 89% homology, respectively, for the rat and mouse AMHR2 sequences (Figure 3B). Moreover, by using Alphafold2, the peptide is predicted to fold in a 3D structure almost superimposable with the one adopted by the same region in the entire protein, with a root mean square deviation (RMSD) of 0.933 A (Data not Shown). We then conducted a detailed mapping of the atomic interactions between the human peptide sequence and the ligand AMH. Using the crystal structure of human AMH as a template for our modeling (Data not Shown), we found it to have high homology with the structural model of murine AMH-AMHR2 (Data not Shown). In both human and murine AMHR2, the residues involved in hydrophobic contacts with the ligand are conserved (F62, 164, M76, and L106; Data not Shown). The residues forming hydrogen bonds and salt bridges, including R97, crucial non-covalent interactions that stabilize the binding of a ligand to its receptor, are also identical between the two species (Data not Shown). Next, we modeled the interaction of the antigenic AMHR2 peptide with human and murine AMH (Data not Shown). The modeled human peptide-AMH complex largely mirrors the binding mode of the murine AMH. These models indicate that the interactions stabilizing the binding between the ligand and the antigenic peptide occur at residues LI 06 and R97 of the peptide-AMH complex (Data not Shown).The synthesized peptide was used to immunize Balb / c females (n = 3, 5-6 weeks-old) and to generate a mouse monoclonal IgM anti-AMHR2 antibody, that we named Hal3, with a KD of 37.1 nM, suggesting a relatively strong binding affinity between the ligand and its receptor (Figure 3C). The protein sequences of variable domains of the antibody are represented in Table 1.

[0287] Next, we performed qRT-PCR experiments to assess Amhr2 expression respectively in the ovary, visceral WAT, pancreas and liver of adult diestrous female mice (P90). We confirmed expression of Amhr2 transcript in the ovarian tissue (positive control) as well as in the visceral WAT but not in the liver and pancreas (negative controls; Figure 3D). As a prelude to the testing of AMHR2-Ab in mice, we then determined the specificity of Hal 3 for the murine AMHR2 using western blot and immunofluorescence. Immunoblotting with Hal 3 revealed a ~70-kDa specific band in protein extracts of the ovaries, while a very faint band was detectable in the liver and absent in the pancreas (Figure 3E). Immunofluorescence experiments using the Hal3 antibody confirmed the spatial distribution of AMHR2 in the granulosa cell layer of the ovarian follicles and in the ovarian stroma (Data not Shown), as well as in the adipocytes of the visceral fat (Data not Shown).

[0288] In order to further establish whether Hal3 antibody binds to AMHR2, we then used an immortalized in vitro model of murine ovarian granulosa cells, KK1, previously shown to be a suitable model to study AMH signalling as these cells retain AMHR2 expression as well as endogenous secretion of AMH40,41. We immunostained KK1 cells using a previously validated anti-AMHR2 antibody10and the Hal3 antibody directly conjugated with a fluorescent dye (ATTO-red). These experiments confirmed the co-localization between AMHR2 and Hal 3 (Data not Shown).

[0289] After establishing the specificity of the antibody and its capacity to specifically bind to AMHR2, we next performed in vitro functional experiments to assess the blocking-function capability of Hal 3. To this purpose, we used an immortalized in vitro model of murine granulosa cells, KK1, previously shown to be a suitable model to study AMH signalling as these cells retain AMHR2 expression as well as endogenous secretion of AMH40,41. Treatment of KK1 cells with two concentrations of Hal3 antibody, 50 pg / ml and 100 pg / ml, significantly reduced phosphorylation of SMAD1 / 5 by half as compared to untreated cells (CNTR) or cells treated with the same concentrations of an IgM antibody (Figure 3F, 3G). These experiments revealed that the antibody successfully inhibited the autocrine stimulation of the specific AMH-AMHR2 signalling pathway.We have previously shown that AMH potently induces the neuronal electrical activity of GnRH neurons, which express AMHR210, and that PCOS-like mice generated by prenatal exposure to high AMH (PAMH mice) have a persistent hyperactivation of GnRH neurons as compared to controls21. Therefore, to validate the efficacy of Hal3 to inhibit hypophysiotropic GnRH neurons, we first generated P AMH, Gnrhl-g mice21. Subsequently, brain coronal slices were prepared for in vitro electrophysiological recordings of GnRHrPOAneurons from CNTR;Gw7i7-g^? and P AMH, Gnrhl-gfp adult females (Figure 3H, 31). Whole-cell pathclamp recordings revealed that the administration of Hal 3 significantly reduced GnRH neuronal firing rate in hypothalamic slice preparations from PAMH mice (Figure 31, 3J).

[0290] Consistent with previous findings21, GnRH neurons from PAMH mice showed a significant increase in their spontaneous action potential firing rate, as compared with control animals (CNTR basal; Figure 3J). Whole-cell path-clamp recordings revealed that the administration of Hal3 significantly reduced GnRH neuronal firing rate in hypothalamic slice preparations from PAMH mice to levels comparable to control conditions (Figure 31, J). After washing out the neutralizing antibody from the bath, the firing rate of each recorded cell rose again to the initial levels supporting the specificity of Hal 3 antibody on the inhibition of GnRH neuronal activity (Figure 3J).

[0291] Overall, these data show that the Hal 3 antibody specifically binds and inhibits AMHR2 in cell-based assays, both in vitro and ex-vivo, a prelude to further preclinical testing.

[0292] Hal3 treatment during mini-puberty prevents PCOS-like reproductive and neuroendocrine defects

[0293] Building upon our findings illustrating on one side the presence of higher levels of AMH during mini-puberty in PAMH females as compared to controls and, on the other side, the significant PCOS-programming effect triggered by AMH exposure during mini-puberty in otherwise healthy mice, we opted to administer daily intraperitoneal injections of either Hal3 or mouse IgM (4 mg / Kg / inj ection) to infantile PAMH female mice from PIO to Pl 5 (Figure 4A) to assess the therapeutic potential of Hal 3.

[0294] We first analyzed estrous cyclicity of adult female mice, at P60, over 15 days (Figure 4B-4D) We monitored daily vaginal cytology of control + vehicle, PAMH + vehicle, PAMH + IgM and PAMH + Hal3 animals. The disrupted estrous cycle of PAMH animals21was normalized by Hal 3 treatment (Figure 4B-4D). Ovarian histology of PAMH animals showed abnormalities consistent with their oligo-anovulatory phenotype, with the presence of fewer post-ovulation corpora lutea as compared to control animals, whereas Hal 3 treatment at minipuberty prevented these defects in adulthood (Data not Shown). In addition, Hal 3 rescued thehyperandrogenic phenotype (Data not Shown), while it did not affect circulating levels of E2 and progesterone (Data not Shown). Finally, the AMHR2-Ab treatment over mini-puberty prevented the PCOS-like pulsatile LH hypersecretion and the elevation in mean LH levels in adulthood (Data not Shown).

[0295] Hal3 treatment during mini-puberty prevents PCOS-like metabolic defects We next characterized the effects of the Hal 3 treatment on the metabolic outcome of prenatally AMH-treated mice (PAMH). We previously reported that PAMH females manifest PCOS-like metabolic derangements by the age of Pl 8019. Here, we showed that AMHR2-Ab administration to PAMH female mice during mini-puberty prevented the appearance at Pl 80 of exacerbated body weight and adiposity (Figure 5A), as well as glucose intolerance (Figure 5B) and insulin insensitivity (Figure 5C).

[0296] Previous evidence suggested that impaired brown adipose tissue (BAT) / WAT function and mass may contribute to the link between metabolic disturbances and reproductive issues in PCOS42'45. We evaluated the histology of BAT and visceral WAT in the different animal groups: control + vehicle, PAMH + vehicle, PAMH + IgM and PAMH + Hal3. PAMH females had more lipid accumulation in the BAT than in controls (Data not Shown and Figure 5D).

[0297] Because BAT whitening may induce tissue dysfunction and impaired thermogenesis, we measured the internal body temperature of these animals (Figure 5E). PAMH animals, treated with either vehicle or IgM at mini-puberty, had a significant decrease in body temperature at thermoneutrality as compared to control animals (Figure 5E). The rectal temperature of PAMH animals treated with Hal 3 at mini-puberty was normalized to control levels (Figure 5E).

