Anti-FSH receptor single-domain antibody, or VHH, and uses thereof

Abstract

The present invention discloses new anti-FSH receptor (FSHR) VHH, and uses thereof. In particular, the VHHs of the present invention are directed against and / or specifically bind to FSHR ectodomain in a region located between leucine-rich repeat 3 (LRR 3, H98) and leucine- rich repeat 5 (LRR 5, D153), are negative allosteric modulators of FSHR, do not block the binding between FSH and FSHR, and do not bind to LH / CG receptor and TSH receptor.

Description

[0001] Anti-FSH receptor single-domain antibody, or VHH, and uses thereof

[0002] Technical field

[0003] The invention relates to the field of biomedical and biopharmaceutical technology relative to follicle-stimulating hormone receptor (FSHR)-binding molecules and uses thereof. More particularly, the invention relates to Variable Domain of Camelid Heavy Chain-only (VHH) molecules, which bind specifically to FSHR, and uses thereof, e.g., to control mammalian fertility or to prevent or treat FSH-dependent diseases and disorders. Conjugates and pharmaceutical compositions comprising said VHHs are also disclosed.

[0004] Mammalian reproduction is a major concern, as attempts are made to control and promote mammalian fertility, including human fertility.

[0005] The follicle-stimulating hormone receptor (FSHR) plays crucial roles in reproduction of mammals, since FSH is required for normal growth and maturation of ovarian follicles in females and for normal spermatogenesis in males. FSHR is present in granulosa cells of the ovary and Sertoli cells of the testis. In the ovary, the expression of FSHR is dependent on the developmental stage of follicles during the ovulation cycle. Therefore, this receptor is directly correlated with the granulosa cell differentiation, follicular development and ovulation.

[0006] FSHR agonists with varying pharmacological profiles are envisioned to help improving the overall efficiency of medically-assisted procreation. On the contrary, FSHR antagonists aimed at inhibiting the FSHR-dependent signaling cascade have been envisioned as a strategy for preventing fertility status. Due to the highly restricted expression of FSHR in the granulosa cells of the ovary, an FSHR antagonist is anticipated to be devoid of effects on other tissues. Recently, small molecules that act as negative allosteric modulators (NAM) of FSHR (e.g., ADX68692, ADX68693) have been described, that could be used as a nonsteroidal contraceptive. However, cross -reactivity of these NAM on LH / CGR has been reported, so that their specificity is questionable (M. A. Ayoub et al. Mol Cell Endocrinol 2016).

[0007] On the other hand, the FSHR overexpression has been identified in the peripheral vessel of most of the human tumors and suggests its role in angiogenesis. FSHR activity has been further investigated for its role during the development of different tumor pathologies. FSHR antagonists have been suggested for inhibiting the development of ovarian cancer, as well as for inhibiting the FSH action at the ovary in estrogen-dependent diseases such as endometriosis. The discovery of FSH regulatory mechanisms has made possible new opportunities for the development of effective treatments of various disorders related to the reproductive process. However, despite progress made in the field of FSHR antagonists, there is still an unsatisfied need for highly specific molecules able to negatively modulate the FSH-induced signal.

[0008] It has been described in the art that camelid antibodies have properties that render them particularly suitable for therapeutic use. In addition to conventional antibodies, which each have two heavy and two light chains, camelids also produce antibodies that exclusively consist of heavy chains. These so-called heavy chain antibodies, or heavy chain-only antibodies, are homodimers of two identical heavy chains that interact with the antigen through a single variable domain called VHH (variable domain of the heavy chain antibodies). VHHs are more stable and about ten times smaller than conventional antibodies and therefore exert a high solubility and significantly improved tissue penetration properties. For this reason, VHH have been proposed for the treatment of various diseases.

[0009] VHHs correspond to the variable region of heavy chain only camelid antibodies that are naturally devoid of light chains. VHHs have a very small size of around 15 kDa. They contain a single chain molecule that can bind its cognate antigen using a single domain. More specifically, VHHs are composed of four framework regions (or FRs) whose sequences and structures are defined as conserved and three complementarity determining regions (or CDRs) with high variability in both sequence content and structural conformation, which are involved in antigen binding and ensure antigen specificity. The VHH domain of a heavy chain antibody hence forms a small polypeptide unit with high antigen-binding capacity. Due to an exceptionally long CDR3 region with a length of 7 to 25 amino acids, the VHH domain can bind in cavities of protein antigens, unlike conventional antibodies. VHH domains can be produced recombinantly as soluble proteins in bacteria or mammalian cells. Such recombinantly produced VHH domains are also called single-domain antibodies (sdABs) or nanobodies.

[0010] The inventors have now made a significant technical contribution to the art in developing single domain antibodies specifically binding to FSHR and acting as negative allosteric modulators (NAM) of FSHR to inhibit FSH-mediated effects. The single domain antibodies of the present invention are particularly suited for contraception or fertility regulation. The single domain antibodies of the present invention are also suitable for treating or preventing diseases and disorders associated to FSH-induced signal dysregulation, such as ovarian cancers, uterine fibroids, etc. of the invention

[0011] The present invention concerns an anti-FSH receptor (FSHR) VHH, wherein said VHH (i) is directed against and / or specifically binds to FSHR ectodomain in a region located between leucine-rich repeat 3 (LRR 3, H98) and leucine-rich repeat 5 (LRR 5, DI 53);

[0012] (ii) is a negative allosteric modulator of FSHR;

[0013] (iii) does not block the binding between FSH and FSHR; and

[0014] (iv) does not bind to LH / CG receptor and TSH receptor.

[0015] In a particular embodiment, the anti-FSHR VHH comprises complementary determining regions wherein

[0016] CDR1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0017] CDR2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0018] CDR3 comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 7, 8, 9, and 10, with optionally 1, 2, 3 or 4 amino acid modifications, selected from addition, deletion and / or substitution, wherein the CDRs are determined by IMGT.

[0019] In a particular embodiment, the anti-FSHR VHH comprises complementary determining regions wherein CDR1 consists of 8 amino acids, CDR2 consists of 7 amino acids and CDR3 consists of 16 amino acids, wherein the CDRs are determined by IMGT.

[0020] In a particular embodiment, the anti-FSHR VHH comprises complementary determining regions wherein the amino acid sequence of CDR1 consists of SEQ ID NO: 5 or a variant thereof having at least 80% preferably at least 85%, 90%, 95% amino acid identity to SEQ ID NO: 5 over the entire length thereof, the amino acid sequence of CDR2 consists of SEQ ID NO: 6 or a variant thereof having at least 80% preferably at least 85%, 90%, 95% amino acid identity to SEQ ID NO: 6 over the entire length thereof, the amino acid sequence of CDR3 is selected from SEQ ID NOs: 7, 8, 9 and 10 or a variant thereof having at least 80% preferably at least 85%, 90%, 95% amino acid identity to any one of said sequences over the entire length thereof.

