WO2011045202A1 - Selective antagonist or agonist of amhrii for modulating fertility - Google Patents

Abstract

The present invention relates to the use of selective anti-Mϋllerian Hormone type II receptor (AMHRII) antagonists for improving female fertility and/or treating female infertility and to the use of selective AMHRII agonists for prolonging fertile period and postponing menopause or for inhibiting male or female fertility.

Description

SELECTIVE ANTAGONIST OR AGONIST OF AMHRII FOR MODULATING

FERTILITY

Related Applications

The present application claims priority to European Patent Application No. EP 09 305 968 filed on October 12, 2009. The European patent application is incorporated herein by reference in its entirety.

Background of the Invention

Anti-Miillerian hormone (AMH), also called Miillerian inhibiting substance (MIS), is a 140 kDa glycoprotein hormone, which belongs to the transforming growth factor (TGF)- family of proteins involved in the regulation of cell growth and differentiation (reviewed in Josso et al, Pediatr. Endocrinol. Rev., 2006, 3: 347-358).

AMH signals by assembling a transmembrane serine/threonine kinase receptor complex of type I and type II components, resulting in the phosphorylation and activation of the latent type I receptor kinase by the constitutively active kinase domain of the type II receptor (Shi et al, Cell, 2003, 113: 685-700). The type II receptor, AMHRII, is AMH specific (Imbeaud et al, Nat. Genet., 1995, 11: 382-388; Mishina et al, Genes Dev., 1996, 10: 2577-2587), while two BMP type I receptors, activin receptor- like kinase (ALK)-2 (Clarke et al, Mol. Endocrinol., 2001, 15: 936- 945; Visser et al, Mol. Endocrinol., 2001, 15: 936-945) and ALK-3 (Jamin et al, Nat. Genet., 2002, 32: 408-410) can also serve as redundant type I receptors for AMH (Orvis et al, Biol. Reprod., 2008, 78: 994-1001). When activated by the type II receptor, the type I receptor phosphorylates the cytoplasmic Smad proteins 1, 5 or 8, which migrate into the nucleus and in concert with other transcription factors, regulate responsive genes, ultimately producing an AMH biological response (reviewed in Josso et al, Trends Endocrinol. Metab., 2003, 14: 91-97).

AMH has an important role in sexual differentiation during development. AMH is produced by the Sertoli cells of the testis in the male (Baarends et al, Endocrinology, 1995, 136: 5614-5622; Hoshiya et al, Mol. Cell Endocrinol., 2003, 211: 43-49), and by ovarian granulose cells of growing follicles in the female (Arango et al, Mol. Reprod. Dev., 2008, 75: 1154-1162; Bakkum-Gamez et al, Gynecol. Oncol., 2008, 108: 141-148). During fetal development in males, secretion of AMH from testicular Sertoli cells is essential for the regression of the Miillerian ducts, and thus the normal male reproductive tract development (Josso et al, Pediatr. Endocrinol. Rev., 2006, 3: 347-358). In the male, secretion of AMH by the Sertoli cells starts during embryogenesis and continues throughout life. Levels drop following puberty, decreasing slowly to a relatively low post-puberty value. The Miillerian ducts are the primordium for the uterus, Fallopian tubes, and upper part of the vagina (Behringer et al, Cell, 1994, 79: 415-425). In the female, serum AMH is maintained at relatively low levels when compared to the male. After puberty, when menstrual cycling begins, circulating AMH slowly decreases throughout life and becomes undetectable at menopause.

Behringer et al. studied the function of AMH with the help of an AMH null (AMH-) mouse model (Behringer et al, Cell, 1994, 79: 415-425). AMH null males develop Miillerian duct derivatives, including oviducts, a uterus, and a vagina, in addition to a complete male reproductive system. AMH null females have macroscopically normal uteri, oviducts and ovaries. Furthermore, these females are fertile and have litters of normal size. Based on the above (Behringer et al, 1994) and other mouse knockout models (AMH-/- and/or AMHRII-/- mice) (Mishina et al, Genes Dev., 1996, 10: 2577-2587; Mishina et al, Endocrinology, 1999, 140: 2084- 2088), it has been concluded that AMH does not play a prominent role in the control of follicle development or other aspects of ovarian function.

In contrast, W088/00054 describes that AMH has been found to reversely inhibit the maturation of oocytes in vitro and suggests that a blocking antibody capable of binding AMH may be used for inducing fertility in a female. However, Ueno et al. (Endocrinology, 1988, 123: 1652-1659) showed that the partially purified recombinant AMH produced from a human AMH genomic construct caused inhibition of oocyte meiosis, but when purified to homogeneity, the effect was lost.

Finally, using a AMH-/- mouse model, EP 1074265 reports that AMH is an important regulator of primordial follicle recruitment and suggests to use AMH antagonists, (defined as any fragment or derivative of AMH antagonising the function of regulating primordial follicle recruitment of AMH), for inducing fertility in a female.

The mechanism of AMH inhibitory effect on recruitment of primordial follicle into the pool of growing follicles has not yet been elucidated. Previous publications (Durlinger et al, Endocrinology, 1999, 140: 5789-5796; Visser et al, Reproduction, 2006, 131: 1-9) have reported that: (1) AMH mRNA expression is detected in ovarian granulosa cells from postnatal 3 days onward, (2) immunohistochemistry and mRNA in situ hybridization revealed specific expression of AMHRII in granulosa cells of mainly nonatretic preantral and small antral follicles, which are recruited primordial follicles, and (3) AMHRI expression is detected in all types of follicles, in granulosa cells and in oocytes. Durlinger et al. suggested that the primary action of AMH may be autocrine in nature, as granulosa cells of small follicles express both AMH and its type II receptor. The small follicles may be stimulated by AMH to produce a factor that acts on primordial follicles. As no information is available showing the expression of AMHRII in these primordial follicles, no direct effect of AMH on primordial follicles can be envisaged (Durlinger et ah, 1999; Visser et ah, 2006).

Summary of the Invention

For the first time, the present inventors have found that AMHRII is expressed in the oocytes from primordial (germ cells) to antral follicles in the ovary and in male gonocytes and in spermatogonia (immature germ cells) at all stages. More specifically, the present inventors have developed a new monoclonal antibody directed against the extracellular domain of human AMHRII and used it on archival samples to characterize the precise expression pattern of AMHRII during gonadogenesis. The AMHRII expression pattern observed suggests a direct role of AMH on the recruitment of primordial follicles.

AMHRII therefore represents a potential target for medical intervention with the goal of fertility treatment or fertility management. Accordingly, the present invention generally relates to the use of selective AMHRII antagonists and agonists for modulating female and male fertility. Indeed, since AMH inhibits the recruitment of primordial follicles into the pool of growing follicles (Durlinger et ah, 2002), AMHRII antagonists may be of great interest for improving female fertility and/or for treating female infertility disorders by increasing the recruitment of primordial follicles. In contrast, AMHRII agonists may be useful for prolonging the fertile period and postponing the menopause or for inhibiting female fertility, i.e., for contraception, by preventing recruitment of primordial follicles. Similarly, since AMH is known to inhibit meiosis (Takahashi et ah, Mol. Cell Endocrinol., 1986, 47: 225), AMHRII agonists may also find applications in male contraception. Thus, more specifically, in one aspect, the present invention relates to a selective anti-Miillerian Hormone type II receptor (AMHRII) antagonist for use in improving female fertility and/or treating female infertility and/or treating male infertility. In a related aspect, the present invention provides a method for improving female fertility and/or for treating female fertility disorders, said method comprising a step of: administering to a subject in need thereof, an efficient amount of at least one selective anti-Miillerian Hormone type II receptor (AMHRII) antagonist.

In this use and method, the selective AMHRII antagonist is not a fragment or a derivative of AMH.

In one embodiment, said AMHRII antagonist is an antibody. In another embodiment, said antagonist is an antibody directed to the extracellular domain of AMHRII.

In another embodiment, said AMHRII antagonist comprises the extracellular domain of AMHRII. In another embodiment, said AMHRII antagonist is a fusion protein comprising the extra-cellular domain of AMHRII.

In another embodiment, said AMHRII antagonist is a siRNA, shRNA, an antisense or a DNAzyme targeting AMHRII gene sequence.