[0298] PAMH females presented larger white adipocytes in the visceral fat, and increased visceral fat mass, compared to controls (Data not Shown and Figure 5F, 5G), suggesting that PAMH mice have hyperlipidemia and lipid accumulation in white adipocytes. Interestingly, these defects were prevented by Hal 3 treatment (Data not Shown and Figure 5F, 5G).

[0299] To shed some light on the adipose tissue dysfunctions detected in PCOS-like mice, we next investigated the expression of proteins involved in lipolysis or de novo lipogenesis in the visceral fat. Because PAMH animals injected with either saline or IgM did not show significant differences in any of the reproductive and metabolic parameters described above, for the subsequent analyses we only used three animal groups: CNTR, PAMH, PAMH + Hal3.

[0300] In agreement with increased adiposity and lipid accumulation in white adipocytes, PAMH mice showed a marked increase in protein levels of phosphorylated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), enzymes crucial for de novo lipogenesis (Data not Shown and Figure 5H). These defects were prevented by Ha 13 (Data not Shownand Figure 5H). The expression of the key lipolytic enzyme, hormone-sensitive lipase (HSL), as well the phosphorylated form of HSL did not change between the different groups (Data not Shown and Figure 5H).

[0301] Overall, these results suggest that prenatal exposure to abnormal levels of AMH promotes lipid accumulation by enhancing lipogenesis in the white adipose tissue of female offspring, and increases lipid droplet depot in the BAT, ultimately affecting thermogenesis. Notably, administering Hal3 during mini-puberty can counteract all PCOS-like metabolic defects.

[0302] Hal3 treatment during mini-puberty prevents intergenerational transmission of PCOS-like traits to female offspring

[0303] Given the strong heritability of PCOS and the well-documented transmission of the cardinal neuroendocrine and metabolic features observed in daughters and sons of PCOS women17,46, as well as in PCOS-like animals17,19,46, we sought to test whether the AMHR2-Ab treatment of PAMH offspring during mini-puberty could prevent the transmission of PCOS-like traits to the subsequent generation. Female PAMH offspring (Fl) of gestating mice prenatally exposed to high AMH (F0) were injected intraperitoneally with either Ha 13 (4 mg / Kg / inj ection) or PBS from PIO to Pl 5 and then mated at P60 with CNTR males (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5) to produce PAMH F2 (intergenerational) offspring (Figure 6A). CNTR F2 female offspring was generated by breeding CNTR Fl females (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5 and treated at mini-puberty with PBS) with CNTR Fl unrelated males (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5; Figure 6A).

[0304] F2 female offspring were then subjected to phenotypic testing. In agreement with our previous findings19, at P60, PAMH F2 female offspring displayed hyperandrogenism (Figure 6B), oligoanovulation (Figure 6C, 6D) and a significant increase in LH pulsatility as compared with CNTR F2 females (Figure 6E). In addition, at Pl 50 PAMH F2 females showed a significant elevation in body weight in comparison with CNTR F2 females (Figure 6F) and altered insulin sensitivity (Figure 6G). Treating PAMH Fl females with Hal 3 during mini-puberty prevented the transmission of all PCOS-like reproductive, neuroendocrine and metabolic traits to their female offspring to the next generation (Figure 6B-6G).

[0305] Bio-distribution of Hal3

[0306] To identify the possible target tissues of Hal3 treatment during mini-puberty, we finally investigated the biodistribution of the neutralizing antibody administered intravenously to female mice at P16. To this purpose, the neutralizing antibody was labeled with a fluorescentmolecule and retro-orbitally injected (Data not Shown). The animals were euthanized after 5 minutes and the ovaries, visceral fat, brain pancreas and brain were dissected and processed for immunostaining and confocal microscopy imaging. We detected a significant presence of fluorescently -labeled Hal 3 within the developing ovarian follicles and the stroma (Data not Shown) as well as in the adipocytes of visceral fat (Data not Shown). Consistent with the low expression levels of Amhr2 mRNA (Figure 3J) and protein (Figure 3K) in the murine pancreas, the Hal3-ATTO fluorescence was found only in the vessels and not in the islets of Langerhans immunostained for insulin (Data not Shown), thus excluding the pancreas as a possible target of the Hal 3 treatment at mini-puberty. In the medio-basal hypothalamus (Data not Shown), the antibody specifically bound GnRH terminals projecting to the median eminence (Data not Shown), ME tanycytes (Data not Shown) and the endothelial cells of the brain vasculature (Data not Shown). Within the rostral preoptic area (POA) (Data not Shown), the antibody bound GnRH neurons (Data not Shown) and endothelial cells of the brain vasculature (Data not Shown).

[0307] Collectively, these results show that Ha 13 binds the ovaries and the adipocytes of the visceral fat, in the periphery, and GnRH neurons and tanycytes in the central nervous system.

[0308] Hal3 treatment rectifies ovulation and hyperandrogenism in PCOS-like animals Our results support the view that Hal 3 may be an effective pharmacological intervention to prevent PCOS defects when administered during the infantile period. We then wondered, whether Hal 3 might also hold therapeutic potential to treat PCOS in adulthood. To test that, PAMH adult females were injected intraperitoneally with either Hal 3 or mouse IgM three times a week for one month (Figure 7A). Estrous cyclicity was examined in control and PAMH mice injected or not with IgM or Hal 3. Consistent with our data presented above, PAMH mice displayed an oligo-ovulatory phenotype (Figure 7B), characterized by sporadic ovulations, resulting in a significant reduction in the number of complete estrous cycles in 15 days as compared to control animals (Figure 7C). Notably, Hal3 administration fully restored the ovulatory pattern of PCOS-like animals back to control conditions (Figure 7B, 7C) and it rescued the hyperandrogenic phenotype, as shown by the normalization of aberrant circulating T levels of PAMH mice after 30 days of Hal3 treatment (Figure 7D). Ovarian histology of PAMH animals showed abnormalities consistent with their oligo-anovulatory phenotype (Data not Shown), with the presence of fewer post-ovulation corpora lutea as compared to control animals, whereas Ha 13 treatment prevented these defects in adulthood (Data not Shown).

[0309] DiscussionPrevious studies have demonstrated that fetal exposure to elevated levels of androgens and / or AMH during prenatal development may significantly contribute to the programming of PCOS, potentially predisposing individuals to develop the condition later in life17,19,21. However, it remains unclear whether the developmental window for PCOS programming extends into early postnatal life. Our findings show that the PAMH mouse model has higher circulating AMH levels during mini-puberty compared to controls. We also demonstrate that aberrant AMH exposure during this period in otherwise healthy pups drives key reproductive and metabolic PCOS features in female mice. These data are consistent with recent clinical data showing that both females and males born to mothers with PCOS have significantly higher serum levels of AMH during mini-puberty compared to those born to non-PCOS mothers13,23'25. In addition, previous studies have proposed the existence of a male metabolic phenotype of PCOS40'42and a recent investigation reported that sons of women affected by PCOS are frequently obese and exhibit dyslipidemia39. In line with this evidence, we show that elevated AMH exposure during mini-puberty in male mice contributes to long-term metabolic issues, including increased body weight, adiposity, and altered glucose metabolism, that emulate traits found in the male close relatives of women with PCOS39'42. However, in contrast with the exposed females, mAMH males do not show gross gonadal alterations nor changes in circulating levels of testosterone.

[0310] Our results also highlight that mAMH females have lower Esrl expression in the ARC / MBH than controls, potentially causing LH hypersecretion, hyperandrogenism, and metabolic issues. Indeed, hyperandrogenemia, acting through the androgen receptor (AR), has been shown to reduce ERa expression in the arcuate nucleus of the MBH43. This contributes to diminished estradiol-mediated hypothalamic negative feedback, perpetuating LH hypersecretion and ovarian hyperandrogenism in both PCOS women and animal models alike44'47Additionally, decreased estradiol-mediated ERa activity in the MBH has been linked to metabolic dysfunctions48,49. These findings suggest that disruptions in sex hormone signaling and ERa expression in the hypothalamus contribute to reproductive and metabolic dysfunctions in PCOS.