[0021] In a particular embodiment, the anti-FSHR VHH comprises an amino acid sequence comprising or consisting of 4 framework regions (FR1 to FR4) and 3 complementary determining regions (CDR1 to CDR3) according to the formula (1):

[0022] FR 1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 ( 1 ) wherein

[0023] FR1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 11, with optionally 1, 2, 3, 4 or 5 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0024] FR2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 15, with optionally 1, 2, 3, 4 or 5 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0025] FR3 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 13, with optionally 1, 2, 3, 4, 5, 6, 7 or 8 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0026] FR4 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 14, with optionally 1, 2, 3 or 4 amino acid modifications, selected from addition, deletion and / or substitution wherein the FRs are determined by IM GT.

[0027] In an embodiment, the amino acid sequence of FR2 comprises or consists of SEQ ID NO: 16, wherein X may be any amino acid, and is preferably selected from A and G.

[0028] In an embodiment, the amino acid sequence of FR1 comprises or consists of SEQ ID NO: 11, the amino acid sequence of FR2 comprises or consists of SEQ ID NO: 12 or SEQ ID NO: 15, the amino acid sequence of FR3 comprises or consists of SEQ ID NO: 13, and the amino acid sequence of FR4 comprises or consists of SEQ ID NO: 14.

[0029] In a particular embodiment, the VHH comprises or consists of - an amino acid sequence of SEQ ID NO:1 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:1 over the entire length thereof, or

[0030] - an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:2 over the entire length thereof, or

[0031] - an amino acid sequence of SEQ ID NO:3 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:3 over the entire length thereof, or

[0032] - an amino acid sequence of SEQ ID NO:4 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:4 over the entire length thereof.

[0033] In a particular embodiment, the VHH specifically binds to human FSHR. Advantageously, said VHH binds at least to amino acid residue H142 of the human FSHR ectodomain. Said VHH may further bind to the amino acid residues DI 37 of the human FSHR ectodomain. In an embodiment, said VHH specifically binds at least to amino acid residues H142, D137 and / or V166.

[0034] In a preferred embodiment, the VHH is specific to human FSHR. Preferably said VHH binds at least to amino acid residue H142 of the human FSHR ectodomain. Said VHH may further bind to the amino acid residues DI 37 of the human FSHR ectodomain, and / or amino acid residue VI 66 of the human FSHR. Advantageously, the VHH binds FSHR with a Kd comprised between 8 nM and 20 nM, preferably between 10 nM and 18 nM.

[0035] It is a further object of the present invention to provide an antigen-binding polypeptide comprising a VHH as described above conjugated to at least one stabilizing group, preferably selected from an antibody or a fragment thereof such as a Fc fragment of IgG or IgA.

[0036] Advantageously, the antigen-binding polypeptide binds FSHR with a Kd comprised between 2 nM and 10 nM, preferably between 3.5 nM and 7.5 nM.

[0037] It is another object of the invention to provide a pharmaceutical composition comprising a VHH or an antigen-binding polypeptide as described above, and a pharmaceutically acceptable carrier. It is another object of the invention to provide a polynucleotide, wherein said polynucleotide encodes the protein consisting of the VHH or of the VHH-Hinge-Fc of the present invention.

[0038] It is another object of the invention to provide an expression vector, wherein said vector contains said polynucleotide.

[0039] The present invention further provides a host cell, wherein said host cell contains the expression vector of the invention or the polynucleotide of the invention is integrated within the genome of the host cell.

[0040] The invention further provides a method for producing an anti-FSHR VHH comprising the steps of:

[0041] (a) culturing the host cell as described above under conditions suitable for production of nanobodies, thereby obtaining a culture containing said anti-FSHR nanobody; and

[0042] (b) isolating and recovering said anti-FSHR nanobody from said culture.

[0043] It is another object of the present invention to provide an anti-FSHR VHH, or an antigenbinding polypeptide, or a pharmaceutical composition of the invention for use as a medicament.

[0044] In particular, the anti-FSHR VHH, antigen-binding polypeptide, or pharmaceutical composition may be used for contraception or fertility regulation.

[0045] It is a further object of the present invention to provide a method for contraception or fertility regulation of a subject, comprising the administration of an anti-FSHR VHH, antigen-binding polypeptide, or pharmaceutical composition of the present invention to said subject.

[0046] Alternatively or in addition, the anti-FSHR VHH, antigen-binding polypeptide, or pharmaceutical composition may be used in the treatment or prevention of ovarian cancer, in the treatment or prevention of pre-menopausal and peri-menopausal hormone-dependent breast cancer, in the treatment or prevention of uterine fibroids or other menstrual-related disorders, or in the treatment or prevention of an estrogen dependent disease.

[0047] It is also an object of the present invention to propose to use of the anti-FSHR VHH, antigenbinding polypeptide, or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of ovarian cancer, in the treatment or prevention of pre-menopausal and peri-menopausal hormone-dependent breast cancer, in the treatment or prevention of uterine fibroids or other menstrual-related disorders, or in the treatment or prevention of an estrogen dependent disease. It is a further object of the present invention to provide a method of treatment or prevention of ovarian cancer of a subject in need thereof, and / or of pre-menopausal and peri-menopausal hormone-dependent breast cancer of a subject in need thereof, and / or of uterine fibroids or other menstrual-related disorders of a subject in need thereof, and / or of an estrogen dependent disease in a subject in need thereof, wherein the anti-FSHR VHH, antigen-binding polypeptide, or pharmaceutical composition of the invention is administered to said subject. VHH from a synthetic library (A, B) Thermal Shift Assay of purified VHH (A) and VHH-Hinge-Fc (B) P52E8, P52E12, P52B7 and P52D6. Melting temperatures (Tm) are indicated on each graph. (C, D) Binding of 300nM VHH on Flag-FSHR, Flag-LH / CGR, TSHR, Flag-mFSHR expressed at CH0-K1 cell surface. Revelation by anti- His-APC labelling (VHH) for TSHR, and by double labelling anti-Flag-PE (receptor) and anti- His-APC (VHH) for Flag-tagged FSHR, LH / CGR and mFSHR.

[0048] Binding characteristics of anti-FSHR candidates VHH (A) Binding of different concentrations of VHH on Flag-tagged FSHR expressed at CH0-K1 cell surface. Fluorescence intensity to VHH concentration curves were plotted to determine VHH binding EC50 (Figure 3, table A). Data were expressed in percentage of the maximum. N=3, means ± SEM. (B) Binding of different concentrations of VHH-Hinge-Fc on FSHR expressed at CH0-K1 cell surface. Fluorescence intensity to VHH-Hinge-Fc concentration curves were plotted to determine VHH-Hinge-Fc binding EC50 (Figure 3, Table A). Data were expressed in percentage of the maximum. N=3, means ± SEM. (C) Binding kinetics of 30nM or lOOnM VHH-LgBiT on SmBiT-FSHR expressed at HEK293A cell surface, measured in luminescence by luciferase complementation. Anti-HEL-LgBiT and LgBiT alone were used as negative controls. N=3, means ± SEM. (D, E) Binding of FSH-LgBiT on SmBiT-FSHR expressed at HEK293A cell surface, measured in luminescence by luciferase complementation. Binding of FSH-LgBiT was assessed at different concentrations, in presence of lOOnM non-relevant Anti- HEL or anti-FSHR VHH (D) or VHH-Hinge-Fc (E). Data represented as area under the curve (AUC) of response measured between 0 and 60 minutes and expressed as the percentage of control condition’s maximum. N=3, means ± SEM. Two-ways ANOVA. (F) Binding of different concentrations of VHH on HEK293A cell membranes containing Flag-tagged FSHR. HTRF signal to VHH concentration curves were plotted to determine VHH binding EC50. Data were expressed as the percentage of maximum binding for each VHH. Binding EC50 of VHH are reported together with the EC50 95% confidence interval (CI). N=3, means ± SEM. (G) VHH and VHH-Hinge-Fc association and dissociation kinetics on biotinylated FSHR ectodomain coupled to FSH, measured by BioLayer Interferometry (BLI). For each VHH or VHH-Hinge-Fc, kinetics constants (Kd, Kon, Koff) (see Figure 3, Table B) were determined after data normalisation to Anti-HEL or Anti-HEL-Hinge-Fc association to the receptor ectodomain and to the OnM VHH or VHH-Hinge-Fc condition.