A selective AMHRII antagonist according to the present invention may be useful for improving the fertility and/or for treating fertility disorders in any suitable mammal female, including women. Thus, in certain embodiments, the AMHRII antagonist is used for improving the fertility and/or for treating a fertility disorder in a woman (in particular a woman during her reproductive years). In other embodiments, the AMHRII antagonist is used for improving female fertility and/or for treating a female fertility disorder in domesticated animals, in particular cattle, sheep, goats, horses, and the like. In certain preferred embodiments, the AMHRII antagonist is used for improving female bovine infertility. In yet other embodiments, the AMHRII antagonist is used for improving female fertility and/or for treating a female fertility disorder in a companion animal, such as a dog, a cat, and the like.

In certain embodiments, a selective AMHRII antagonist of the invention is used in combination with sexual hormones for improving female fertility and/or for treating female infertility. Similarly, in certain embodiments, the method for improving female fertility and/or for treating female fertility disorders further comprises a step of administering to the subject an efficient amount of at least one sexual hormone.

The AMHRII antagonist of the invention may be administered per se or as pharmaceutical compositions. Accordingly, in another aspect, the present invention provides for the use of an inventive selective AMHRII antagonist for the manufacture of medicaments, pharmaceutical compositions or pharmaceutical kits for improving female fertility and/or for treating female fertility disorders.

In a related aspect, the present invention provides a pharmaceutical composition comprising an effective amount of an inventive selective AMHRII antagonist and at least one pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition is adapted for administration in combination with an additional biologically active agent, such as a sexual hormone. The pharmaceutical composition may be adapted for simultaneous administration of the AMHRII antagonist and the sexual hormone, or alternatively the pharmaceutical composition may be adapted for sequential administration of the AMHRII antagonist and the sexual hormone. Thus, in particular, the invention provides a kit for improving female fertility and/or treating female infertility, comprising as separated components: a selective AMHRII antagonist according to the present invention, and at least one sexual hormone.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

Brief Description of the Drawing

Figure 1 presents a time-course of hAMHR-II expression. Archival human samples were subjected to immunohistochemistry using a rabbit polyclonal anti- AMHRII antibody (see Examples section for details). The human samples tested are described in Table 1. Female samples (A- #5874, C- #7913 and E- #1321) ranged from 28 weeks of gestation to 8 years and male samples (B- #3422, D- #7904, and F- #7356) ranged from 17 weeks of gestation to 12 years.

Figure 2 presents data showing that mAb 13H8 and the polyclonal antibody N2 can bind to AMHR-II. (A) hEC-AMHR-II, or (B) mEC-AMHR-II were coated on a plate and four different antibodies were added at different concentrations. An HRP- coupled secondary antibody was added and a colorimetric reaction developed and was measured at 450 nm.

Figure 3 presents data showing that mAb 13H8 can detect both human and mouse AMHR-II. COS cells were transfected with different cDNAs: hAMHR-II-HA, mAMHR-II-His, mAMHR-II-GFP and mBMPR-II-GFP. (A) COS protein lysates were prepared and 30 μg of proteins were analyzed. The N2 polyclonal antibody (top panel) and the 13H8 monoclonal antibody (middle panel) can detect both the human (lane 2) and the mouse (lanes 3 and 4) AMHR-II. The previously characterized 12G4 monoclonal antibody can only reveal the human protein. There are no cross-reactions with either of the antibodies with BMPR-II (lane 5). No specific band was detected in the mock- transfected cells (lane 1). (B) Membrane fractions were prepared from the cell lines AT, SMAT, CHO, CHO-3W, uterus and spleen and subjected to a western blot. The detection was achieved with the mAbl3H8 monoclonal antibody.

Figure 4 presents data showing that mAb 13H8 can detect both human and mouse AMHR-II in AMHR-II overexpressing cells. (A-D) COS cells were analyzed by immunocytochemistry using the 13H8 monoclonal antibody. The signal was amplified using the Vectastain® ABC kit and the substrate was DAB. (A) Mock- transfected cells and (D) BMPR-II-transfected cells did not reveal any positive signal. (B) hAMHR-II-transfected cells and (C) mAMHR-II-transfected cells were strongly positive indicating that the 13H8 monoclonal antibody can detect both the human and the mouse AMHR-II proteins and does not generate a false positive signal with BMPR-II protein. (E-G) COS cells were analyzed by immunofluorescence using either the 13H8 monoclonal antibody (F) or the rabbit polyclonal AMH antibody (G). hAMHRII transfected cells were incubated (G) with human recombinant plasmin- cleaved AMH.

Figure 5 presents data showing that mAb 13H8 displays the same specificity than mAb 12G4 and polyclonal antibody N2 on human samples. Archival human samples were subjected to immunohistochemistry using the different anti-AMHRII antibodies: (A) N2 polyclonal antibody, (B) mAb 12G4 monoclonal antibody, (C) mAb 13H8 monoclonal antibody and (D) c kit antibody. A to D refer to sample #1321. Figure 6 presents data showing the expression of AMHR-II in the male rat. Newborn (A, C and E) and adult (B, D and F) rat testes were fixed and embedded in paraffin prior to being sectioned and subjected to immunohistochemistry. Samples A and B were exposed to mouse Ig in place of the primary antibody. Samples C and D were incubated with mAb 13H8 antibody (4 mg/ml) while samples E and F were incubated with polyclonal antibody N2 (4 μg/ml).

Figure 7 presents data showing the expression of AMHR-II in the female rat. Adult rat ovaries were fixed and embedded in paraffin prior being sectioned and subjected to immunohistochemistry. (A) The sample was incubated with mAb 13H8 (1 μg/ml) - (B) is an enlargement of (A) showing a dark brown primordial follicle. (D) and (F) The samples were incubated with mAb 13H8 (4 μg/ml). (C) and (E) The samples were incubated with the control Ig isotype.

Figure 8 presents data showing that EC-hAMHRII can block the AMH signaling pathway. SMAT cells were incubated with recombinant plasmin-cleaved AMH to activate the Smadl pathway (lanes 1, 2, and 3). Cell lysates were prepared and subjected to a western blot using the P-Smadl/5/8 antibody. When the cells were pre-incubated with a control peptide (lane 2), the signal was identical to that observed in the presence of AMH alone (lane 1). By contrast, after a pre-incubation of AMH with EC-hAMHRII, the signal was diminished, which indicates that the transduction was partially blocked (lane 3).

Figure 9 shows a representation of the location of the different putative sites of interaction mapped onto a three dimensional model of the extracellular domain of AMHRII. The Van der Waals surface is represented in transparency; spheres on the left upper part of the model correspond to the atoms of the amino acids that are part of the epitope. The side chain of Arginine 54 is also represented in spheres on the right upper part of the model to illustrate the possible sites of interaction of AMH with this domain of AMHRII.

Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

The terms "Anti-Miillerian Hormone" and "AMH" are used herein interchangeably. They refer to a 140 kDa glycoprotein hormone. AMH is synthesized as a large precursor with a short signal sequence followed by the pre-pro hormone that forms homodimers. Prior to secretion, the mature hormone undergoes glycosylation and dimerization to produce a 140-kDa dimer of identical disulphide- linked 70-kDa monomer subunits, each monomer contains an N-terminal domain (also called the "pro" region) and a C-terminal domain (also called the "mature" region).

As used herein, the term "AMHRII" corresponds to type II receptor of AMH. AMHRII polypeptides are described in WO95/16709. The mRNA sequence that encodes human AMHRII is known under accession number NM_020547.

The terms "AMHRII antagonist" and "selective AMHRII antagonist" are used herein interchangeably. They encompass any compound, naturally- occurring or net synthetic, that specifically inhibits or neutralizes (partially or totally) the biological activation of AMHRII, thereby reducing or inhibiting AMHRII biological activity. The terms "AMHRII agonist" and "selective AMHRII agonist" are used herein interchangeably. They encompass any compound, naturally-occurring or synthetic, that specifically activates or promotes the biological activation of AMHRII, thereby increasing AMHRII biological activity. In the context of the present invention, the term "selective" more specifically means that the AMHRII antagonists and agonists do not have an effect on AMHRI.

As used herein, the term "AMHRII biological activity" refers to an activity exerted by AMHRII in response to its specific interaction with AMH. Such biological activity can be determined in vivo, in situ or in vitro, according to standard techniques and procedures. Such an activity can be a direct activity, such as an association with, or an enzymatic activity on a second protein, or an indirect activity, such as a cellular process mediated by interaction of AMHRII with a second protein or a series of interactions as in intracellular signalling.