[0311] We have previously shown that AMHR2 is expressed by GnRH neurons throughout the lifespan in both rodents and humans as well as in other neuronal and non-neuronal populations of the MBH10,12. Furthermore, our studies have revealed that AMH acts as a potent stimulator of GnRH neuronal activity, driving increased LH secretion, which in turn exacerbates ovarian androgen production10. AMH over-exposure during mini-puberty may create a feed-forward loop to GnRH neurons, leading to ovarian hyperandrogenism and metabolic dysfunction laterin life. This supports a causal role for elevated AMH levels during early life in the pathophysiology of PCOS and offers a window for testing pharmacological interventions. To this purpose, we designed an anti-AMHR2 -blocking antibody aiming to reduce AMH action by preventing its binding.

[0312] Functional validation in vitro and ex vivo led to in vivo studies in a PCOS-like mouse model (PAMH), demonstrating significant improvements in reproductive, endocrine, and metabolic systems. Indeed, treatment with the Hal 3 antibody during mini -puberty prevented the manifestation of reproductive and metabolic cardinal defects of PCOS in adult females.

[0313] Here, we demonstrate that Hal 3 treatment during mini-puberty not only prevents PCOS-like defects in females but it also eliminates transmission of these traits to the F2 generation, highlighting the potential of early intervention to reduce long-term PCOS-related health impacts over multiple generations.

[0314] Importantly, Hal 3 was observed to accumulate in the hypothalamus, in the ovaries and in the visceral fat, suggesting that the beneficial effects of Hal 3 could operate through both central and peripheral mechanisms. Within the brain, we detected fluorescently-labeled Hal 3 in various brain cell types, including GnRH neurons, endothelial cells of the brain vasculature and tanycytes of the ME. These findings align with prior research on AMHR2 expression in these cells, confirming the targeting specificity of the antibody10,12.

[0315] Previously, we demonstrated that circulating AMH can reach GnRH terminals through the fenestrated vessels of the ME21. In this study, we peripherally injected the Hal 3 antibody which, classified as an IgM, possesses a pentameric structure approximately 35 nm in size. Due to its dimensions, we hypothesize that it can traverse the trans-endothelial pores (range: 30 to 80 nm in diameter) found in circumventricular organs50such as the ME51,52. By binding to GnRH terminals, the antibody may counteract AMH's excitatory effects on GnRH neuronal activity, a mechanism observed in PCOS-like animals21. In addition, we recently showed that AMH promotes profound micro- structural changes in the murine hypothalamic ME, inducing the retraction of AMHR2-expressing tanycytes and thus creating a permissive environment for GnRH secretion12. These data, together with recent clinical trial research point to the neuroendocrine system of PCOS as a promising therapeutic target53'55. While our study found no changes in kissl mRNA expression in PCOS-like models, inhibiting Neurokinin B (NKB) signaling in kisspeptin neurons shows therapeutic promise. This is important as arcuate kisspeptin neurons regulate pulsatile GnRH secretion56and since Neurokinin 3 Receptor (NK3R) antagonists reduce LH pulse frequency and testosterone levels in women with PCOS53,54,57Peripherally, the Hal 3 antibody likely exerts its effects on the ovaries, counteracting the aberrant AMH signaling. Indeed, ovarian AMH over- secretion, along with elevated testosterone, promotes pre-antral follicle survival and inhibits FSH-induced antral follicle growth58, contributing to oligo-anovulation, PCOM, and type-2 diabetes in PCOS59.

[0316] Furthermore, our study provides evidence for AMHR2 expression in the mouse visceral adipocytes at both transcriptional and protein levels. Consistent with these data, the fluorescently -tagged Hal 3 antibody successfully binds to adipocytes in the visceral fat.

[0317] Enlarged adipocytes contribute to PCOS metabolic issues by driving insulin resistance37, with adipocytes from women with PCOS having higher lipid content60,61. Androgen excess promotes de novo lipogenesis, reduces P-oxidation and lipolysis, and causes lipotoxicity62, leading to lipid accumulation in tissues like muscle and liver1, which contributes to insulin resistance. Accordingly, in PAMH animals, increased body weight, greater adiposity, enlarged adipocytes, and elevated lipogenic enzyme expression were observed. Previous studies have also shown that androgen excess hinders adipose stem cell differentiation into preadipocytes and mature adipocytes38,63,64. These mechanisms are thought to trigger compensatory adipocyte hypertrophy, contributing to dyslipidemia in PCOS65. It is thus plausible that AMH could play a role in adipocyte cellular differentiation, although further research is needed to elucidate how AMH might influence adipose tissue biology under both physiological and pathological conditions.

[0318] Taken together, these findings suggest that Ha 13 protects against PCOS by inhibiting ovarian androgen production, restoring ovulation, and also mitigating hyperandrogenism's impact on adipose tissue. This dual action, along with its central effects, highlights Hal3's therapeutic potential for both reproductive and metabolic aspects of PCOS.

[0319] Our findings underscore the pivotal role of early postnatal hormonal exposure in the development of PCOS, emphasizing the significance of abnormal AMH signaling as a crucial factor in the syndrome's pathophysiology. Gestational exposure to elevated levels of AMH in mice has been shown to induce PCOS-like traits in female offspring, with elevated AMH levels persisting across multiple generations19,21. Here, we show that abnormal hormonal exposure in utero also leads to increased circulating AMH levels during mini-puberty. Based on the minipuberty blocking strategy applied in PAMH animals, it thus appears that the key endocrine change driving adult PCOS-like traits could be the rise in AMH during this postnatal window, suggesting that mini-puberty is a critical developmental stage contributing to the reproductive and metabolic traits of adult females. This is in agreement with recent studies revealing a minipuberty in infant girls, characterized by two peaks of elevated AMH66, ovarian hormones, andgonadotrophins67at 1 and 4-6 months after birth. During this period, higher AMH levels correlate with larger ovaries and larger follicles66,68.

[0320] Overall, we show that the Hal 3 antibody show benefits in preventing PCOS traits when given during mini-puberty and treating PCOS when applied in adulthood, thus supporting future clinical trials to evaluate the therapeutic potential of this approach in women with PCOS.

[0321] EXAMPLE 2: Development and characterization of AMHR2 blocking-function antibodies

[0322] (Stepl) Identification ofAMHR2 sequences for Ab production

[0323] As previously developed in Exampel, a study has been carried out to identify on the N-terminal region of AMHR2, involved in the interaction with its ligand AMH, two aminoacid sequences with antigenic features and high homology among different mammalian species (such as human, mouse and rats). The peptide sequences were identified as the most promising for the production of antibodies with blocking activity:

[0324] Peptide 3-GCESLHCDPSPRAHPSPGSTLFTCSCGT (28 aminoacids) (SEQ ID NO: 1) Peptide 4-CNANYSHLPPP (11 aminoacids) (SEQ ID NO: 12) (Step2)Immunization ofBalb / c females

[0325] The peptides synthetized were exploited with a >90% purity and the KLH-conjugated forms of each one was used to immunize 3 5-6 weeks old Balb / c females. 5 injections of each peptide per mouse were performed.

[0326] An ELISA test with the unconjugated peptides was carried out on the serum of the mice to identify the animal with the higher antibody titre for each peptide. The lymphocytes isolated from the spleen of the selected mice were fused with NSO myeloma cells

[0327] (Step3) Selection of clones of anti-AMHR2

[0328] The lymphocytes isolated from the spleen of the selected mice were fused with NSO myeloma cells three days after the final boost, according to the Kblher and Milstein (Nature, 1975) protocol.

[0329] • For each peptide 3 different dilutions of the fused cells were distributed in 4 24- multiwell plates (1 for 1:2 dilution, 1 for 1:4 dilution and 2 for 1:8 dilution). The supernatants of the different wells were analysed by ELISA test with the unconjugated peptides identifying the positive clones.