[0049] Figure 3: EC50 and kinetics constants of VHH and VHH-Hinge-Fc binding on FSHR. (Table A) Binding EC50 of VHH and VHH-Hinge-Fc on FSHR expressed at CHO-K1 cell surface, determined by flow cytometry. The EC50 95% confidence interval (CI) was indicated below the EC50 for each VHH and VHH-Hinge-Fc. (Table B) VHH and VHH-Hinge-Fc binding kinetics constants (Kd, Kon, Koff) on biotinylated FSHR ectodomain coupled to FSH, determined by BioEayer Interferometry (BEI). For each VHH or VHH-Hinge-Fc, Kd was determined after data normalisation to Anti-HEE or Anti-HEE-Hinge-Fc association to the receptor ectodomain and to the OnM VHH or VHH-Hinge-Fc condition.

[0050] Figure 4: Negative modulation of FSHR signaling by VHH-Hinge-Fc. Modulation of FSHR signaling by anti-FSHR P52E8, P52E12, P52B7 and P52D6 or non-relevant Anti-HEE VHH in HEK293A cells. (A) Measurement of cytosolic kinetics of cAMP production induced by 0. InM or 0.3nM FSH, with or without IpM VHH, using Camyel cAMP BRET sensor. N=3, means ± SEM. (B, C) Cytosolic measurement of cAMP production induced by different FSH concentrations (0.1 to lOOnM) in presence of different concentrations of VHH-Hinge-Fc (lOnM to IpM). (B) Representative kinetics at InM FSH with different concentrations of P52E12- Hinge-Fc. (C) Representation at each FSH concentration of the area under the curve from cAMP response measured between 0 and 60 minutes. N=3-6, means ± SEM. (D) Measurement of mGs recruitment kinetics at FSHR induced by lOnM FSH, without VHH or with different VHH concentrations (300nM, IpM). N=3-4, means ± SEM. (E) Measurement of mGs recruitment at FSHR induced by lOnM or 30nM FSH, with or without different concentrations of VHH-Hinge-Fc. Representation at each FSH concentration of area under the curve from mGs recruitment measured at different VHH-Hinge-Fc concentrations. N=3-6, means ± SEM. (F) Statistical results of Figure 4E. (G) Measurement of P-arrestin2 recruitment kinetics at FSHR induced by 30nM FSH, without VHH or with different VHH concentrations (300nM, IpM). N=3, means ± SEM. (H) Measurement of P-arrestin2 recruitment at FSHR induced by lOnM or 30nM FSH, with or without different concentrations of VHH-Hinge-Fc. Representation at each FSH concentration of area under the curve from P-arrestin2 recruitment measured at different VHH-Hinge-Fc concentrations. N=3-6, means ± SEM. (I) Statistical results of Figure 4H. (J) Measurement of the CRE promotor-controlled firely luciferase reporter gene expression after 6 hours stimulation by different FSH concentrations (0.03 to 30nM) and 300nM VHH- Hinge-Fc. Non-relevant Anti-HEE VHH-Hinge-Fc was used as control condition. Data were expressed as the percentage of control condition’s maximal response. N=3, means ± SEM. Two- ways ANOVA. Figure 5: Anti-FSHR VHH-Hinge-Fc are likely to exert their NAM effect on FSHR signaling by stabilizing the receptor in an inactive conformation. (A) Modulation of agonist-induced FSHR internalization by anti-FSHR P52E8, P52E12, P52B7 and P52D6 VHH- Hinge-Fc in HEK293A cells. Non-relevant Anti-HEL VHH-Hinge-Fc was used as control condition. Measurement of FSHR location at plasma membrane after lOOnM FSHR stimulation in presence of IpM VHH-Hinge-Fc, using Lyn-Ypet and FSHR-RLuc8 (ebBRET). N=3, means ± SEM. (Bl, B2) Modulation of FSHR blocked at plasma membrane cAMP response by anti- FSHR P52E8, P52E12, P52B7 and P52D6 VHH-Hinge-Fc. Cytosolic measurement of cAMP production induced by FSH in presence of 300nM VHH-Hinge-Fc. (Bl) Cytosolic cAMP response induced by InM FSH, in presence of 30pM Dyngo4a or 30pM PitStop2 in HEK293A cells. N=3, means ± SEM. (B2) Cytosolic cAMP response induced by 0.3nM FSH in HEK293AParrl / 2 cells. VHH-Hinge-Fc were added either 5 minutes or 15 minutes after FSH stimulation. Non-relevant Anti-HEL VHH-Hinge-Fc was used as control condition. N=3, means ± SEM. (Cl, C2) Modulation of FSHR blocked at plasma membrane cAMP response by anti-FSHR P52E12 VHH-Hinge-Fc in HEK293AParrl / 2 cells. Non-relevant Anti-HEL VHH-Hinge-Fc was used as control condition. Cytosolic cAMP production induced by 0.3nM FSH (Cl) or IpM B3 (C2). 300nM VHH-Hinge-Fc were added either 5 minutes before (-5’), at the same time (O’), 5 minutes after (+5’) or 15 minutes after (+15’) agonist stimulation. N=3, means + SEM. (D) Modulation of mGs recruitment at FSHR by anti-FSHR P52E8, P52E12, P52B7 and P52D6 or non-relevant Anti-HEL VHH-Hinge-Fc, in presence of DMSO (control) or 30pM Dyngo4a. Measurement of mGs recruitment at FSHR induced by lOnM FSH in HEK293A cells. VHH-Hinge-Fc were added 10 minutes after FSH stimulation. N=3, means + SEM. (E) Modulation of mGs recruitment at FSHR blocked at plasma membrane by anti-FSHR P52E12 VHH-Hinge-Fc in HEK293AParrl / 2 cells. Non-relevant Anti-HEL VHH-Hinge-Fc was used as control condition. mGs recruitment induced by lOnM FSH. 300nM VHH-Hinge- Fc were added either 5 minutes before (-5’), at the same time (O’), 5 minutes after (+5’) or 15 minutes after (+15’) FSH stimulation. N=3, means + SEM.