AMHRII antagonists and agonists according to the present invention are not AMH or AMH fragments or AMH derivatives. As used herein, the term "fragment of AMH" refers to a polypeptide comprising an amino acid sequence of several consecutive amino acid residues of the amino acid sequence of AMH. Thus, for example, the sequence of human AMH (accession number NP_000470) contains 560 amino acids. A fragment of human AMH encompasses any portion of the sequence of human AMH that is less than 560 amino acid long. Preferred fragments of AMH show the function of regulating primordial follicle recruitment or of antagonising said function of AMH, as defined in EP 1074265 (which is incorporated herein by reference in its entirety). As used herein the term "derivative of AMH" refers to any mutant of AMH obtained by deletion, substitution, insertion, alteration of the DNA encoding AMH, as defined in EP 1074265 (which is incorporated herein by reference in its entirety).

As used herein, the term "subject" denotes a human or another mammal (e.g. , primate, dog, cat, goat, horse, pig, bovine, mouse, rat, rabbit, etc). In certain embodiments of the present invention, the subject is a female, in particular a female during her reproductive years. Non-human subjects may be transgenic or otherwise modified animals. In certain embodiments, the subject is a woman. In such embodiments, the subject may be referred to as an "individual".

As used herein, the terms "antibody" and "immunoglobulin" are used herein interchangeably. They refer to any immunoglobulin (i.e. , an entire immunoglobulin molecule or an active portion of an immunoglobulin molecule) that specifically binds to an antigen. The terms encompass monoclonal antibodies or mAbs (i.e., antibodies of a single amino acid composition which are directed against a specific antigen and which are produced by a identical immune cells that are all clones of a unique parent cell) and polyclonal antibodies (i.e., antibodies which are obtained from different immune cells. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope). All derivatives and fragments thereof, which maintain specific binding ability, are also included in the terms. The terms also cover any protein having a binding domain, which is homologous or largely homologous to an immunoglobulin-binding domain. These proteins may be derived from natural sources, or may be partly or wholly synthetically produced.

In naturally-occuring 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 types of light chain, lambda (λ) and kappa (κ). 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 nonhypervariable 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 three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

A biologically active fragment or portion of an inventive monoclonal antibody may be, for example, a Fab fragment or portion, a F(ab')2 fragment or portion, a Fab' fragment or portion, or a single chain Fv (scFv) polypeptide. As used herein, the term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 Da 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 papaine (a protease) are bound together through a disulfide bond. The term "F(ab')₂" refers to an antibody fragment having a molecular weight of about 100,000 Da 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 pepsin (a protease). The term "Fab"' refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')₂. 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. The human scFv fragment of the invention includes CDRs that are held in appropriate conformation, preferably by using gene recombination techniques.

The term "chimeric antibody" refers to a monoclonal antibody which comprises a VH domain and a VL domain of an antibody derived from a non-human animal mammal (e.g., mouse, rat, hamster, rabbit, and the like), a CH domain and a CL domain of a human antibody.

The term "humanized antibody" refers to antibodies in which the framework or complementarity determining regions" (CDR) have been modified to comprise the CDR from a donor immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In certain preferred embodiments, a "humanized antibody" is prepared by grafting a mouse CDR into the framework region of a human antibody.

The term "specific binding", when used in reference to an antibody, refers to an antibody binding to a predetermined antigen. Typically, the antibody binds with an

7 -1

affinity of at least 1 x 10 M^" , and binds to the predetermined antigen with an affinity that is at least two-fold greater than the affinity for binding to a non-specific antigen (e.g. , BSA, casein).

As used herein, the term "improving female fertility" generally refers to increasing the chance of conception. In certain preferred embodiments of the present invention improving female fertility is achieved by increasing primordial follicle recruitment.

As used herein, the term "inhibiting fertility" generally refers to reducing the chance of conception. In certain preferred embodiments of the present invention inhibiting fertility is achieved by reducing or preventing primordial follicle recruitment.

In the context of the present invention, a "pharmaceutical composition" comprises an effective amount of at least one AMHRII antagonist or agonist of the invention and at least one pharmaceutically acceptable carrier or excipient.

As used herein, the term "effective amount" refers to any amount of a compound, agent, antibody or composition that is efficient to fulfil its intended purpose(s), e.g., a desired biological or medicinal response in a cell, tissue, system or subject. In the context of the present invention, the purpose is generally to modulate male or female fertility. More specifically, the purpose(s) may be: to improve female fertility, to treat a female fertility disorder, to extend the fertility period of a female, to inhibit female fertility, or to achieve male contraception.

The term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The use of such media and agents for formulating pharmaceutically active substances is well known in the art (see for example "Remington's Pharmaceutical Sciences", E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention generally relates to the use of selective AMHRII antagonists and agonists for modulating female and male fertility.

I - Selective AMHRII Antagonists and Agonists

The present invention relates to the use of selective AMHRII antagonists and AMHRII agonists for modulating male and female fertility. AMHRII antagonists according to the present invention encompass any natural or synthetic compound that specifically inhibits or neutralizes (partially or totally) the biological activation of AMHRII and that is not AMH or a fragment or derivative of AMH. AMHRII agonists according to the present invention encompass any natural or synthetic compound that specifically activates or promotes the biological activation of AMHRII and that is not AMH or a fragment or derivative of AMH. A. Identification of Selective AMHRII Antagonists and AMHRII Agonists

Selective AMHRII antagonists and agonists may be found within numerous classes of compounds, including small molecules, antibodies, peptides, nucleic acid molecules, saccharides, steroids, and the like. A candidate compound may be a synthetic or natural compound; it may be a single molecule or a mixture or complex of different molecules. AMHRII antagonists and agonists may be found by screening collections or libraries of compounds or by synthesizing compounds and testing them. Both natural and chemical compounds may be tested. Natural product collections are generally derived from microorganisms, animals, plants, or marine organisms. Chemical libraries often consist of structural analogs of known compounds or compounds that are identified as "hits" or "leads" via natural product screening. Chemical libraries are relatively easy to prepare by traditional automated synthesis, PCR, cloning or proprietary synthetic methods. Both natural compound collections and chemical libraries are commercially available.

Determination of the ability of a candidate compound to specifically inhibit or promote the biological activation of AMHRII may be performed using any suitable method. For instance, one such method is based the use of the mouse Sertoli cell line, SMAT1 (Dutertre et al, Mol. Cell Endocrinol., 1997, 136: 57-68), which has been used by the present inventors (see Examples section). The SMAT1 cell line expresses AMHRII and all three type I receptors, and has been shown to be responsive to AMH as evidenced by phosphorylation of Smadl, 5, or 8, the first components in the signal transduction pathway of AMH (Belville et al, Oncogene, 2005, 24: 4984-4992). Comparison of phosphorylation of Smads between control condition and test condition allows the determination of the agonistic or antagonistic activity of the candidate compound. Blockade of Smad phosphorylation is indicative of an antagonistic activity, whereas increase in Smad phosphorylation is indicative of an agonistic activity.

B. Selective AMHRII Antagonists

In particular, in certain embodiments, a selective AMHRII antagonist according to the invention is a polypeptide comprising or consisting of the extra-cellular domain of AMHRII (residues 18-145 of the sequence Q16671, SEQ ID NO: 1) or a functional-conservative variant or fragment thereof that retains the biological function of the extra-cellular domain of AMHRII, i.e., the ability to specifically bind to AMH.

According to the invention, a functional-conservative variant may be (1) a polypeptide consisting of the extra-cellular domain of AMHRII, said polypeptide comprising one or more mutation, deletion, or addition, provided that said polypeptide retains the ability to specifically bind to AMH; or (2) a polypeptide consisting of the extracellular domain of AMHRII flanked by a N-terminal amino acid sequence and/or a C-terminal amino acid sequence. For example, certain embodiments of the invention, the polypeptide may consist of the extra-cellular domain of AMHRII (SEQ ID NO: 1) having an additional 1 to 50 amino acids in the N-terminal region and/or an additional 1 to 50 amino acids in the C-terminal region. In other embodiments of the invention, the polypeptide may consist of the extra-cellular domain of AMHRII (SEQ ID NO: 1) having an additional 1 to 30 additional amino acids in the N-terminal region and/or an additional 1 to 30 additional amino acids in the C-terminal region. In yet other embodiments of the invention, the polypeptide may consist of the extracellular domain of AMHRII (SEQ ID NO: 1) having an additional 1 to 10 additional amino acids in the N-terminal region and/or an additional 1 to 10 additional amino acids in the C-terminal region.