[0330] • For each peptide were selected at least 15 clones with ELISA test results >0,8, as reported in the following table:Table 2

[0331]

[0332] (Step4)Cloning cells peptide 3 anti-AMHR2

[0333] The cells from hybridoma 3 -Peptide 3 (anti-AMHR2) were subjected to cloning in soft agar. 94 different clones collected were amplified and their supernatants were analysed by ELISA test with the unconjugated peptides. 30 clones showing ELISA test results >1,8 were identified (see Table 3)

[0334] Table 3

[0335]

[0336] (Step4)Test neutralizing activity different clones Peptide 3 anti-AMHR2 Densitometric analysis of Western Blot results. Data of 3 independent experiments were analyzed using ImageJ, and graphs were plotted by GraphPad Prism are reported (data not shown and Figure 8; all the samples are significantly different from control (CONT).P = peptide number.The inhibition level for each sample analyzed reach the stastical significance (Step6) Purification of monoclonal anti-AMHR2, hybridoma clone 1 -Peptide 3 (HA13) Supernatants obtained from hybridoma clone 1 -Peptide 3 were collected from growing cells. The two clones were adapted to the following growth conditions:

[0337] clone 1- Peptide 3 DMEM High glucose, glutamine 400 mM, Fetal Clone serum 5% The supernatant were exchanged with Phosphate buffer 20mM pH 8,0 by HiPrep 26 / 10 Desalting column using the liquid chromatography system Akta pure 25 and the obtained samples were purified by affinity chromatography. The purification was performed on Akta pure 25 instrument with HiTrap Protein A HP columns, containing the Protein A, produced by a selected strain of Staphylococcus aureus, able to specifically bind to Fc-part of IgG. The monoclonal antibody were eluted from column by decreasing of pH, using a O,1M citric acid buffer pH 4,0 immediately neutralized with IM Tris-HCl pH 9,0.

[0338] (Step 7) Validation of neutralizing activity of monoclonal antibodies: anti-AMHR2 To further confirm the Clone 1 peptide 3 (producing anti-AMHR2 antibody) ability to inhibit AMH-AMHR2 interaction, the purified monoclonal antibodies were again tested, using KK1 cell model (mouse ovarian granulosa cell line), characterized by an autocrine stimulation of the specific AMH-AMHR2 signalling pathway. Each purified antibody was tested toevaluate its ability of inhibiting the phosphorylation (thus the activation) of SMAD proteins, one of the key protein family characterizing the AMH-AMHR2 downstream cascade. KK1 granulosa cell model (Kananen et al., 1995; Mol Endocrinol).

[0339] EXAMPLE 3: Anti AMHR2 scFv

[0340] (Stepl) Anti AMHR2 scFv assembly, production and in vitro testing

[0341] To generate the engineered scFv antibody, the VH and VL sequences from anti-AMHR2 monoclonal antibody were joined in the VH-linker-VL arrangement by inserting the commonly used (Gly4Ser)3 linker peptide sequence between the variable domains. The joining was obtained by SOE per and the scFv sequences, controlled by Sanger sequencing, were inserted by SLIC method (Li and Elledge 2012) into the E. coli expression plasmid pET26b+. The plasmid carries an N-terminal pelB signal sequence, for the periplasmic localization of the ricombinant protein, and a C-terminal 6xHis-Tag sequence. The oxidizing ambient of periplasmic compartment allows the correct folding of the antibody molecules, containing disulfide bridges, while the His-Tag at the C-terminal of the recombinant proteins is used to purify and indentify the expressed scFvs. Once obtained, the vectors with the scFv DNA were controlled by sequencing and used to transform BL21 (DE3) E. coli cells, commonly used for the expression of recombinant proteins.

[0342] The BL21 (DE3) bacterial cells containing the plasmid with the anti AMHR2 scFv encoding sequence (pET26-AMHR2scFv) were tested to verify the expression of the recombinant antibody. Different conditions of growth and induction were tested to set the protocol for the higher production of anti-AMHR2 scFv. A protein of the expected size (about 30 KDa, as calculated by Expasy ProtParam Tool) was obtained in the periplasm of BL21 by growing bacterial cells at 37°C in LB medium supplemented with 50 mg / ml kanamycin. The expression was induced when the optical density at 600 nm (OD600) reached to 0,6-0, 8 by adding 0,lmM isopropyl-b-D-l-thiogalactopyranoside (IPTG) and growing bacterial cells for further 3 hours.

[0343] After isolation of periplasmic fraction, Akta pure 25 instrument was used to purify the protein of interest by affinity chromatography with HisTrap HP columns.

[0344] (Step 2) Antibody Variable Domain Sequencing of anti-AMHR2 ell P 3 Hybridomas Total RNA was isolated from frozen cl5 Pl and ell P3 hybridoma cell pellets, following the technical manual of TRIzol® Reagent. Total RNA was then treated with DNAse I and reverse transcribed into cDNA using oligo-dT, to specifically select mature mRNA.The primers used for the variable domains' isolation were modified from Wang et al., 2000.

[0345] For anti AMHR2 (ell P3) antibodies, k chain isotype-specific reverse and degenerate forward primers were used to isolate VL sequences, by amplification with high fidelity Taq polymerase. Isotype IgG2a and IgM reverse and 5 different degenerate forward primers were used for the isolation of VH sequences of anti AMHR2 antibodies. The amplification products of the expected size (350-400 bp) for each variable domain were cloned separately in pCR-blunt vector. Colony PCR screening and / or enzyme restriction analysis on extracted plasmids were carried out to identify clones with inserts of the correct size. The selected ones were sequenced for the identification of VL and VH in each clone.

[0346] Identification of VL sequence

[0347] The sequencing results of 15 different clones selected to contain an insert of the right size were analyzed by IMGT / V Quest program and, after identification of the productive rearranged sequence, the aminoacid sequence was also analyzed with the Kabat scheme (www.bioinf.org.uk)

[0348] Identification of VH sequence

[0349] Amplification products of the expected size were obtained using only one of the 5 different degenerate forward primers together with the isotype specific reverse primer. More than 80 different clones selected to contain into the pCR-blunt vector an insert of the right size were analyzed by IMGT / V Quest program to obtain the productive rearranged sequence. Most of them indeed displayed aspecific sequences (no result on IMGT / V Quest program) or no rearranged sequence, devoid of CDR3 and with an internal stop codon. Once identified the productive rearranged sequence, it was also analyzed with the Kabat scheme (www.bioinf.org.uk).

[0350] DNA and protein sequences of variable domains of antibody produced by ell P3 (anti AMHR2) hybridomas are reported in table 1 (see below).

[0351] (Step 3) Anti AMHR2 scFv production

[0352] The BL21 (DE3) bacterial cells containing the plasmid with the anti AMHR2 scFv encoding sequence (pET26-AMHR2scFv) were tested to verify the expression of the recombinant antibody.

[0353] Different conditions of growth and induction were tested to set the protocol for the higher production of anti-AMHR2 scFv. A protein of the expected size (about 30 KDa, as calculated by Expasy ProtParam Tool) was obtained in the periplasm of BL21 by growing bacterial cells at 37°C in LB medium supplemented with 50 mg / ml kanamycin. The expressionwas induced when the optical density at 600 nm (OD600) reached to 0,6-0, 8 by adding 0,lmM isopropyl-b-D-1- thiogalactopyranoside (IPTG) and growing bacterial cells for further 3 hours.

[0354] After isolation of periplasmic fraction, Akta pure 25 instrument was used to purify the protein of interest by affinity chromatography with HisTrap HP columns.

[0355] In order to test the ability of the anti-AMHR2 scFv antibody to inhibit AMH-AMHR2 interaction the antibody purified after expression in bacterial cells was tested, using one of the Dival cell model, characterized by an autocrine stimulation of the specific AMH-AMHR2 signalling pathway. Different concentrations of protein were tested in vitro to evaluate the ability of inhibiting the phosphorylation (thus the activation) of SMAD, one of the key protein characterizing the AMH- AMHR2 downstream cascade. Experiments were performed focusing at short lead time of inhibition (< 2h), after evaluating the time course of SMAD phosphorilation in Dival experimental model. All the Western Blot data obtained by each concentration of scFv were statistically analyzed using Student t-test (n = 3). The results are shown (Figure 9A-9C).

[0356] The anti-AMHR2 scFv antibody shows statistically significant levels of inhibition at the higher concentrations used (12,5 mg / ml, 25 mg / ml and 50 mg / ml), reaching a maximal inhibition of 41,8% with 50 mg / ml concentration (panel B and C of Figure 9), thus indicating a corresponding inhibition of the AMH-AMHR2 interaction.Table 4: Useful amino acid sequences and nucleotidic sequence for practicing the invention

[0357]

[0358] REFERENCES:

[0359] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0360] 1. Stener-Victorin, E., Teede, H., Norman, R.J., Legro, R., Goodarzi, M.O., Dokras, A., Laven, J., Hoeger, K., and Piltonen, T.T. (2024). Polycystic ovary syndrome. Nat Rev Dis Primers 10, 27. 10.1038 / s41572-024-00511-3.