[0051] Figure 6: Determination of anti-FSHR VHH epitope. (A) Modulation of human and mouse FSHR cAMP response by anti-FSHR P52E8, P52E12, P52B7 and P52D6 or non-relevant Anti- HEL VHH-Hinge-Fc in HEK293A cells. Cytosolic measurement of cAMP production induced by InM FSH in presence of 300nM VHH-Hinge-Fc. N=3, means + SEM. (B) Binding of 300nM non-relevant Anti-HEL or anti-FSHR VHH (P52E8, P52E12, P52B7 or P52D6) on Flag-tagged wild-type (WT) and mutants 1 to 11 of human FSHR expressed at HEK293A cell surface. Revelation by double labelling anti-Flag-PE (receptor) and anti-His-APC (VHH). (C) Modulation of wild-type (WT) and mutants 1 to 11 human FSHR cAMP response by anti-FSHR P52E8, P52E12, P52B7 and P52D6 VHH-Hinge-Fc in HEK293A cells. Non-relevant Anti-HEL VHH-Hinge-Fc was used as control condition. Cytosolic measurement of cAMP production induced by 0.3nM (WT and mutants 1, 2, 7), InM (mutants 4, 5, 6), 3nM (mutants 3, 8, 9) or lOnM (mutants 10, 11) FSH in presence of 300nM VHH-Hinge-Fc. N=3, means ± SEM.

[0052] Detailed description of the invention

[0053] Definitions

[0054] As used herein, “heavy-chain antibodies” (HCAb) refer to immunoglobulins which are devoid of light chains and consist in two heavy chains. Each heavy chain comprises a constant region (CH) and a variable domain (VH) which enables the binding to a specific antigen, epitope or ligand. As used herein, HCAbs encompass heavy chain antibodies of the camelid-type in which each heavy chain comprises a variable domain called VHH and two constant domains (CH2 and CH3). Noteworthy, camelid HCAbs lack the first constant domain (CHI). Such heavychain antibodies directed against a specific antigen can be obtained from immunized camelids.

[0055] As used herein, a “single-domain antibody” (sdAb) refers to a single-variable domain, derived from a heavy-chain only antibody, which is able to bind an antigen, an epitope or a ligand alone, that is to say, without the requirement of another binding domain. A single domain antibody may derive from, or consists in, a VHH, also called "nanobody". It is the smallest antigenbinding fragment with complete function, derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. VHH refers to the variable domain found in HCAb of Camelidae (Hamers-Casterman et al. 1993; Desmyter et al. 1996). In the family of "Camelidae" immunoglobulins devoid of light polypeptide chains are found. As used herein, “Camelidae” encompass dromedary, camel, lama, vigugna and alpaca. Camelid HCAbs have been described by Hamers-Casterman et al., Nature, 1993, 363:446.

[0056] An "epitope”, as used herein, refers to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and multidimensional nuclear magnetic resonance.

[0057] A "conformational epitope” , as used herein, refers to an epitope comprising amino acids in a spacial conformation that is unique to a folded 3-dimensional conformation of the polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3-dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. Similarly, conformational epitopes may here also consist of a linear sequence of amino acids of one or more polypeptides that come together and adopt a conformation that is unique to the quaternary structure.

[0058] The term "specificity”, as used herein, refers to the ability of a binding domain, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigen, versus a different antigen, and does not necessarily imply high affinity (as defined further herein). A binding domain, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a nanobody, that can specifically bind to and / or that has affinity for a specific antigen or antigenic determinant (e.g. epitope) is said to be "against” or "directed against” said antigen or antigenic determinant. A binding domain as described herein is said to be "cross-reactive" for two different antigens or antigenic determinants if it is specific for both these different antigens or antigenic determinants.

[0059] The terms "specifically bind” and "specific binding” , as used herein, generally refers to the ability of a binding domain, in particular an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a nanobody, to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

[0060] The term “identity” in relation to an amino acid sequence as used herein refers to the degree of correspondence between two amino-acid sequences (no gaps between the sequences). In other terms, it is the extent, expressed as a percentage, to which two amino acid sequences have the same amino acid at equivalent positions. The “identity” of the "percentage identity” between two amino acid sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool “Emboss needle” for pairwise sequence alignment of proteins provided by EMBL-EBI and available on: http: / / www.ebi. ac.uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, using default settings: (I) Matrix: BLOSUM62, (ii) Gap open: 10, (iii) gap extend: 0.5, (iv) output format: pair, (v) end gap penalty: false, (vi) end gap open: 10, (vii) end gap extend: 0.5.

[0061] As used herein, by “amino acid modification” is meant a change in the amino acid sequence of a polypeptide. "Amino acid modifications" which may be also termed "amino acid changes" , herein include amino acid mutations such as substitution, insertion, and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign “+” indicates a combination of substitutions. In the present document, the following terminology is used to designate a substitution: L62A denotes that amino acid residue (Leucine, L) at position 62 of the parent sequence is substituted by an Alanine (A). A97V / I / M denotes that amino acid residue (Alanine, A) at position 97 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M). In particular embodiments, substitutions are silent substitutions. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well- described in the state of the art. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine). By "amino acid insertion" or "insertion" is meant the addition of at least one amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of at least one amino acid at a particular position in a parent polypeptide sequence.

[0062] The terms “wild-type” FSH receptor or “parent” FSH receptor refer to the non-mutated version of the FSH receptor (FSHR) as it appears naturally. In the present case, the parent FSH receptor refers to the human FSH receptor having the amino acid sequence as set forth in SEQ ID N°21.

[0063] The term "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and / or a prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and / or eliminating, reducing and / or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly. A prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and / or delaying the progression and / or the incidence of a disease or the risk of its occurrence. In certain embodiments, such a term refers to the improvement or eradication of a disease, a disorder, an infection or symptoms associated with it. In other embodiments, this term refers to minimizing the spread or the worsening of cancers. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.

[0064] As used herein, the term “disorder" or “disease” refers to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavourable environmental factors. Preferably, these terms refer to a health disorder or disease e.g. an illness that disrupts normal physical or mental functions.

[0065] The term "cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body, for example in metastasis.

[0066] As used herein, the term “subject”, “host”, “individual,” or “patient” refers to human, including adult and child, and veterinary subjects particularly non-human mammal.

[0067] As used herein, a “pharmaceutical composition” refers to a preparation of one or more of active agents, such as comprising one or more VHHs and / or antigen-binding polypeptide according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical or veterinary composition is to facilitate administration of the active agent to an organism. Compositions of the present invention can be in a form suitable for any conventional route of administration or use. In one embodiment, a “pharmaceutical composition” typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.

[0068] An "acceptable vehicle” or “acceptable carrier” as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical or veterinary compositions. A "therapeutically effective amount" is an amount which, when administered to a subject, is the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The “effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, and characteristics of the subject to be treated.

[0069] As used herein, the term “medicament” refers to any substance or composition with curative or preventive properties against disorders and / or diseases.