The capacity of a variant or a fragment of the extra-cellular domain of AMHRII to specifically bind AMH may be assessed by any conventional techniques known in the art. Examples of such conventional techniques are include, but are not limited to, precipitation experiments and ELISA experiments as described in the Examples section.

In certain embodiments, an AMHRII antagonist is a fusion protein comprising the extra-cellular domain of AMHRII fused to another protein directly or indirectly (e.g. , through a peptide linker such as IEGRMD). Techniques for producing and purifying fusion proteins are well known in the art. Typically, an example of such a fusion protein is an AMHRII-Fc fusion protein comprising the extra-cellular domain of AMHRII as defined here above, fused to at least a domain of the Fc region of an immunoglobulin. In such a AMRHII-Fc fusion protein, the extra-cellular domain of AMHRII forms the amino-terminal domain of the fusion protein and the Fc region forms the carboxy-terminal domain of the fusion protein. The extra-cellular domain of AMHRII may be fused directly or indirectly to the Fc region.

Another example of a fusion protein according to the present invention is a fusion protein comprising the mature extra-cellular domain of AMHRII as defined here above, fused to the CH2 and CH3 domains and the hinge region of IgGl (residues 104-330 of Swiss Prot Accession P01857).

In other embodiments, a selective AMHRII antagonist according to the invention binds to AMHRII with sufficient affinity and specificity so as to neutralize the biological effect of AMHRII. Such AMHRII antagonists may be antibodies, antibody fragments, or synthetic compounds. In particular, such AMHRII antagonists may be antibodies directed to the extracellular domain of AMHRII or biologically active fragments of such antibodies. According to the invention, the extra-cellular domain of AMHRII corresponds to residues 18-145 of Swiss Prot Accession Q16671.

Antibodies according to the present invention may be prepared using any suitable method known in the art.

For example, polyclonal and monoclonal antibodies may be prepared using standard methods employing an isolated AMHRII polypeptide, or an antigenic portion thereof such as the extra-cellular domain, as immunogen. As known in the art, either the full-length polypeptide or protein or antigenic peptide fragments of the full length polypeptide or protein can be used as immunogens. AMHRII antigenic peptides suitable for use in such a method may be of any length, and, for example, comprise at least 8 (preferably 10, 15, 20, or 30 or more) amino acid residues. Furthermore, the antigenic peptides are such that they encompass an epitope of the protein in order for the antibody raised against the peptide to form a specific immune complex with the protein.

Typically in such as method, the immunogen is used to immunize a suitable (i.e., immunocompetent) subject, such as a rabbit, goat, mouse, or other mammal or vertebrate. An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide(s). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immuno stimulatory agent. Antibody-producing cells can be obtained from the peripheral blood or, preferably, the spleen or lymph nodes of humans or other suitable animals that have been immunized with the immunogen of interest. Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present invention. The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA). In one approach, a hybridoma is produced by fusing a suitable immortal cell line (e.g. , a myeloma cell line, such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS 1 , NS2, AE- 1 , L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS I , Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1 , JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMALWA, NEURO 2A, or the like), or heteromyelomas, fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line as known in the art (see, e.g., www.atcc.org, www.lifetech.com, and the like), with antibody producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof.

Methods for the production and isolation of monoclonal antibodies from hybridoma cultures are well known in the art. Hybridoma cells are grown using standard methods, in suitable culture media such as, for example, D-MEM and RPMI- 1640 medium. A monoclonal antibody against the extracellular domain of AMHRII can be recovered and purified from hybridoma cell cultures by any suitable method known in the art.

Other suitable methods for producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibodies from a peptide or polypeptide library that are capable of producing a repertoire of human antibodies. Such techniques include, but are not limited to, ribosome display, single cell antibody producing technologies, gel microdroplet and flow cytometry, B-cell selection.

Methods for engineering or humanizing non-human or human antibodies can also be used for preparing antibodies according to the present invention. Such methods are well known in the art. Generally, a humanized or engineered antibody has one or more amino acid residues from a source that is not human, including, but not limited to, mouse, rat, rabbit, non-human primate or other mammal. The human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable, constant or other domain of a known human sequence. Known human Ig sequences are disclosed and known by the person skilled in the art (see, for example www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast; www . atcc . org/phage/hdb .html ; www.kabatdatabase.com/top.html; www.sciquest.com; www.abcam.com; www . antibodyresource . c om/onlinecomp .html ;

mcb.harvard.edu/BioLinks/lmmunology.html; www.immunologylink.com).

Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. Generally, part or all of the non- human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids. Antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of antibodies of the present invention can be performed using any known method.

AMHRII antibodies according to the invention can also be optionally generated by immunization of a transgenic animal {e.g., mouse, rat, hamster, non-human primate, and the like) capable of producing a repertoire of human antibodies, as described herein and/or as known in the art. Cells that produce a human AMHRII antibody can be isolated from such animals and immortalized using suitable methods, such as the methods described herein. Transgenic mice that can produce a repertoire of human antibodies that bind to human antigens can be produced by known methods. Generally, these mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement. The endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the capacity of the animal to produce antibodies encoded by endogenous genes. Antibodies of the present invention can also be prepared in milk by administering at least one anti- AMHRII antibody encoding nucleic acid to transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce antibodies in their milk. Such animals can be provided using known methods.

Antibodies of the present invention can additionally be prepared using at least one AMHRII antibody encoding nucleic acid to provide transgenic plants and cultured plant cells (including, but not limited to, tobacco and maize) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured therefrom.

The term "antibody" is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to mammalian AMHRII and in particular AMHRII extra-cellular domain. Antibody fragments of the present invention may be produced by any suitable method known in the art including, but not limited to, enzymatic cleavage (e.g. , proteolytic digestion of intact antibodies) or by synthetic or recombinant techniques).

As mentioned above, an antibody of the present invention specifically binds to AMHRII (in particular human AMHRII) so as to neutralize the biological effect of AMHRII. Thus, antibodies of the present invention may have a wide range of affinities (KD). In certain preferred embodiments, human monoclonal antibodies of the present invention bind human AMHRII with high affinity. For example, a human mAb can bind human AMHRII with a KD equal to or less than about 10⁷ M, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or any range or value therein. The affinity or avidity of an antibody (or fragment thereof) for an antigen can be determined experimentally using any suitable method. The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Ka, Kd) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

In yet other embodiments, a selective AMHRII antagonist according to the invention is selected from the group consisting of siRNAs, shRNAs, antisense molecules and DNAzymes targeting the AMHRII gene sequence, and more specifically in the group consisting of siRNAs, shRNAs, antisense molecules and DNAzymes whose effect on the AMHRII gene sequence results in the inhibition or suppression of the expression of AMHRII.

AMHRII gene expression can be modulated in several different ways including by the use of siRNAs, shRNAs, antisense molecules, ribozymes, and DNAzymes. Synthetic siRNAs, shRNAs, ribozymes, and DNAzymes can be designed to specifically target one or more genes and they can easily be delivered to cells in vitro or in vivo.

The present invention encompasses antisense nucleic acid molecules, i.e., molecules that are complementary to a sense nucleic acid encoding an AMHRII polypeptide, e.g., complementary to the coding strand of a double- stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can bind to a sense nucleic acid though a hydrogen bond. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a AMHRII polypeptide. The non-coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences that flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g. , an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. , phosphorothioate derivatives, peptide nucleic acids (PNAs), and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5- methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6- diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected AMHRII polypeptide to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then be administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g. , by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol I or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an [alpha] -anomeric nucleic acid molecule. An [alpha] -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual [alpha] -units, the strands run parallel to each other (Gaultier et ah, Nucleic Acids Res., 1987, 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et ah, Nucleic Acids Res., 1987 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett., 1987, 215: 327- 330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a AMHRII polypeptide can be designed based upon the nucleotide sequence of AMHRII. Alternatively, an mRNA encoding a AMHRII polypeptide can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.