[0361] 2. Teede, H.J., Tay, C.T., Laven, J., Dokras, A., Moran, L.J., Piltonen, T.T., Costello, M.L., Boivin, J., L, M.R., J, A.B., et al. (2023). Recommendations from the 2023 International Evidencebased Guideline for the Assessment and Management of Polycystic Ovary Syndrome, fertility and sterility 120. 767-793. 10.1016 / j.fertnstert.2023.07.025.

[0362] 3. Escobar-Morreale, H.F. (2018). Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment. Nature reviews. Endocrinology 14, 270-284. 10.1038 / nrendo.2018.24.

[0363] 4. Livadas, S., Pappas, C., Karachalios, A., Marinakis, E., Tolia, N., Drakou, M., Kaldrymides, P., Panidis, D., and Diamanti-Kandarakis, E. (2014). Prevalence and impact of hyperandrogenemia in 1,218 women with polycystic ovary syndrome. Endocrine 47, 631-638.

[0364] 10.1007 / S12020-014-0200-7.

[0365] 5. Joham, A.E., Norman, R.J., Stener-Victorin, E., Legro, R.S., Franks, S., Moran, L.J., Boyle, J., and Teede, H.J. (2022). Polycystic ovary syndrome. Lancet Diabetes Endocrinol 10, 668-680.

[0366] 10.1016 / S2213-8587(22)00163-2.

[0367] 6. Pigny, P., Jonard, S., Robert, Y., and Dewailly, D. (2006). Serum anti-Mullerian hormone as a surrogate for antral follicle count for definition of the polycystic ovary syndrome. The Journal of clinical endocrinology and metabolism 91, 941-945. 10.1210 / jc.2005-2076.

[0368] 7. Cook, C.L., Siow, Y., Brenner, A.G., and Fallat, M.E. (2002). Relationship between serum mullerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women. Fertility and sterility 77, 141-146. 10.1016 / s0015-0282(01)02944-2.

[0369] 8. Dietz de Loos, A., Hund, M., Buck, K., Meun, C., Sillman, J., and Laven, J.S.E. (2021). Antimullerian hormone to determine polycystic ovarian morphology. Fertility and sterility 116, 1149-1157. 10.1016 / j.fertnstert.2021.05.094.

[0370] 9. Cate, R.L., Mattaliano, R.J., Hession, C., Tizard, R., Farber, N.M., Cheung, A., Ninfa, E.G., Frey, A.Z., Gash, D.J., Chow, E.P., and et al. (1986). Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal cells. Cell 45, 685-698.

[0371] 0092-8674(86)90783-X [pii],

[0372] 10. Cimino, I., Casoni, F., Liu, X., Messina, A., Parkash, J., Jamin, S.P., Catteau-Jonard, S., Collier, F., Baroncini, M., Dewailly, D., et al. (2016). Novel role for anti-Mullerian hormone in theregulation of GnRH neuron excitability and hormone secretion. Nature communications 7, 10055.

[0373] 10.1038 / ncommsl0055.

[0374] 11. Silva, M.S.B., and Giacobini, P. (2021). New insights into anti-Mullerian hormone role in the hypothalamic-pituitary-gonadal axis and neuroendocrine development. Cellular and molecular life sciences : CMLS 78, 1-16. 10.1007 / s00018-020-03576-x.

[0375] 12. Barbotin, A.L., Mimouni, N.E.H., Kuchcinski, G., Lopes, R., Viard, R., Rasika, S., Mazur, D., Silva, M.S.B., Simon, V., Boursier, A., et al. (2023). Hypothalamic neuroglial plasticity is regulated by anti-Mullerian hormone and disrupted in polycystic ovary syndrome. EBioMedicine 90, 104535. 10.1016 / j.ebiom.2023.104535.

[0376] 13. Crisosto, N., Codner, E., Maliqueo, M., Echiburu, B., Sanchez, F., Cassorla, F., and Sir-Petermann, T. (2007). Anti-Mullerian hormone levels in peripubertal daughters of women with polycystic ovary syndrome. The Journal of clinical endocrinology and metabolism 92, 2739-2743.

[0377] 10.1210 / jc.2007-0267.

[0378] 14. Gorsic, L.K., Kosova, G., Werstein, B., Sisk, R., Legro, R.S., Hayes, M.G., Teixeira, J.M., Dunaif, A., and Urbanek, M. (2017). Pathogenic Anti-Mullerian Hormone Variants in Polycystic Ovary Syndrome. The Journal of clinical endocrinology and metabolism 102, 2862-2872.

[0379] 10.1210 / jc.2017-00612.

[0380] 15. Gorsic, L.K., Dapas, M., Legro, R.S., Hayes, M.G., and Urbanek, M. (2019). Functional Genetic Variation in the Anti-Mullerian Hormone Pathway in Women With Polycystic Ovary Syndrome. The Journal of clinical endocrinology and metabolism 104, 2855-2874. 10.1210 / jc.2018-02178.

[0381] 16. Crisosto, N., Ladron de Guevara, A., Echiburu, B., Maliqueo, M., Cavada, G., Codner, E., Paez, F., and Sir-Petermann, T. (2019). Higher luteinizing hormone levels associated with antimullerian hormone in postmenarchal daughters of women with polycystic ovary syndrome. Fertility and sterility 111, 381-388. 10.1016 / j.fertnstert.2018.10.011.

[0382] 17. Risal, S., Pei, Y., Lu, H., Manti, M., Fomes, R., Pui, H.P., Zhao, Z., Massart, J., Ohlsson, C., Lindgren, E., et al. (2019). Prenatal androgen exposure and transgenerational susceptibility to polycystic ovary syndrome. Nat Med 25, 1894-1904. 10.1038 / s41591 -019-0666- 1.

[0383] 18. Stener-Victorin, E., and Deng, Q. (2021). Epigenetic inheritance of polycystic ovary syndrome - challenges and opportunities for treatment. Nature reviews. Endocrinology 17, 521-533.

[0384] 10.1038 / s41574-021-00517-x.

[0385] 19. Mimouni, N.E.H., Paiva, I., Barbotin, A.L., Timzoura, F.E., Plassard, D., Le Gras, S., Temier, G., Pigny, P., Catteau-Jonard, S., Simon, V., et al. (2021). Polycystic ovary syndrome is transmitted via a transgenerational epigenetic process. Cell metabolism 33, 513-530 e518.

[0386] 10.1016 / j.cmet.2021.01.004.

[0387] 20. Peigne, M., Simon, V., Pigny, P., Mimouni, N.E.H., Martin, C., Dewailly, D., Catteau-Jonard, S., and Giacobini, P. (2023). Changes in circulating forms of anti-Muullerian hormone andandrogens in women with and without PCOS: a systematic longitudinal study throughout pregnancy. Human reproduction 38, 938-950. 10.1093 / humrep / dead050.

[0388] 21. Tata, B., Mimouni, N.E.H., Barbotin, A.L., Malone, S.A., Loyens, A., Pigny, P., Dewailly, D., Catteau-Jonard, S., Sundstrom-Poromaa, I., Piltonen, T.T., et al. (2018). Elevated prenatal anti-Mullerian hormone reprograms the fetus and induces polycystic ovary syndrome in adulthood. Nat Med 24, 834-846. 10.1038 / s41591-018-0035-5.

[0389] 22. Silva, M.S.B., Decoster, L., Trova, S., Mimouni, N.E.H., Delli, V., Chachlaki, K., Yu, Q., Boehm, U., Prevot, V., and Giacobini, P. (2022). Female sexual behavior is disrupted in a preclinical mouse model of PCOS via an attenuated hypothalamic nitric oxide pathway. Proceedings of the National Academy of Sciences of the United States of America 119, e2203503119. 10.1073 / pnas.2203503119.

[0390] 23. Sir-Petermann, T., Codner, E., Maliqueo, M., Echiburu, B., Hitschfeld, C., Crisosto, N., Perez-Bravo, F., Recabarren, S.E., and Cassorla, F. (2006). Increased anti-Mullerian hormone serum concentrations in prepubertal daughters of women with polycystic ovary syndrome. The Journal of clinical endocrinology and metabolism 91, 3105-3109. 10.1210 / jc.2005-2693.

[0391] 24. Tadaion Far, F., Jahanian Sadatmahalleh, S., Ziaei, S., and Kazemnejad, A. (2019). Comparison of the umbilical cord Blood's anti-Mullerian hormone level in the newborns of mothers with polycystic ovary syndrome (PCOS) and healthy mothers. J Ovarian Res 12, 111. 10.1186 / s 13048-019-0583-4.