[0070] VHH molecules

[0071] An object of the invention relates to single domain antibodies (VHHs) that bind a FSHR. More particularly, the VHHs of the present invention advantageously bind to specific regions of the FSHR’s extracellular hydrophilic domain.

[0072] By working on Negative allosteric modulator (NAM) of FSHR, the inventors have developed single domain antibody (VHH) that specifically bind to FSHR and that negatively modulate FSH-mediated effects.

[0073] Allostery is a phenomenon describing the ability of interactions occurring at one site of a protein to modulate interactions at a spatially distinct site of the same molecule in a reciprocal manner. These modulators can either positively or negatively affect the receptor activation. An allosteric ligand that potentiates an agonist mediated response is referred to as a Positive allosteric modulator (PAM), whereas one that inhibits activity is known as a Negative allosteric modulator (NAM).

[0074] It is therefore an object of the present invention to provide an anti-FSH receptor (FSHR) VHH, wherein said VHH

[0075] (i) is directed against and / or specifically binds to FSH ectodomain in a region located between leucine-rich repeat 3 (LRR 3, H98) and leucine-rich repeat 5 (LRR 5, DI 53);

[0076] (ii) is a negative allosteric modulator of FSHR;

[0077] (iii) does not block the binding between FSH and FSHR; and

[0078] (iv) does not bind to LH / CG receptor and TSH receptor.

[0079] The FSHR structure comprises an extracellular hydrophilic domain (ECD) composed of a long N-terminal part and three loops. This ECD connects to a hydrophobic region comprising seven transmembrane domains (TMDs) and intracellular hydrophilic regions composed of three intracellular loops and the C-terminal part (Jiang et al., 2012, Jiang et al. ,2014a, Fan and Hendrickson, 2005, Ulloa-Aguirre and Zarinan, 2016, Duan et al. 2023). The ECD can be functionally divided into two subdomains, the hormone binding domain (HBD) consisting of ten consecutive leucine-rich repeats domains (LRR) and the hinge region located between the HBD and the first TMD. The hinge region contains two additional LRR, followed by a hairpin loop and an alpha helix flanked on both sides by two cysteine box motifs that are able to form cysteine bonds between themselves. The hinge ends with a highly conserved decapeptide located close to the first TMD reported to be necessary for receptor activation (Bruser et al., 2016).

[0080] The inventors have identified a specific region in the FSHR’s ECD located between residue H98 and residue D153 of the amino acid sequence of FSHR, which forms the LRR3 to LRR5 of FSHR’s ECD, and that is particularly suited as target for VHH in order to negatively modulate the FSHR activation while binding between FSH and FSHR is not inhibited. Therefore, the epitope recognized by the VHH of the present invention is located between residues H98 and D153, more preferably between residues K104 and D153.

[0081] Interestingly, the VHHs developed by the inventors are very specific to FSHR and do not bind to LH / CG receptor nor to TSH receptor.

[0082] Advantageously, the VHH of the present invention negatively modulate FSH-mediated cAMR

[0083] Alternatively or in addition, the VHH of the invention may negatively modulate Gs protein recruitment at FSHR.

[0084] Alternatively or in addition, the VHH of the invention may negatively modulate P-arrestin recruitment at FSHR.

[0085] Advantageously, the VHH of the present invention has no effect on steroidogenesis, the process by which cholesterol is converted to biologically active steroid hormones, in particular ovarian and testicular steroidogenesis.

[0086] The VHH of the invention advantageously comprises three complementary determining regions (CDRs) which determine its binding specificity. Preferably, the CDRs are distributed between framework regions (FRs). The CDRs have generally a length of 5 to 40 amino acids. Preferably, the VHH comprises four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1 " or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively. These framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region 1" or "CDR1 as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. These framework regions and complementary determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxy terminus).

[0087] The CDRs of a given VHH can be determined by any method available to those skilled in the art. For example, and in a non-limiting manner, the Chothia or the Kabat method can be used to determine the CDRs (Chothia et al., Nature 342, 877-883; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). Alternative method of determining CDRs can also be used such as the intermediate method between Chothia and Kabat called AbM (Oxford Molecular AbM antibody modelling software) or the so-called "Contact" method based on an analysis of available complex structures (Saerens et al, Mol Biol. 2005) or on the IMGT method, such as disclosed in Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme).

[0088] In the context of the present invention, unless otherwise specified, the CDRs are determined based on the IMGT method.

[0089] It is an object of the present invention to provide an anti-FSH receptor (FSHR) VHH, wherein said VHH

[0090] (i) comprises a CDR2 having the amino acid sequence as set forth in SEQ ID NO: 6, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, wherein the CDR2 are determined by IMGT

[0091] (ii) is a negative allosteric modulator of FSHR;

[0092] (iii) does not block the binding between FSH and FSHR; and

[0093] (iv) does not bind to LH / CG receptor and TSH receptor.

[0094] According to the present invention, the VHH may comprise CDRs, wherein

[0095] CDR1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0096] CDR2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, and / or CDR3 comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 7, 8, 9, and 10, with optionally 1, 2, 3 or 4 amino acid modifications, selected from addition, deletion and / or substitution, wherein the CDRs are determined by IMGT.

[0097] Preferably, such amino acid modifications do not significantly affect the antigen binding capacity of the resulting VHH, nor its NAM’s properties. Preferably, such amino acid modifications are substitutions such as silent substitutions.

[0098] In particular embodiments, CDR1 comprises from 6 to 10 amino acids, such as 8 amino acids, and / or the CDR2 comprises from 5 to 9 amino acids, such as 7 amino acids and / or the CDR3 comprises from 10 to 20 amino acids, preferably from 14 to 18 amino acids, such as 16 amino acids. Advantageously, the CDR1 consists of 8 amino acids, the CDR2 consists of 7 amino acids and the CDR3 consists of 16 amino acids.

[0099] In some particular embodiments, the VHH of the present invention comprises CDRs, wherein the amino acid sequence of CDR1 comprises or consists of SEQ ID NO: 5 or a variant thereof having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity to SEQ ID NO: 5 over the entire length thereof, and / or the amino acid sequence of CDR2 comprises or consists of SEQ ID NO: 6 or a variant thereof having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity to any one of said sequences over the entire length thereof, and / or the amino acid sequence of CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NOs: 7, 8, 9 and 10 or a variant thereof having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity to any one of said sequences over the entire length thereof.

[0100] As exposed above, the VHH advantageously has the formula FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4 (1).

[0101] In some particular embodiment,

[0102] FR1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 11, with optionally 1, 2, 3, 4 or 5 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0103] FR2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 15, with optionally 1, 2, 3, 4 or 5 amino acid modifications, selected from addition, deletion and / or substitution, and / or FR3 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 13, with optionally 1, 2, 3, 4, 5, 6, 7 or 8 amino acid modifications, selected from addition, deletion and / or substitution, and / or

[0104] FR4 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 14, with optionally 1, 2, 3 or 4 amino acid modifications, selected from addition, deletion and / or substitution, wherein the FRs are determined by IM GT.