The invention also encompasses ribonucleic acid molecules which are complementary, antisense, double stranded homologues, siRNA, or are sequence specific single-stranded RNAs which form short hairpin structures, shRNA (collectively, interfering RNA), that can be used to down-modulate specific gene expression, in this case, AMHRII, and therefore to inhibit protein expression. In another example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g. , DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols.

In another embodiment, the invention relates to AMHRII antagonists or agonists, as described herein, which are modified by the covalent attachment of a moiety. Such modification can produce an AMHRII antagonist or agonist with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group. In particular embodiments, the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms. As used herein, the term "fatty acid" encompasses mono-carboxylic acids and di- carboxylic acids. Fatty acids and fatty acid esters suitable for modifying antibodies of the invention can be saturated or can contain one or more units of unsaturation. Fatty acids that are suitable for modifying antibodies of the invention include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C2o, arachidate), n-docosanoate (C22, behenate), n- triacontanoate (C30), n-tetracontanoate (C40), cis- delta 9-octadecanoate (C18, oleate), all c/s-delta5,8, l 1 , 14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably, one to about six, carbon atoms.

The modified human polypeptides and antibodies can be prepared using suitable methods, such as by reaction with one or more modifying agents. A "modifying agent" as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An "activating group" is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.

II - Methods for Modulating Fertility

Selective AMHRII antagonists and agonists of the present invention may be administered for modulating male or female fertility.

A. Indications

AMHRII antagonists according to the present invention can be used for improving female fertility and/or for treating female infertility disorders and/or for improving or treating male infertility. In preferred embodiments, an inventive AMHRII antagonist exerts its effect by increasing the recruitment of primordial follicles. AMHRII antagonists may also find application in techniques of fertility- preservation in patients with cancer based on ovarian tissue cryopreservation.

AMHRII agonists of the present invention may be used for extending the fertility period and postponing the menopause. AMHRII agonists may also be used for inhibiting female fertility (i.e. , for contraception) by preventing recruitment of primordial follicles. Current hormonal methods for contraception do not inhibit the recruitment of primordial follicles. Therefore, one advantage of the inventive approach to female contraception is the conservation of the pool of primordial follicles. AMHRII agonists may also find applications in male contraception.

The methods of treatment of the present invention comprise a step of administering an effective amount of at least one AMHRII antagonist or at least one AMHRII agonist to a subject in need thereof. The methods of treatment of the present invention may be accomplished using an inventive AMHRII antagonist or agonist or a pharmaceutical composition comprising thereof (see below). These methods generally comprise administration of an effective amount of at least one inventive AMHRII antagonist or agonist, or a pharmaceutical composition thereof, to a subject in need thereof. Administration may be performed using any of the methods known to one skilled in the art. In particular, the AMHRII antagonist or agonist, or composition thereof, may be administered by various routes including, but not limited to, aerosol, parenteral, oral or topical route.

In general, an inventive AMHRII antagonist or agonist or composition will be administered in an effective amount, i.e. an amount that is sufficient to fulfill its intended purpose. The exact amount of AMHRII antagonist or agonist or pharmaceutical composition to be administered will vary from subject to subject, depending on the age, sex, weight and general health condition of the subject to be treated, the desired biological or medical response {e.g., recruitment of primordial follicles, improvement of female fertility, and the like). In many embodiments, an effective amount is one that increases the recruitment of primordial follicles. In other embodiments, an effective amount is one that prevents the recruitment of primordial follicles.

Subjects that may receive a treatment for improving fertility or for postponing menopause according to the invention may be females of any mammal species, including humans. In certain embodiments, an AMHRII antagonist is administered to a woman, and in particular to a woman during her reproductive years. In other embodiments, the AMHRII antagonist is administered to female domesticated animal {e.g., cattle, sheep, goats, horses, and the like) or to a female companion animal (e.g., dog, cat, and the like). Subjects that may receive a treatment for inhibiting fertility according to the invention may be females or males of any mammal species, including humans.

In certain embodiments, an inventive AMHRII antagonist (or agonist) or composition is administered alone according to a method of treatment of the present invention. In other embodiments, an inventive AMHRII antagonist (or agonist) or composition is administered in combination with at least one additional biologically active agent. The inventive AMHRII antagonist (or agonist) or composition may be administered prior to administration of the biologically active agent, concurrently with the biologically active agent, and/or following administration of the biologically active agent.

Biologically active agents that may be administered in combination with an inventive AMHRII antagonist include, in particular, sexual hormones. Biologically active agents that may be administered in combination with an inventive AMHRII agonist for inhibiting fertility include, in particular, contraceptive, such as progestins and/or oestrogens.

B. Administration

An inventive AMHRII antagonist or agonist, (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), in a desired dosage can be administered to a subject in need thereof by any suitable route. Various delivery systems are known and can be used to administer AMHRII antagonists or agonists of the present invention, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include intravenous administration of a liquid composition, transdermal administration of a liquid or solid formulation, oral, topical administration, or interstitial or inter-operative administration. Administration may be affected by the implantation of a device whose primary function may not be as a drug delivery vehicle. Administration may also be performed by incubation in an ex-vivo sample (ex. ovarian biopsy). As will be appreciated by those of ordinary skill in the art, in embodiments where an inventive AMHRII antagonist or agonist is administered in combination with an additional biologically active agent, the antagonist or agonist and biologically active agent may be administered by the same route (e.g. , orally) or by different routes (e.g., intravenously and orally). C. Dosage

Administration of an inventive AMHRII antagonist or agonist of the present invention will be in a dosage such that the amount delivered is effective for the intended purpose. In the case of administration of an antibody, generally, the dosage range is from about 0.05 mg/kg to about 12.0 mg/kg. This may be as a bolus or as a slow or continuous infusion which may be controlled by a microprocessor controlled and programmable pump device. Alternatively, DNA encoding preferably a fragment of a monoclonal antibody may be isolated from hybridoma cells and administered to a mammal. The DNA may be administered in naked form or inserted into a recombinant vector, e.g., vaccinia virus, in a manner which results in expression of the DNA in the cells of the patient and delivery of the antibody.

When a AMHRII antagonist or agonist is to be administered to an animal (e.g., a human) in order to modulate expression or activity of AMHRII, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. IV - Pharmaceutical Compositions

As mentioned above, the AMHRII antagonists or agonists of the invention may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising an effective amount of an inventive AMHRII antagonist or AMHRII agonist described herein and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biologically active agents.

A. Formulation

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intraperitoneal, intradermal, subcutaneous, oral (e.g., inhalation or buccal), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediamine-tetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions, suitable for injectable use, include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. 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. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. 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. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Pharmaceutical excipients and additives useful in stabilizing the present composition include, but are not limited to, polypeptides, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary but non-limiting polypeptide excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine. Sterile injectable solutions can be prepared by incorporating the active compound (e.g. , a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, PA) 1990.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel, or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams, as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. For antibodies, the preferred dosage is about 0.1 mg/kg to 100 mg/kg of body weight (generally about 10 mg/kg to 20 mg/kg). Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, the use of lower dosages and less frequent administration is often possible. Modifications, such as lipidation, can be used to stabilize antibodies and to enhance uptake and tissue penetration.

The AMHRII antagonist nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration or by stereotactic injection. The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations for the delivery of antibodies can be found, for example, in "Remington's Pharmaceutical Sciences", E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA.

Thus, for example, for ease of administration, a monoclonal antibody of the present invention will typically be combined with a pharmaceutically acceptable carrier, such as water, physiological saline, or oils. Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents. Except insofar as any conventional medium is incompatible with the active ingredient and its intended use, its use in any compositions is contemplated. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.

The pharmaceutical compositions of the present invention can be included in a container, pack, or dispenser together with instructions for administration.

B. Additional Biologically Active Agents

In certain embodiments, an inventive selective AMHRII antagonist or agonist is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, in particular, sexual hormones and contraceptives.

In such pharmaceutical compositions, the AMHRII antagonist (or agonist) and additional biologically active agent(s) may be combined in one or more preparations for simultaneous, separate or sequential administration of the AMHRII antagonist (or agonist) and therapeutic agent(s). More specifically, an inventive composition may be formulated in such a way that the AMHRII antagonist (or agonist) and therapeutic agent(s) can be administered together or independently from each other. For example, an AMHRII antagonist (or agonist) and a biologically active agent can be formulated together in a single composition. Alternatively, they may be maintained (e.g. , in different compositions and/or containers) and administered separately, for example at different times of the menstrual cycle.