[0392] 25. Zhou, S., Lu, D., Wen, S., Sheng, Y ., Kang, D., and Xu, L. (2022). Elevated Anti-Mullerian Hormone Levels in Newborns of Women with Polycystic Ovary Syndrome: a Systematic Review and Meta-analysis Based on Observational Studies. Reprod Sci 29, 301-311. 10.1007 / s43032-021-00652-w.

[0393] 26. Rohayem, J., Alexander, E.C., Heger, S., Nordenstrom, A., and Howard, S.R. (2024). Mini-Puberty, Physiological and Disordered: Consequences, and Potential for Therapeutic Replacement. Endocrine reviews. 10.1210 / endrev / bnae003.

[0394] 27. Chachlaki, K., Messina, A., Delli, V., Leysen, V., Maumyi, C., Huber, C., Temier, G., Skrapits, K., Papadakis, G., Shruti, S., et al. (2022). NOS1 mutations cause hypogonadotropic hypogonadism with sensory and cognitive deficits that can be reversed in infantile mice. Sci Transl Med 14, eabh2369. 10.1126 / scitranslmed.abh2369.

[0395] 28. Chachlaki, K., Le Due, K., Storme, L., and Prevot, V. (2024). Novel insights into minipuberty and GnRH: Implications on neurodevelopment, cognition, and COVID-19 therapeutics. Journal of neuroendocrinology, el3387. 10.1111 / jne.13387.

[0396] 29. Prevot, V., Rio, C., Cho, G.J., Lomniczi, A., Heger, S., Neville, C.M., Rosenthal, N.A., Ojeda, S.R., and Corfas, G. (2003). Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes. J Neurosci 23, 230-239. 23 / 1 / 230 [pii] .

[0397] 30. Tschop, M., Smiley, D.L., and Heiman, M.L. (2000). Ghrelin induces adiposity in rodents. Nature 407, 908-913. 10.1038 / 35038090.31. Bruning, J.C., and Fenselau, H. (2023). Integrative neurocircuits that control metabolism and food intake. Science 381, eabl7398. 10.1126 / science.abl7398.

[0398] 32. Simerly, R.B., and Young, B.J. (1991). Regulation of estrogen receptor messenger ribonucleic acid in rat hypothalamus by sex steroid hormones. Molecular endocrinology 5, 424-432.

[0399] 10.1210 / mend-5-3-424.

[0400] 33. Moore, A.M., Prescott, M., Marshall, C.J., Yip, S.H., and Campbell, R.E. (2015). Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome. Proceedings of the National Academy of Sciences of the United States of America 112, 596-601. 10.1073 / pnas.1415038112.

[0401] 34. McQuillan, H.J., Clarkson, J., Kauff, A., Han, S.Y., Yip, S.H., Cheong, I., Porteous, R., Heather, A.K., and Herbison, A.E. (2022). Definition of the estrogen negative feedback pathway controlling the GnRH pulse generator in female mice. Nature communications 13, 7433.

[0402] 10.1038 / s41467-022-35243-z.

[0403] 35. Dubois, S.L., Acosta-Martinez, M., DeJoseph, M.R., Wolfe, A., Radovick, S., Boehm, U., Urban, J.H., and Levine, J.E. (2015). Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor alpha in kisspeptin neurons. Endocrinology 156, 1111-1120.

[0404] 10.1210 / en.2014-1851.

[0405] 36. McCartney, C.R., Campbell, R.E., Marshall, J.C., and Moenter, S.M. (2022). The role of gonadotropin-releasing hormone neurons in polycystic ovary syndrome. Journal of neuroendocrinology 34, el3093. 10.1111 / jne.13093.

[0406] 37. Stener- Victorin, E., Padmanabhan, V., Walters, K.A., Campbell, R.E., Benrick, A., Giacobini, P., Dumesic, D.A., and Abbott, D.H. (2020). Animal Models to Understand the Etiology and Pathophysiology of Polycystic Ovary Syndrome. Endocrine reviews 41. 10.1210 / endrev / bnaa010.

[0407] 38. Hevener, A.L., Clegg, D.J., and Mauvais-Jarvis, F. (2015). Impaired estrogen receptor action in the pathogenesis of the metabolic syndrome. Molecular and cellular endocrinology 418 Pt 3, 306-321. 10.1016 / j.mce.2015.05.020.

[0408] 39. Hart, K.N., Stocker, W.A., Nagykery, N.G., Walton, K.L., Harrison, C.A., Donahoe, P.K., Pepin, D., and Thompson, T.B. (2021). Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-beta family. Proceedings of the National Academy of Sciences of the United States of America 118. 10.1073 / pnas.2104809118.

[0409] 40. Kevenaar, M.E., Laven, J.S., Fong, S.L., Uitterlinden, A.G., de Jong, F.H., Themmen, A.P., and Visser, J.A. (2008). A functional anti-mullerian hormone gene polymorphism is associated with follicle number and androgen levels in polycystic ovary syndrome patients. The Journal of clinical endocrinology and metabolism 93, 1310-1316. 10.1210 / jc.2007-2205.

[0410] 41. Hoyos, L.R., Visser, J.A., McLuskey, A., Chazenbalk, G.D., Grogan, T.R., and Dumesic, D.A. (2020). Loss of anti-Mullerian hormone (AMH) immunoactivity due to a homozygousAMH gene variant rsl0417628 in a woman with classical polycystic ovary syndrome (PCOS). Human reproduction 35, 2294-2302. 10.1093 / humrep / deaal99.

[0411] 42. Oliveira, F.R., Mamede, M., Bizzi, M.F., Rocha, A.L.L., Ferreira, C.N., Gomes, K.B., Candido, A.L., and Reis, F.M. (2019). Brown adipose tissue activity is reduced in women with polycystic ovary syndrome. European journal of endocrinology / European Federation of Endocrine Societies 181, 473-480. 10.1530 / EJE-19-0505.

[0412] 43. Li, L., Scotti, A., Fang, J., Yin, L., Xiong, T., He, W., Qin, Y., Liew, C., Khayyat, N., Zhu, W., and Cai, K. (2020). Characterization of brown adipose tissue (BAT) in polycystic ovary syndrome (PCOS) patients by Z-Spectral Imaging (ZSI). Eur J Radiol 123, 108777.

[0413] 10.1016 / j.ejrad.2019.108777.

[0414] 44. Manneras-Holm, L., Leonhardt, H., Kullberg, J., Jennische, E., Oden, A., Holm, G., Hellstrom, M., Lonn, L., Olivecrona, G., Stener-Victorin, E., and Lonn, M. (2011). Adipose tissue has aberrant morphology and function in PCOS: enlarged adipocytes and low serum adiponectin, but not circulating sex steroids, are strongly associated with insulin resistance. The Journal of clinical endocrinology and metabolism 96, E304-311. 10.1210 / jc.2010-1290.

[0415] 45. Echiburu, B., Perez-Bravo, F., Galgani, J.E., Sandoval, D., Saldias, C., Crisosto, N., Maliqueo, M., and Sir-Petermann, T. (2018). Enlarged adipocytes in subcutaneous adipose tissue associated to hyperandrogenism and visceral adipose tissue volume in women with polycystic ovary syndrome. Steroids 130, 15-21. 10.1016 / j. steroids.2017.12.009.

[0416] 46. Risal, S., Li, C., Luo, Q., Fomes, R., Lu, H., Eriksson, G., Manti, M., Ohlsson, C., Lindgren, E., Crisosto, N., et al. (2023). Transgenerational transmission of reproductive and metabolic dysfunction in the male progeny of polycystic ovary syndrome. Cell Rep Med 4, 101035.

[0417] 10.1016 / j.xcrm.2023.101035.

[0418] 47. Chen, X., Koivuaho, E., Piltonen, T.T., Gissler, M., and Lavebratt, C. (2021). Association of maternal polycystic ovary syndrome or anovulatory infertility with obesity and diabetes in offspring: a population-based cohort study. Human reproduction 36, 2345-2357.

[0419] 10.1093 / humrep / deab 112.