[0105] In a particular embodiment, the amino acid sequence of FR2 comprises or consists of SEQ ID NO: 16, wherein X is preferably selected from A and G.

[0106] According to the invention, the VHH may comprise FRs wherein the amino acid sequence of FR1 comprises or consists of SEQ ID NO: 11, the amino acid sequence of FR2 comprises or consists of SEQ ID NO: 12 or SEQ ID NO: 15, the amino acid sequence of FR3 comprises or consists of SEQ ID NO: 13, and the amino acid sequence of FR4 comprises or consists of SEQ ID NO: 14.

[0107] In some embodiments, the anti-FSHR VHH of the invention comprises or consists essentially of or consists of an amino acid sequence defined in any of the sequence SEQ ID NO:1 (QVQLQESGGGLVQAGGSLRLSCAASGYILSINGMGWYRQAPGKERELVAAIGSGGIT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGAGYRSASGAAHFYWG QGTQVTVSS), SEQ ID NO:2

[0108] (QVQLQESGGGLVQAGGSLRLSCAASGYILSINGMGWYRQAPGKERELVAAIGSGGIT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGAGLRSAVAAAHFYWG QGTQVTVSS), SEQ ID NOG

[0109] (QVQLQESGGGLVQAGGSLRLSCAASGYILSINGMGWYRQAPGKERELVAGIGSGGIT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGAALRYTSGTAHFYWG QGTQVTVSS) and SEQ ID NO:4

[0110] (QVQLQESGGGLVQAGGSLRLSCAASGYILSINGMGWYRQAPGKERELVAAIGSGGIT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGAGLRSTVAAAHFYWG QGTQVTVSS), or a sequence having at least 80% sequence identity thereto, preferably at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more amino-acid sequence identity thereto.

[0111] In some embodiments, the anti-FSHR VHH of the invention comprises or consists essentially of or consists of an amino acid sequence defined in any of the sequence SEQ ID NO: 1, SEQ ID NOG, SEQ ID NOG and SEQ ID NOG, with optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications, selected from addition, deletion and / or substitution. In a particular embodiment, the VHH comprises or consists of amino acid sequence defined in any of the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Advantageously, said VHH specifically binds to human FSHR.

[0112] In some particular embodiments, the VHH of the invention has a molecular weight from about 11 kDa to about 18 kDa, for instance from 13 kDa to 17 kDa such as from 15 to 17 kDa or from 15.5 to 16.5 kDa such as about 16 kDa.

[0113] The present invention discloses particular VHHs directed against the FSHR showing a strong affinity for FSHR, in the nanomolar range.

[0114] The term "affinity", as used herein, refers to the degree to which a nanobody binds to an antigen so as to shift the equilibrium of antigen and nanobody toward the presence of a complex formed by their binding. Thus, for example, where an antigen and nanobody are combined in relatively equal concentration, a nanobody of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex.

[0115] The affinity of an antibody for FSHR may be determined by well-known methods described in the prior art. The dissociation constant (Kd) is commonly used to describe the affinity between the antibody and the antigenic target, defined by the equation Kd =[Ag][Ab] / [Ag Ab], which represents the affinity of the antibody-combining site; where [Ag] is the concentration of free antigen (M), [Ab] is the concentration of free antibody (M) and [Ag Ab] is the concentration (M) of the a antigen-antibody complex. Where the antigen and antibody or sdAb react strongly together there will be very little free antigens or free antibody or sdAb, and hence the equilibrium constant or affinity of the antibody or a sdAb will be low. For instance, the Kd of an antibody can be determined by surface plasmon resonance assay in which the antibody is immobilized on the biosensor chip and the solubilized FSHR is passed over the immobilized antibody under flow conditions leading to the measurements of konand koff and thus Kd. Alternatively, biolayer interferometry (BEI) can be used. Alternatively, FSHR may be immobilized on the biosensor chip and the antibody of interest is passed over said immobilized FSHR. As another alternative, the Kd of the antibody can be estimated with flow cytometry or Time Resolved-Fluorescence Energy Transfer (TR-FRET) that measures the EC50. In TR- FRET assays, ligands are usually coupled to conventional fluorescein-like or dy647-like fluorophores (acceptor species), whereas receptors are labelled with lanthanide cryptates (donor species). The FRET signal between the donor and the acceptor (for instance between terbium and d2) can be measured. This signal occurs when the antibody binds to the FSHR only. The FRET signal can be plotted against antibody concentrations so as to obtain a dose-response curve from which the binding affinity of antibody for FSHR can be determined. Preferably, the anti-FSHR VHH according to the invention have a Kd of at most 30 nM, preferably at most 25 nM, even more preferably at most 20 nM.

[0116] In certain aspects, the VHH of the invention binds FSHR, more particularly region between LRR3 and LRR5 of FSHR, with a Kd value comprised between 8 nM and 20 nM, preferably between 10 nM and 18 nM, in particular with a Kd of about 10.5 nM, of about 11.7 nM, of about 12.7 nM or of about 17.6 nM. Advantageously, said Kd is determined by BioLayer Interferometry on purified and biotinylated FSHR ectodomain coupled with FSH.

[0117] Production of VHH

[0118] The VHH molecules described herein in their broadest sense are not limited to a specific biological source or to a specific method of preparation. For example, the VHH can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi- synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0119] Although naive or synthetic libraries of immunoglobulin single variable domains may contain conformational binders against the target FSHR, a preferred example includes the immunization of a Camelidae with a target FSHR to expose the immune system of the animal with the conformational epitopes that are unique to the FSHR. Animals can be immunized with mixtures of the interacting monomers. Thus, such VHH sequences can generally be generated or obtained by suitably immunizing a species of Camelid with a target FSHR, by obtaining a suitable biological sample from said Camelid (such as a blood sample, or any sample of B- cells), and by generating VHH sequences directed against a target FSHR, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person. Alternatively, such naturally occurring VHH domains can be obtained from naive libraries of Camelid VHH sequences, for example by screening such a library using a target FSHR or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries may be used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling. Yet another technique for obtaining VHH sequences directed against a target involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and / or heavy chain antibodies directed against a target), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, or any sample of B-cells), and then generating VHH sequences directed against a target starting from said sample, using any suitable technique known per se. Alternatively, synthetic phage library, designed in silico based on Next Generation Sequencing (NGS) analyses of camelids immune repertoires may be used.

[0120] The invention also relates to antigen-binding polypeptides (also interchangeably called herein “conjugates”) comprising one or more anti-FSHR VHH as defined above, conjugated to at least one molecule or functional group.

[0121] For example, such a modification may involve the introduction (e.g. by covalent linking or in another suitable manner) of one or more functional groups, residues or moieties into or onto the VHH, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the VHH. Examples of such functional groups and of techniques for introducing them can generally comprise all functional groups and techniques mentioned in the art, as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (e.g., Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980).

[0122] Such molecule or functional group may for example be linked directly (for example covalently) to the VHH, or optionally via a suitable linker or spacer.