As used herein, the term "androgen" has its art understood meaning and encompasses any natural or synthetic compound that stimulates or controls the development and maintenance of male characteristics in vertebrates by binding to androgen receptors. Compositions comprising an AMHRII antagonist and an androgen may be used for improving female fertility and/or treating female infertility and/or prolonging the fertile period and postponing menopause. Indeed, androgens are known to promote the growth of small follicles in the primary ovary (Weil et ah , J. Clin. Endocr. Metabol., 1998 , 83: 2479-2485).

As used herein, the term "contraceptive" refers to female oral contraceptives, in particular to progestins and to combination of oestrogens and progestogens. Administration of an AMHRII agonist and a contraceptive, either in a single composition or different compositions may be used for inhibiting fertility. C. Pharmaceutical Packs of Kits

In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g. , vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of an AMHRII antagonist (or agonist) of the present invention.

Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g. , lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale. In certain embodiments, a pharmaceutical pack or kit includes one or more additional therapeutic agent(s) (e.g. , sexual hormones or contraceptives, as described above). Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.

An identifier, e.g. , a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained. Materials and Methods

Antibody Preparation . The recombinant extracellular part of His-tagged hAMHR-II protein (EC-hAMHR-II) was used as an antigen. BalB/c mice were immunized with 3 intraperitoneal injections of 50 μg of purified protein and one intravenous injection with 10 μg of purified protein. Cells from the mouse myeloma cell line NSI 9046 were the fused to the spleen cells to generate hybridomas. The first screening was achieved using ELISA on hybridomas supernatants. Each well was coated with 250 ng of EC-hAMHR-II and assayed with 100 μΐ of supernatant. The second screening included a western blot on transfected cell lines and an ELISA, as described below. The selected clones were then amplified, purified and called mAbl3H8. Epitope Mapping. The sepharose activated NHS Hitrap was purchased from GE Healthcare. The EnzyBeads™ Trypsin and EnzyBeads™ Chymotrypsin were purchased from Agro-Bio (France). The MB-HIC 18, peptides for MALDI-TOF calibration and a-cyano-4-hydroxycinnamic acid (HCCA) were obtained from Bruker Daltonics (Germany). The trypsin (sequencing grade modified) and chymotrypsin (sequencing grade modified) were obtained respectively from Promega (USA) and Roche Diagnostics (Germany).

Preparation of Affinity Columns for Immunoadsorption. The affinity columns were prepared according to the supplier user guide. Briefly, the antibodies were bound to the sepharose activatived NHS Hitrap at a concentration of 1 mg of 13H8 mAb/1 mL volume of beads. The NHS-functions were activated by 1 mM of HC1. Immediately after the activation, the 13H8 Mab was incubated for 30 minutes at room temperature (RT) under gentle agitation. The quantity of immobilized-antibodies was estimated by measuring the optical density at 280 nm.

Epitope Mapping by Epitope Extraction. For epitope extraction, 1 mg of antigen (lmg/mL in 25 mM NH₄HCO₃ buffer, pH 8) was reduced with 10 mM DTT (Sigma- Aldrich) for 5 minutes at 100°C. The antigen was then alkylated with 55 mM iodoacetamide for 45 minutes at 57°C in the absence of light. The antigen was digested with EnzyBeads™ Trypsin or EnzyBeads™ Chymotrypsin according to the supplied user guide. Briefly, the antigen was mixed with 400μί of EnzyBeads™ Trypsin or 400μΕ of EnzyBeads™ Chymotrypsin and incubated 2 hours at 37°C. To determine trypsin or chymotrypsin autodigest products, a sample was prepared similar to the antigen sample but lacking the antigen. The digested samples were loaded on affinity columns. The microcolumns with the antigen digests were incubated for 30 minutes at 25°C under gentle agitation. After the incubation, the columns were washed with 3 x 1 mL of 25 mM NH₄HCO₃ buffer (pH 8) and 3 x 1 mL of 0.15 M PBS/0.5 M NaCl (pH 7.2). The peptide bounds were eluted with 50 mM Glycine buffer (pH 2.5). Eluats were desalted and concentrated with MB-HIC 18 according to the supplied user guide.

Epitope Mapping by Epitope Excision. For Epitope Mapping by epitope excision, four immobilized-antibodies column were used. These immobilized-antibodies columns were incubated with either 1 mg of antigen (lmg/mL in 0.15MPBS pH 7.2) for 1 hour at RT. Each column was washed with 6 x 1 mL of 25 mM NH₄HCO₃ buffer, pH 8. The immobilized mAb 13H8 with affinity-bound antigen was then reduced with 1 mL of 10 mM DTT for 1 hour at RT and alkylated with 1 mL of 55 mM iodoacetamide for 1 hour at RT in the absence of light. Each column was washed with 5 x 1 mL of 25 mM NH₄HC0₃ buffer, pH 8.

For trypsin digestion, samples were incubated with 20 μg of proteinase in 1 mL of 25 mM NH₄HC0₃ buffer, pH 8 for 30 minutes or 2 hours at 37°C. The chymotrypsin digestions were performed with 20 μg of chymotrypsin in 1 mL of 25 mM NH₄HC0₃ buffer, pH 8 for 30 minutes or 2 hours at 37°C. These columns were washed with 5 x 1 mL of 25 mM NH₄HC0₃ buffer, pH 8. The retained peptides corresponding to the epitope peptides were eluted with 3 x 1 mL of 50 mM glycine buffer pH2.5. MALDI-TOF Mass Spectrometry. MALDI-TOFMS and MALDI-TOFMS/MS were performed on an Autoflex TOF/TOF (BrukerDaltonics, Germany) equipped with a nitrogen laser (337 nm). The instrument was run in positive ionization mode and measurements were performed in the reflector mode (m/z 750 to 4000). The matrix solution was prepared from a saturated solution of a-cyano-4-hydroxycinnamic acid in H₂0/TFA/ACN (49.9/0.1/50) diluted 3 times. Samples were prepared using the dried droplet method by spotting 0.5 μΐ_^ of matrix solution and 0.5 μΐ_^ of sample solution onto a MTP 384 ground steel MALDI target. For calibration in the reflector mode, signals of angiotensin II ([M + H]⁺ _mono at m/z 1046.5418), angiotensin I ([M + H]⁺ _mono at m/z 1296.6848), substance P ([M + H]⁺ _mono at m/z 1347.7354), bombesin ([M + H]⁺ _mono at m/z 1619.8223), ACTH_Clip [1-17] ([M + H]⁺ _mono at m/z 2093.0862) and ACTH_Clip [18-39] ([M + H]⁺ _mono at m/z 2465.1983) were employed. Data acquisition and data processing were performed using the FlexControl software version 2.2 and FlexAnalysis software version 2.2.

Cell Line and Transfection. The green monkey kidney cell line COS-7 was grown as described previously in Dulbecco's modified Eagle's medium (Life Technologies, Rockville, MD, USA) (Gluzman, Cell, 1981, 23: 175-182). For transient expression experiments, 60-mm tissue culture plates or LabTek chamber slides were seeded with 3 x 10⁵ cells and incubated in Dulbecco modified Eagle medium at 37°C overnight. The next day, the cells were rinsed with phosphate-buffered saline (PBS) and transfected with the Lipofectamine™ Plus® reagent according to the manufacturer's protocol (Invitrogen, USA). Plasmid DNA containing human and mouse Amhr2 cDNAs and mouse Bmpr2 cDNA were either prepared in the inventors' laboratory (hAmhr2) or kindly provided by Dr. Richard Behringer (mAmhr2 and mBmpr2).