[0420] 48. Recabarren, S.E., Sir-Petermann, T., Rios, R., Maliqueo, M., Echiburu, B., Smith, R., Rojas-Garcia, P., Recabarren, M., and Rey, R.A. (2008). Pituitary and testicular function in sons of women with polycystic ovary syndrome from infancy to adulthood. The Journal of clinical endocrinology and metabolism 93, 3318-3324. 10.1210 / jc.2008-0255.

[0421] 49. Recabarren, S.E., Smith, R., Rios, R., Maliqueo, M., Echiburu, B., Codner, E., Cassorla, F., Rojas, P., and Sir-Petermann, T. (2008). Metabolic profde in sons of women with polycystic ovary syndrome. The Journal of clinical endocrinology and metabolism 93, 1820-1826. 10.1210 / jc.2007-2256.

[0422] 50. Crisosto, N., Echiburu, B., Maliqueo, M., Luchsinger, M., Rojas, P., Recabarren, S., and Sir-Petermann, T. (2017). Reproductive and metabolic features during puberty in sons of women with polycystic ovary syndrome. Endocr Connect 6, 607-613. 10.1530 / EC-17-0218.51. Lopez, M., and Tena-Sempere, M. (2015). Estrogens and the control of energy homeostasis: a brain perspective. Trends in endocrinology and metabolism: TEM 26, 411-421.

[0423] 10.1016 / j.tem.2015.06.003.

[0424] 52. Willis, C.L., Garwood, C.J., and Ray, D.E. (2007). A size selective vascular barrier in the rat area postrema formed by perivascular macrophages and the extracellular matrix. Neuroscience 150, 498-509. 10.1016 / j.neuroscience.2007.09.023.

[0425] 53. Monroe, B.G., and Holmes, E.M. (1983). The freeze-fractured median eminence. II. Developmental changes in the neurohemal contact zone of the median eminence of the rat. Cell and tissue research 233, 81-97. 10.1007 / BF00222234.

[0426] 54. Ciofi, P., Garret, M., Lapirot, O., Lafon, P., Loyens, A., Prevot, V., and Levine, J.E. (2009). Brain-endocrine interactions: a microvascular route in the mediobasal hypothalamus. Endocrinology 150, 5509-5519. 10.1210 / en.2009-0584.

[0427] 55. George, J.T., Kakkar, R., Marshall, J., Scott, M.L., Finkelman, R.D., Ho, T.W., Veldhuis, J., Skorupskaite, K., Anderson, R.A., McIntosh, S., and Webber, L. (2016). Neurokinin B Receptor Antagonism in Women With Polycystic Ovary Syndrome: A Randomized, Placebo-Controlled Trial. The Journal of clinical endocrinology and metabolism 101, 4313-4321. 10.1210 / jc.2016-1202.

[0428] 56. Skorupskaite, K., George, J.T., Veldhuis, J.D., Millar, R.P., and Anderson, R.A. (2020). Kisspeptin and neurokinin B interactions in modulating gonadotropin secretion in women with polycystic ovary syndrome. Human reproduction 35, 1421-1431. 10.1093 / humrep / deaal04.

[0429] 57. Abbara, A., and Dhillo, W.S. (2021). Targeting Elevated GnRH Pulsatility to Treat Polycystic Ovary Syndrome. The Journal of clinical endocrinology and metabolism 106, Q4215-Q4211.

[0430] 10.1210 / clinem / dgab422.

[0431] 58. Goodman, R.L., Herbison, A.E., Lehman, M.N., and Navarro, V.M. (2022). Neuroendocrine control of gonadotropin-releasing hormone: Pulsatile and surge modes of secretion. Journal of neuroendocrinology 34, el3094. 10.1111 / jne.13094.

[0432] 59. Fraser, G.L., Obermayer-Pietsch, B., Laven, J., Griesinger, G., Pintiaux, A., Timmerman, D., Fauser, B., Lademacher, C., Combalbert, J., Hoveyda, H.R., and Ramael, S. (2021). Randomized Controlled Trial of Neurokinin 3 Receptor Antagonist Fezolinetant for Treatment of Polycystic Ovary Syndrome. The Journal of clinical endocrinology and metabolism 106, e3519-e3532.

[0433] 10.1210 / clinem / dgab320.

[0434] 60. Abbott, D.H., Hutcherson, B.A., and Dumesic, D.A. (2024). Anti-Mullerian Hormone: A Molecular Key to Unlocking Polycystic Ovary Syndrome? Seminars in reproductive medicine 42, 41-48. 10.1055 / S-0044-1787525.

[0435] 61. Legro, R.S., Driscoll, D., Strauss, J.F., 3rd, Fox, J., and Dunaif, A. (1998). Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc Natl Acad Sci U S A 95, 14956-14960. 10.1073 / pnas.95.25.14956.62. Durlinger, A.L., Gruijters, M.J., Kramer, P., Karels, B., Kumar, T.R., Matzuk, M.M., Rose, U.M., de Jong, F.H., Uilenbroek, J.T., Grootegoed, J.A., and Themmen, A.P. (2001). Anti-Mullerian hormone atenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 142, 4891-4899. 10.1210 / endo.l42.11.8486.

[0436] 63. Fisch, S.C., Nikou, A.F., Wright, E.A., Phan, J.D., Leung, K.L., Grogan, T.R., Abbot, D.H., Chazenbalk, G.D., and Dumesic, D.A. (2018). Precocious subcutaneous abdominal stem cell development to adipocytes in normal-weight women with polycystic ovary syndrome. Fertility and sterility 110, 1367-1376. 10.1016 / j.fertnstert.2018.08.042.

[0437] 64. Dumesic, D.A., Tulberg, A., Leung, K.L., Fisch, S.C., Grogan, T.R., Abbot, D.H., Naik, R., and Chazenbalk, G.D. (2021). Accelerated subcutaneous abdominal stem cell adipogenesis predicts insulin sensitivity in normal-weight women with polycystic ovary syndrome. Fertility and sterility 116, 232-242. 10.1016 / j.fertnstert.2020.10.003.

[0438] 65. O'Reilly, M.W., Kempegowda, P., Walsh, M., Taylor, A.E., Manolopoulos, K.N., Allwood, J.W., Semple, R.K., Hebenstreit, D., Dunn, W.B., Tomlinson, J.W., and Arlt, W. (2017). AKR1C3-Mediated Adipose Androgen Generation Drives Lipotoxicity in Women With Polycystic Ovary Syndrome. The Journal of clinical endocrinology and metabolism 102, 3327-3339.

[0439] 10.1210 / jc.2017-00947.

[0440] 66. Chazenbalk, G., Singh, P., Irge, D., Shah, A., Abbot, D.H., and Dumesic, D.A. (2013). Androgens inhibit adipogenesis during human adipose stem cell commitment to preadipocyte formation. Steroids 78, 920-926. 10.1016 / j.steroids.2013.05.001.

[0441] 67. Blouin, K., Nadeau, M., Perreault, M., Veilleux, A., Drolet, R., Marceau, P., Mailloux, J., Luu-The, V., and Tchemof, A. (2010). Effects of androgens on adipocyte differentiation and adipose tissue explant metabolism in men and women. Clinical endocrinology 72, 176-188. 10.1111 / j .1365-2265.2009.03645.x.

[0442] 68. Bril, F., Ezeh, U., Amiri, M., Hatoum, S., Pace, L., Chen, Y.H., Bertrand, F., Gower, B., and Azziz, R. (2023). Adipose Tissue Dysfunction in Polycystic Ovary Syndrome. The Journal of clinical endocrinology and metabolism 109, 10-24. 10.1210 / clinem / dgad356.

[0443] 69. Chin, H.B., Krall, J.R., Goldberg, M., Stanczyk, F.Z., Darge, K., Stallings, V.A., Rogan, W.J., Umbach, D.M., and Baird, D.D. (2023). Early Life Anti-Mullerian Hormone Trajectories in Infant Girls. Epidemiology 34, 568-575. 10.1097 / EDE.0000000000001610.

[0444] 70. Ljubicic, M.L., Busch, A.S., Upners, E.N., Fischer, M.B., Petersen, J.H., Raket, L.L., Frederiksen, H., Johannsen, T.H., Juul, A., and Hagen, C.P. (2022). A Biphasic Patern of Reproductive Hormones in Healthy Female Infants: The COPENHAGEN Minipuberty Study. The Journal of clinical endocrinology and metabolism 107, 2598-2605. 10.1210 / clinem / dgac363.