[0123] Such conjugate can be prepared using a coupling between a VHH and a molecule by any methods known in the art, preferably by a chemical, biochemical or enzymatic pathway, or by genetic engineering.

[0124] The molecule or functional group conjugated to the VHH may be for example any active compound useful in medicine, such as a drug, an imaging molecule, a diagnostic agent, a tracer, a tag or a dye.

[0125] In a particular embodiment, the anti-FSHR VHH of the invention may be fused or conjugated to a labelling mean, e.g. a molecule or a protein selected from an enzyme such as horseradish peroxidase or alkaline phosphatase, a fluorescent protein such as GFP, a fluorescent label such as fluorescein rhodamine label, a chemiluminescent label or bioluminescent label such as luminal, a chromophore, a radio-isotope e.g. suitable for in vivo, ex vivo or in vitro imaging or diagnosing.

[0126] The conjugate may also contain, in addition to or instead of said active compound, a stabilizing group (e.g., an Fc from an IgG, or albumin for instance) to increase the plasma half-life of the VHH or conjugate.

[0127] One of the most widely used techniques for increasing the half-life of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Alternatively, or in addition, the VHH may be fused to an antibody’ fragment.

[0128] Therefore, it is an object of the present invention to provide an antigen-binding polypeptide comprising a VHH as defined above, conjugated to at least one stabilizing group, preferably selected from an antibody or a fragment thereof such as a Fc fragment of IgG or IgA (e.g., VHH-Hinge-Fc).

[0129] Alternatively or in addition, in order to prolonged half-life of the VHH, a binding moiety that recognizes albumin may be conjugated to the VHH.

[0130] In a particular embodiment, said VHH-Hinge-Fc binds FSHR with a Kd comprised between 2 nM and 10 nM, preferably between 3.5 nM and 7.5 nM, such as a Kd of about 3.75 nM, about 4.49 nM or about 7.02 nM. Advantageously, said Kd is determined by BioLayer Interferometry on purified and biotinylated FSHR ectodomain coupled with FSH.

[0131] In a particular embodiment, the VHH-Hinge-Fc of the present invention comprises a VHH sequence as defined in any one of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG and SEQ ID NO:4 and a Hinge-Fc sequence as defined in SEQ ID NO:22. The VHH-Hinge-Fc may further comprise a Flag tag (SEQ ID NO:23). Preferably, the Flag tag is at the C-terminus of the VHH-Hinge-Fc sequence. The VHH-Hinge-Fc may further comprise a signal peptide of SEQ ID NO:24. Preferably, the signal peptide is at the N-terminus of the VHH-Hinge-Fc sequence. In some embodiment, the VHH-Hinge-Fc comprises or consists of the amino acid sequence as defined in any one of the sequences SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. In a particular embodiment, the VHH-Hinge-Fc consists of the amino acid sequence as defined in any one of the sequences SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, with the signal peptide of SEQ ID NO:24 at the N-terminus of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. In some embodiments, said VHH-Hinge-Fc has a molecular weight from about 70 kDa to about 100 kDa, more preferably from 80 kDa to 90 kDa, in particular about 85 kDa.

[0132] Advantageously, the VHH-Hinge-Fc of the present invention negatively modulate FSH- mediated cAMP.

[0133] Alternatively or in addition, the VHH-Hinge-Fc of the invention may negatively modulate Gs protein recruitment at FSHR.

[0134] Alternatively or in addition, the VHH-Hinge-Fc of the invention may negatively modulate P- arrestin recruitment at FSHR.

[0135] Advantageously, the VHH-Hinge-Fc of the present invention has no effect on steroidogenesis, the process by which cholesterol is converted to biologically active steroid hormones, in particular ovarian and testicular steroidogenesis.

[0136] It is a further object of the present invention to provide VHH multimers comprising at least two VHHs of the present invention link together via linker(s). In a particular embodiment, the conjugate comprises two VHHs of the present invention (VHH dimer) said VHHs being same or different. In another embodiment, the conjugate comprises three VHHs, four VHHs, five VHHs, etc., said VHHs being same or different, and being linked via linkers of same or different length. Alternatively, the conjugate may comprise at least one VHH of the present invention and at least one VHH targeting another epitope.

[0137] Nucleic acids, vectors and host cells

[0138] A further aspect of the invention relates to an isolated nucleic acid construct or a polypeptide construct encoding a VHH as defined above. The nucleic acid may be single- or doublestranded or a mixture of the two. The nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture thereof. It can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to one skilled in the art, including chemical synthesis, recombination, and / or mutagenesis.

[0139] The nucleic acid according to the invention may be deduced from the amino acid sequence of the VHH molecules according to the invention and codon usage may be adapted according to the host cell in which the nucleic acid shall be transcribed. These steps may be carried out according to methods well known to one of skill in the art and some of which are described in the reference manual Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Cold Spring Harbor). The invention also relates to a vector containing such an isolated nucleic acid, optionally under control of regulatory sequences (e.g., promoter, terminator, etc.). The vector may be for example a plasmid, virus, cosmid, phagemid or artificial chromosome.

[0140] The present invention further relates to the use of a nucleic acid or vector according to the invention to transform, transfect or transduce a host cell.

[0141] The present invention thus also provides a host cell comprising one or several nucleic acids of the invention and / or one or several vectors of the invention and / or one or several polypeptides encoding the VHH of the invention.

[0142] The host cell may be any host cell capable of expressing or producing a VHH of the invention, including e.g. a prokaryotic host cell, such as e.g., E. coli, or a (cultured) mammalian, plant, insect, fungal or yeast host cell, including e.g. CHO-cells, BHK-cells, human cell lines (including HeLa, COS and PER C6), Sf9 cells and Sf+ cells. An appropriate host cell encompasses a cell of a eukaryotic microorganism such as yeasts and filamentous fungi. Preferred yeast host cell includes Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and Kluyveromyces lactis. The term "host cell" also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. Preferably, the cell is not a human embryonic stem cell. The nucleic acid encoding the VHH may also be transferred in vivo, such as by viral transduction or by transfection, e.g., through nanoparticles, electroporation, microinjection, transcutaneous transfection, etc.

[0143] A further object of the invention is a method for producing a VHH according to the invention, wherein the method comprises the steps of: a) culturing a host cell as previously-defined and b) recovering the said nucleic acid, vector or polypeptide encoding the VHH as defined hereabove from the cell culture.

[0144] It goes without saying that step a) is performed under conditions allowing the expression of the desired nucleic acid, vector or polypeptide by the host cell. Suitable expression conditions may include the use of a suitable medium, the presence of a suitable source of food and / or suitable nutrients, a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled artisan in the art.

[0145] Under such conditions, the VHH of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced. The VHH of the invention may then be isolated from the host cell and / or from the culture medium in which said host cell was cultivated, using protein isolation and / or purification techniques known per se, such as chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques and the like. The VHH may also comprise a tag such as a tag comprising 6 histidines or a streptavidin tag for purification purposes.