Western Blot Analysis. For total protein extracts, COS cells from 60-mm tissue culture plates were washed and solubilized in 400 μΐ_^ lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton, 1 mM phenylmethylsulfonylfluoride, lx proteinase inhibitor mixture (Sigma- Aldrich, USA)). Membrane fractions were prepared from cells and fresh tissue. Tissues were homogenized in cold H buffer (50 mM Tris-HCl/ 5 mM EDTA) containing a protease inhibitor cocktail and centrifuged at 20400 g for 15 minutes. The supernatant SI was kept in ice and the pellet was resuspended in H buffer and the suspension was centrifuged at 20400 g for 15 minutes at 4°C. The supernatant S2 was combined to SI and centrifuged at 48400 g for 1 hour at 4°C. The pellet containing the membrane protein extract is resuspended in H buffer. Protein concentration was determined using the BCA assay (Pierce Chemical Co., USA). 20 μg of cell lysates were subjected to Western blot analysis after 7.5% SDS-PAGE (Bio-Rad Laboratories, USA) using the indicated primary antibodies (1 mg/ml) and peroxidase-labeled secondary antibodies at 1:5000 as described previously (Faure et al., J. Biol. Chem., 1996, 27-30571-305751). Proteins were visualized using the ECL Plus Kit Detection System (Amersham Pharmacia Biotech, USA). The signal was captured using the BioDoc-It system and a monochrome CCD camera (UVP). Competition Assay. Smad phosphorylation was determined as previously described (Gouedard et al, J. Biol. Chem., 2000, 275: 27973-27978. Briefly, SMAT1 cells were seeded into 6 well tissue culture plates at approximately 50% density in Dulbecco's modified Eagle's medium (Life Technologies, USA) containing 10% fetal bovine serum (FBS; Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). The next day, the cells were washed, starved during 1 hour with medium without serum and incubated or not during 30 minutes with 13H8 (100μg/ml). Then AMH (2 μg/ml) pre-incubated or not during 30 minutes with EC-AMHRII (50 μg/ml) was added in culture medium without serum for 1 hour. The cells were washed and solubilized in 200 μΐ of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (V/V) Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride, a proteinase inhibitor mixture (Sigma-Aldrich) and a phosphatase inhibitor cocktail (Calbiochem Merck Biosciences, Germany). The lysates were cleared by centrifugation and supernatants were analyzed by SDS-PAGE followed by western blotting with a rabbit anti-phosphoSmadl, 5 MAb (Cell Signaling; 1: 1000 dilution) and a goat anti-rabbit IgG antibody conjugated to HRP (Jackson ImmunoResearch Laboratories; 1:5000 dilution). The membranes were stripped and re-probed with a mouse anti-tubulin MAb (clone B-5-1-2, Sigma- Aldrich). ELISA. Dynatech Immulon 2 (96 well) ELISA plates were coated with either mouse extracellular AMHR-II fragment or human extracellular AMHR-II fragment overnight at 4°C in PBS (50 μίΛνεΙΙ). The plates were washed six times with water and then blocked for 2 hours at room temperature using 150 μίΛνεΙΙ of block buffer (PBS; 1% BSA). The block buffer was discarded and the antibodies were serially diluted down the plate by a factor of two starting at 200 ng/mL. Plates were incubated for 1 hour, followed by four washes with PBS/0.05% Tween 20. 50 μΐ of anti-rabbit-HRP or anti-mouse-HRP (dilution 1/2500, Jackson Laboratories) were added to each well and incubated for 1 hour. The plates were washed six times with PBS/0.05% Tween-20 and 50 of TMB substrate were added to each well. The reactions were quenched by the addition of 50 μίΛνεΙΙ of 2 M sulfuric acid. Absorbance at 450 nm was read in a plate reader.

Immunohistochemistry. Most human samples used in the present study were from archives from the Pathology Department of the Necker/Enfants Malades Hospital, and originated from autopsy material, biopsies, or castration in genetic males raised as females. The age of fetuses was evaluated from the last menstrual period. Tissues were fixed in 10% formalin and embedded in paraffin. All the samples are listed in Table 1.

Rat samples were fixed in Bouin overnight at 4°C. They were rinsed in phosphate buffered saline (PBS), then dehydrated in ethanol and embedded in paraffin and serially sectioned at 7 μιη. For immunohistochemistry, tissue sections were de- paraffinized, rehydrated and washed in tris buffered saline (TBS) containing 0.1% Tween-20. After endogenous peroxydase inactivation, tissue sections were blocked in 5% horse serum and subsequently incubated with primary antibodies. The secondary antibody was biotinylated and the revelation was achieved using the ABC kit (Vector laboratory, USA). Specificity of staining was confirmed using the corresponding IgG from the same species. Immunocytochemistry. COS cells in LabTek chamber slides were fixed for 10 minutes in 4% paraformaldehyde and washed in PBS. Then after endogenous peroxydase inactivation, they were treated as described above with the different primary antibodies. Antibodies. The antibodies against AMHR-II were either a rabbit polyclonal antibody against a histidine-tagged fusion protein corresponding to the extracellular domain of human AMHR-II (Gouedard et al, J. Biol. Chem., 2000, 275: 27973- 27978; Allard et al, Development, 2000, 127: 2249-3360), or two mouse monoclonal antibodies against an epitope of the extracellular domain of human AMHR-II called mAbl2G4 (Salhi et al, Biochem., 2004, 379: 785-793) and mAbl3H8. The following primary antibodies were also used: polyclonal anti-AMH (Josso et al, Trends Endocrinol. Metab., 2003, 14: 91-97), polyclonal rabbit anti-human c-kit (Dako), mouse monclonal anti-His (Invitrogen), mouse monclonal anti-alpha tubulin (Sigma). Results

Time-course of AMHR-II Expression in Male and Female Gonads.

Several human samples from fetal stage to prepubertal stage were collected. The present inventors checked the expression of AMHR-II using the rabbit polyclonal anti-AMHRII antibody. AMHR-II was detected as early as 28 weeks of gestation in the ovary (Figure 1A) and 17 weeks of gestation in the testis (Figure IB). The expression was very well defined in several cell types, somatic cells and germ cells. Shortly after birth, AMHR-II expression persisted clearly in the oocytes (Figure 1C) and in gonocytes (Figure ID). At the prepubertal stage, the same expression pattern (Figure IE and IF) was detected in germ cells. The identity was confirmed using c- kit antibody which is a specific germ cell marker (data not shown). The validation of the results obtained was investigated using a new monoclonal antibody directed against hAMHRII.

Only mAbl3H8 Antibody can interact with Mouse Recombinant AMHR-II.

Using a plate format, the inventors designed an experiment to test the affinity of the 2 selected clones (mAb 13H8 and mAb 13H12) with the mouse or the human extracellular domain of AMHR-II. These affinities were compared to those of the two previously described antibodies (rabbit polyclonal and mAb 12G4). The plate was coated with a fixed amount of either EC-hAMHR-II (Figure 2A) or EC-mAMHR-II (Figure 2B). The antibody was added at various concentrations. As expected, the four antibodies tested were found to bind to EC-hAMHR-II in a dose dependent manner (Figure 2A). The three monoclonal antibodies were raised against the extracellular part of human AMHR-II. Beside the polyclonal antibody, only mAb 13H8 was found to also bind to EC-mAMHR-II (Figure 2B). The binding was dose- dependent and the response was even more efficient at high antibody concentration.

Using two different approaches including trypsin and chymotrypsin digestions followed by mass spectrometry, the inventors were able to identify a conformational epitope. After sequencing, the fragment HCDPSPR was identified as the sequence involved in the antigen-antibody interaction. mAbl3H8 can interact both Human and MouseAMHR-II.

After a first selection, the purified clones were tested by Western Blot and immunocytochemistry. The inventors transfected COS cells with different constructs encoding hAMHR-II, mAMHR-II or mBMPR-II. Each construct was a fusion protein with a Histidine tag (hAMHR-II, mAMHR-II and mBMPR-II). After 48 hours, the cells were analyzed by Western blot or immunocytochemistry. The Western blots obtained showed a specific band for hAMHR-II and mAMHR-II for both the polyclonal antibody and the mAb 13H8 monoclonal antibody (Figure 3A). The previously described mAb 12G4 monoclonal antibody did not detect the mouse AMHR-II. Phylogenetically, the closest member of AMHR-II is BMPR-II so the inventors checked if there could be some cross-reactions between AMHR-II and BMPR-II. None of the antibodies tested, including mAb 13H8, were found to detect BMPR-II. Using anti-His antibody, all type II receptors transfected were detected. Alpha-tubulin was used to normalise the results obtained.