[0445] 71. Fischer, M.B., Mola, G., Rom, A.L., Frederiksen, H., Holm Johannsen, T., Sundberg, K., Hegaard, H.K., Juul, A., and Hagen, C.P. (2024). Ovarian and Uterine Morphology in Minipuberty:Associations with Reproductive Hormones. A COPANA Study of 302 girls. The Journal of clinical endocrinology and metabolism. 10.1210 / clinem / dgae678.

[0446] 72. Kuiri-Hanninen, T., Sankilampi, U., and Dunkel, L. (2014). Activation of the hypothalamic-pituitary-gonadal axis in infancy: minipuberty. Horn Res Paediatr 82, 73-80.

[0447] 10.1159 / 000362414.

[0448] 73. Yoon, H., Enquist, L.W., and Dulac, C. (2005). Olfactory inputs to hypothalamic neurons controlling reproduction and fertility. Cell 123, 669-682. 10.1016 / j .cell.2005.08.039.

[0449] 74. Esparza, L.A., Schafer, D., Ho, B.S., Thackray, V.G., and Kauffman, A.S. (2020). Hyperactive LH Pulses and Elevated Kisspeptin and NKB Gene Expression in the Arcuate Nucleus of a PCOS Mouse Model. Endocrinology 161. 10.1210 / endocr / bqaa018.

[0450] 75. Giton, F., Trabado, S., Maione, L., Sarfati, J., Le Bouc, Y ., Brailly-Tabard, S., Fiet, J., and Young, J. (2015). Sex steroids, precursors, and metabolite deficiencies in men with isolated hypogonadotropic hypogonadism and panhypopituitarism: a GCMS-based comparative study. The Journal of clinical endocrinology and metabolism 100, E292-296. 10.1210 / jc.2014-2658.

[0451] 76. Caldwell, A.S.L., Edwards, M.C., Desai, R., Jimenez, M., Gilchrist, R.B., Handelsman, D.J., and Walters, K.A. (2017). Neuroendocrine androgen action is a key extraovarian mediator in the development of polycystic ovary syndrome. Proceedings of the National Academy of Sciences of the United States of America 114, E3334-E3343. 10.1073 / pnas.1616467114.

[0452] 77. Folgueira, C., Beiroa, D., Porteiro, B., Duquenne, M., Puighermanal, E., Fondevila, M.F., Baqa-Femandez, S., Gallego, R., Hernandez-Bautista, R., Castelao, C., et al. (2019). Hypothalamic dopamine signaling regulates brown fat thermogenesis. Nat Metab 1, 811-829.

[0453] 10.1038 / s42255-019-0099-7.

[0454] 78. Bruss, M.D., Khambatta, C.F., Ruby, M.A., Aggarwal, I., and Hellerstein, M.K. (2010). Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates. American journal of physiology. Endocrinology and metabolism 298, E108-116. 10.1152 / ajpendo.00524.2009.

[0455] 79. Kohler, G., and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497. 10.1038 / 256495a0.

[0456] 80. Galfre, G., and Milstein, C. (1981). Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 73, 3-46. 10.1016 / 0076-6879(81)73054-4.

[0457] 81. Kananen, K., Markkula, M., Rainio, E., Su, J.G., Hsueh, A. J., and Huhtaniemi, I.T. (1995). Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter / simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Molecular endocrinology 9, 616-627.

[0458] 10.1210 / mend.9.5.7565808.

[0459] 82. Chauvin, M., Garambois, V., Colombo, P.E., Chentouf, M., Gros, L., Brouillet, J.P., Robert, B., Jarlier, M., Dumas, K., Martineau, P., et al. (2021). Anti-Mullerian hormone (AMH)autocrine signaling promotes survival and proliferation of ovarian cancer cells. Sci Rep 11, 2231.

[0460] 10.1038 / s41598-021-81819-y.

[0461] 83. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408.

[0462] 10.1006 / meth.2001.1262.

[0463] 84. Manfredi-Lozano, M., Leysen, V., Adamo, M., Paiva, I., Rovera, R., Pignat, J.M., Timzoura, F.E., Candlish, M., Eddarkaoui, S., Malone, S.A., et al. (2022). GnRH replacement rescues cognition in Down syndrome. Science 377, eabq4515. 10.1126 / science.abq4515.

Claims

-n -CLAIMS:

1. An isolated anti-AMHR2 antibody, wherein said antibody specifically binds to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO: 1) of the AMHR2 protein.

2. The isolated anti-AMHR2 antibody according to claim 1, wherein said antibody is a neutralizing antibody3. The isolated anti-AMHR2 antibody according to claim 1 or 2, wherein said antibody is a monoclonal antibody or an antibody fragment selected form the list consisting of Fab, Fab', F(ab')2 Fv, scFv, dsFv, diabodies, tribodies, or tetrabodies.

4. The isolated anti-AMHR2 antibody to any one of claim 1 to 3 wherein the antibody comprising a variable heavy chain having at least 70% of identity with sequence set forth as SEQ ID NO:2 and a variable light chain having at least 70% of identity with sequence set forth as SEQ ID NO:3.

5. The isolated anti-AMHR2 antibody according to claim 4, wherein said antibody has a sequence of variable heavy chain (VH) set forth as SEQ ID NO:2 and has a sequence of variable light chain (VL) set forth as SEQ ID NO:

36. The isolated anti-AMHR2 antibody according to any one of claim 1 to 5 wherein said antibody comprises(a) a heavy chain wherein the variable domain comprisesa VH-CDR1 having at least 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR1 of antibody HA13 (SEQ ID NO:4),a VH-CDR2 having at least 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR2 of antibody HA13 (SEQ ID NO:5) and a VH-CDR3 having at least 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR3 of antibody HA13 (SEQ ID NO:6).(b) a light chain wherein the variable domain comprisesa VL-CDR1 having at least 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VL-CDR1 of antibody HA13 (SEQ ID NO:7),- 78 -a VL-CDR2 having at least, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR2 of antibody HA13 (SEQ ID NO:8) anda VL-CDR3 having at least 9, 8, 7, 6, 5, 4, 3, 2, 1 conservative substitutions within the VH-CDR3 of antibody HA13 (SEQ ID NO:9).

7. The isolated anti-AMHR2 antibody according to claim 6, wherein said antibody comprisea heavy chain having the VH-CDR1 as set forth in SEQ ID NO:4, VH-CDR2 as set forth in SEQ ID NO:5 and VH-CDR3 as set forth in SEQ ID NO: 6 and a light chain having the VL-CDR1, CDR1 as set forth in SEQ ID NO:7, VL- CDR2 as set forth in SEQ ID NO:8 and VL-CDR3 as set forth in SEQ ID NO:9.

8. An isolated antibody, which cross-blocks or is cross-blocked by at least one antibody according to Claim 7, from binding to an epitope comprising residues GCESLHCDPSPRAHPSPGSTLFTCSCGT (SEQ ID NO:1) of the AMHR2 protein.

9. The isolated anti-AMHR2 antibody according to any one of claim 1 to 8 is a humanized antibody or a chimeric antibody.

10. A nucleic acid sequence encoding the isolated anti-AMHR2 antibody according to anyone of claims 1 to 9.

11. A vector comprising a nucleic acid sequence encoding the isolated anti-AMHR2 antibody according to claims 10.

12. A pharmaceutical composition comprising the isolated anti-AMHR2 antibody according to according to anyone of claims 1 to 9, or the vector of claim 11 comprising a nucleic acid sequence encoding the isolated anti-AMHR2 antibody according to claim 10.

13. The pharmaceutical composition according to claims 12; or the isolated anti-AMHR2 antibody according to anyone of claims 1 to 9; or the vector of claim 11 comprising a nucleic acid sequence encoding the isolated anti-AMHR2 antibody according to claim 10, for use in the treatment of PCOS.- 79 -14. The pharmaceutical composition for use according to claims 13, wherein the subject to be treated is a girl, born to a mother with PCOS, at mini-pubertal (1-6 months) or a preadolescent girl (8-10 years old) or an adolescent girl (11-17 years) or an adult woman (more than 18 years old).

15. A method for treating a PCOS comprising administering a subject in need thereof with a therapeutically effective amount of the pharmaceutical composition according to claims 12; or the isolated anti-AMHR2 antibody according to anyone of claims 1 to 9 or the vector of claim 11 comprising a nucleic acid sequence encoding the isolated anti- AMHR2 antibody according to claim 10.

Images