Immunocytochemistry was used to test the ability of mAb 13H8 to react with AMHR-II in a cell context. The hAMHR-II or mAMHR-II-transfected cells (Figure 4B and C) display several brown positive cells. This indicates that immunocytochemistry with mAb 13H8 can be used to detect AMHR-II overexpressing cells. The detection is equally sensitive in human and in mouse. As controls, the mock-transfected cells did not display any positive signal (Figure 4A). Similarly, the mBMPR-II-transfected cells did not react positively in the presence of mAb 13H8 (Figure 4D). Using a binding assay detected by immunofluorescence, a punctuated expression pattern of AMH in AMHR-II transfected cells was previously observed by the inventors. Two days after transfection, cells were incubated with plasmin-cleaved AMH and the binding was detected using a rabbit polyclonal anti-AMH antibody (Figure 4F). Similarly, the mAb 13H8 monoclonal antibody was used on AMHR-II transfected cells (Figure 4G) and the same punctuated expression pattern was observed. As a control, cells were transfected with mock (Figure 4E).

The monoclonal antibody mAbl3H8 was used to check the endogenous expression of AMHR-II in the membrane fraction of different mouse cell lines and tissues (Figure 3B). The positive control, CHO-3W, a stably hAMHRII transfected CHO cell line, displayed a specific band of 70 kDa. Similarly, the expression of AMHRII in AT29C and SMAT1 cell lines was detected using the mAb 13H8 antibody. Lysates from murine uterus and spleen confirmed the specificity of the 70 kDa signal detected. Interestingly, in the uterus an additional smaller band of about 50 kDa was also detected. This shorter form of AMHRII could result from an alternative splicing as described previously in the rabbit (di Clemente et ah, Mol. Endocrinol., 1994, 8: 1006-1020).

Validation of mAbl3H8 on Archival Human Samples.

Immunohistochemistry was performed on a human ovary using the different anti- AMHR-II antibodies (Figure 5). Very clearly, the N2 polyclonal antibody (Figure 5A) is capable of detecting the expression of AMHR-II on primordial follicles of a prepubertal girl (sample #1321, see table 1). Similarly, mAb 12G4 and mAb 13H8 monoclonal antibodies (Figure 5B and C) displayed the same signal pointing to a very specific expression pattern. More precisely, using c-kit antibody (Figure 5D) a specific marker of germ cell, the inventors were able to show that the oocytes of primordial follicles are labelled.

The same analysis was performed in the rat. In the male, at the neonatal stage, AMHR-II was detected in the interstitial tissue and in spermatogonia by mAb 13H8 and by N2 (Figure 6C and 6E, respectively). At the adult stage, AMHR-II was more widely expressed (Figure 6D and 6F) and was also detected in Sertoli cells with the two antibodies. As expected, the controls did not show any signal, confirming the specificity of the staining (Figure 6A and 6B). In the adult female rat, different concentrations of mAb 13H8 antibody were used to check if there was a differential detection at low versus high antibody concentrations. Using mAb 13H8 antibody at a concentration of 1 g/ml, AMHR-II expression was very specific in the oocytes of developing follicles (Figure 7A) and more surprisingly in primordial follicles (Figure 7B). With high antibody concentrations (4 μg/ml), AMHR-II expression level was still very high in the oocytes (Figure 7F), including in primordial follicles and became visible in granulosa cells of the corresponding follicles (Figure 7D and F). The corresponding control tissues (Figure 7 C and E) exposed to the mouse Ig at the same concentration did not exhibit a specific signal.

EC-hAMHRII can block the AMH Signaling Pathway.

SMAT cells were incubated with recombinant plasmin-cleaved AMH to activate the Smadl pathway. Cell lysates were prepared and subjected to a western blot using the P-Smadl/5/8 antibody. When the cells were pre-incubated with EC-hAMHRII, a polypeptide consisting of the extra-cellular domain of AMHRII (residues 18-145 of the sequence Q16671, SEQ ID NO: 1), the signal was diminished (see Figure 8, lane 4, compared to lanes 2 and 3). This indicates that the transduction was partially blocked and that EC-hAMHRII acts as an AMHRII antagonist.

Discussion

Previously, several groups have described AMHR-II expression at the mRNA level in different species. In the rat, AMHR-II mRNAs were detected by RNAse protection in neonatal ovaries, which contain mainly primordial follicles (Baarends et ah, Endocrinology, 1995, 136: 5614-5622). Using in situ hybridization, these mRNAs were not observed on oocytes or on granulosa cells of primordial follicles. These contradictory results were never explained.

The present inventors have conducted a study using a polyclonal antibody named N2, which they had developed, on archival human samples. Surprisingly, they were able to detect AMHR-II protein in male and female germ cells. More precisely, the protein was present in the oocytes of primordial and primary follicles in the ovary and in spermatogonia at all stages. To confirm these results, they developed a monoclonal antibody raised against the extracellular part of human AMHR-II. After three fusions, two clones were obtained that were assessed for their specificity against the human and rodent species. One of the clone (mAb 13H8) proved to be specific for both human and rat and was used on the archival samples. The expression of AMHR- II in germ cells was confirmed, and the identity of the cells was verified using a c-kit antibody. In the rat, mAb 13H8 revealed that AMHR-II is expressed in male gonocytes and later in all stages spermatogonia. In the female, different expression levels were observed depending on the cell type. AMHR-II was detected at high levels in the oocytes of developing follicles, especially in primordial follicles. The somatic expression was confirmed using higher concentrations of mAb 13H8 antibody.

This never before described expression pattern could explain a direct role of AMH on the recruitment of primordial follicles (Durlinger et ah, Endocrinology, 2002, 143: 1076-1084). In addition, in transgenic mice overexpressing AMH, neonatal ovaries contain very few germ cells and the remaining cells get lost during the next two weeks (Behringer et ah, Nature, 1990, 345: 167-170). This indicates that, like in the freemartin condition, female germ cells are destroyed in the presence of AMH (Vigier et al, Reprod. Nutr. Dev., 1988, 28: 1113-1128). The presence of AMHR-II on oocytes could explain the direct deleterious effect of AMH on germ cells.

The results obtained outline a discrepancy between the previously described AMHR-II expression pattern and the AMHR-II expression pattern observed in the present study using a new monoclonal antibody. Using in situ hybridization, AMHR- II mRNA was never detected in oocytes despite the apparent high protein level revealed using mAb 13H8. The results shown in Figure 8 show that it is possible to block AMH signaling pathway, thereby suggesting that it is possible to improve female fertility and/or treat female infertility by EC-hAMHRII, a polypeptide consisting of the extra-cellular domain of AMHRII (residues 18-145 of the sequence Q16671, SEQ ID NO: 1).

A mutation of the AMHR-II gene (called hot mutation) in the medaka, a small freshwater fish has been found to led to (1) excessive proliferation of germ cells which starts at the hatching stage regardless of the genetic sex, (2) initiation of premature meiosis in phenotypically male hot homozygotes, in keeping with the expression of AMHR-II on germ cell of both sexes (Morinaga et ah, Proc. Natl. Acad. Sci. USA, 2007, 104: 9691-9696).

Claims

Claims What is claimed is:

1. A selective anti-Miillerian Hormone type II receptor (AMHRII) antagonist for use in improving female fertility and/or treating female infertility, wherein said antagonist is not a fragment or a derivative of AMH.

2. The selective AMHRII antagonist according to claim 1, wherein said antagonist is an antibody.

3. The selective AMHRII antagonist according to claim 1 or 2, wherein said antagonist is an antibody directed to the extracellular domain of AMHRII.

4. The selective AMHRII antagonist according to claim 1, wherein the antagonist comprises the extracellular domain of AMHRII.

5. The selective AMHRII antagonist according to claim 4, wherein said antagonist is a fusion protein comprising the extracellular domain of AMHRII.

6. The selective AMHRII antagonist according to claim 4 or 5, wherein said antagonist is a protein comprising the amino acid sequence set forth in SEQ ID NO: 1.

7. The selective AMHRII antagonist according to claim 1, wherein said antagonist is a siRNA, shRNA, an antisense or a DNAzyme targeting AMHRII gene sequence.

8. The selective AMHRII antagonist according to any one of claims 1-7, for improving female human infertility.

9. The selective AMHRII antagonist according to any one of claims 1-7 for improving female bovine infertility.

10. The selective AMHRII antagonist according to any one of claims 1-9, wherein said antagonist is used in combination with sexual hormones.

11. A kit for improving female fertility and/or treating female infertility, comprising as separate components: a selective AMHRII antagonist wherein said antagonist is not fragment or a derivative of AMH, and

sexual hormones.

The kit according to claim 11, wherein the selective AMHRII is as defined any one of claims 2-7.