[0146] The invention also provides a method to obtain a VHH against FSHR as defined herein. The method for obtaining and / or selecting a VHH according to the invention may be based on a protein selection technology such as, but without being limited to, cell display, phage display, ribosome display, mRNA display, DNA display or plasmid display. These techniques are well- described in the state in the art. For instance, in order to generate a library of VHHs displayed on bacteriophages, the skilled artisan can refer to Muydermans et al., Molecular Biotechnology, 2001, 74, 277-302, in particular to the section entitled Recombinant VHH, the disclosure of which being incorporated therein by reference.

[0147] Pharmaceutical composition

[0148] It is another object of the present invention to provide a pharmaceutical composition comprising at least one VHH or antigen-binding polypeptide as defined above and optionally one or more pharmaceutically acceptable excipients.

[0149] In a particular embodiment, the composition comprises at least two different VHHs of the present invention, or at least two different antigen-binding polypeptides of the present invention, or at least one VHH of the present invention and at least one antigen-binding polypeptide of the present invention. In another embodiment, the composition comprises one or more VHH multimers, in particular VHH dimers, VHH trimers, as defined above.

[0150] The pharmaceutical composition of the invention may be formulated according to standard methods such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; Twenty first Edition, 2005). Pharmaceutically acceptable excipients that may be used are, in particular, described in the Handbook of Pharmaceuticals Excipients, American Pharmaceutical Association (Pharmaceutical Press; 6th revised edition, 2009).

[0151] In one aspect, the compositions of the invention advantageously comprise a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier can be selected from the carriers classically used according to each mode of administration such as (a) fillers or diluents such as for example, starch, lactose, sucrose, glucose, mannitol, microcrystalline cellulose and silicic acid; (b) binders, such as, carboxymethylcellulose, gelatin, polyvinylpyrrolidone, sucrose; (c) humectants, as for example, glycerol; (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, sodium croscarmellose and sodium carbonate; (e) solution retarders, as for example paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as glycerol monostearate; (h) adsorbents such as kaolin and bentonite; (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, (j) antioxidant agents, (k) buffering agents such as sodium citrate or sodium phosphate, (1) preservatives, (m) flavors and perfumes, etc.

[0152] The pharmaceutical composition of the invention may be obtained by admixing a VHH or an antigen-binding polypeptide of the invention with an appropriate degree of purity with at least one customary excipient (or carrier) as described hereabove. In particular, a VHH or an antigenbinding polypeptide of the invention is the active ingredient of the composition.

[0153] It goes without saying that the excipient(s) to be combined with the active ingredient may vary upon (i) the physico-chemical properties including the stability of the said active ingredient, (ii) the pharmacokinetic profile desired for said active ingredient, (iii) the galenic form and (iv) the route of administration.

[0154] The pharmaceutical compositions of the invention may be formulated to be suitable for administration by any conventional route, including by enteral route (i.e. oral) e.g. in the form of tablets, capsules, by parenteral, intramuscular, transdermal, intravenous route e.g. in the form of injectable solutions or suspensions and by topical route e.g. in the form of gels, ointments, lotions, patches, suppositories and the like.

[0155] For oral administration, the active ingredient may be presented as discrete units, such as tablets, capsules, powders, granulates, solutions, suspensions, and the like.

[0156] For instance, mixed with such pharmaceutically acceptable excipients, the active agent may be compressed into solid dosage units, such as pilis, tablets, or be processed into capsules or suppositories. For making solid dosage units, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general, any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the active agent of the invention can be administered as solid compositions include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.

[0157] By means of pharmaceutically acceptable liquids the active agent can be applied as a fluid composition, e.g. as an injection preparation, in the form of a solution, suspension, emulsion, or as a spray, e.g. a nasal spray. For parenteral administration, aqueous suspensions, isotonic 1 saline solutions and sterile injectable solutions may be used, containing pharmaceutically acceptable dispersing agents and / or wetting agents, such as propylene glycol or butylene glycol.

[0158] For parenteral administration, the pharmaceutical composition of the invention may be presented in unit-dose or multi-dose containers, e.g. injection liquids in predetermined amounts, for example in sealed vials and ampoules. In some particular embodiments, the pharmaceutical composition may also be stored in a freeze dried (lyophilized) condition requiring only the addition of sterile liquid carrier, e.g. water, prior to use.

[0159] The pharmaceutical compositions typically comprise an effective dose of a VHH or antigenbinding polypeptide of the invention. A “therapeutically effective dose” or “therapeutically effective amount" as described herein refers to the dose that gives a therapeutic effect for a given condition and administration schedule. A “therapeutically effective dose” of an active substance does not necessarily cure a disease or disorder but will provide a treatment for this disease or disorder so that its appearance is delayed, impeded or prevented, or its symptoms are attenuated, or its term is modified or is less severe, or the recovery of the patient is accelerated.

[0160] The exact dose and regimen of administration of the active ingredient, or a pharmaceutical composition thereof, will necessarily be dependent upon the therapeutic effect to be achieved, and may vary with the particular compound, the route of administration, and the age and condition of the individual subject to whom the medicament is to be administered.

[0161] The invention also relates to a diagnostic composition characterized in that it comprises a VHH or VHH-diagnostic or medical imaging agent conjugate compound such as defined above.

[0162] Uses according to the invention

[0163] The VHHs, antigen-binding polypeptides, compositions and constructs (i.e. isolated nucleic acids, polypeptides and / or vectors) according to the invention may be used in various fields, including biological research, biochemical industry or medicine.

[0164] It is therefore an object of the present invention to provide anti-FSHR VHH or an antigenbinding polypeptide or a pharmaceutical composition, as defined above, for use as a medicament.

[0165] Particularly the VHHs, antigen-binding polypeptides, compositions and constructs of the present invention find applications in contraception or fertility regulation. In particular, the VHHs, antigen-binding polypeptides, compositions and constructs of the invention may be used as contraceptive for human and non-human mammal. Indeed, it is well known that FSHR activity plays an essential role in fertility, particularly in folliculogenesis in females. Indeed, it has been demonstrated in rodents that the total absence of FSHR expression (FSHR knock-out) leads to sterility in females, due to a blockage of folliculogenesis at the pre-antral stage (Dierich et al. 1998). In addition, a number of FSHR polymorphisms and inactivating mutations have been identified in women. These mutations, which lead to defects in FSH response, are clinically associated with amenorrhea and infertility phenotypes (Doherty et al. 2002; Jiang et al. 1998; Lundin et al. 2022; Rivero-Muller et Huhtaniemi, 2002 ; Tapanainen et al. 1998). In males, FSHR knock-out does not lead to sterility. However, these animals show a partial failure of spermatogenesis (Dierich et al. 1998). In humans, FSHR inactivating mutations have been associated with a quantitative and qualitative reduction in spermatogenesis (Tapanainen et al. 1997). Given that defects in FSHR activity are associated with fertility defects, negatively modulating FSHR activity may inhibit folliculogenesis and spermatogenesis.

[0166] Importantly, the VHHs of the present invention are able to perturbate the cAMP / PKA / CREB signalling pathway, which is activated by FSHR and mediates the late stage of the folliculogenesis. Thereby the ovarian growth and survival, as well as the development of Sertoli cells may be inhibited or at least perturbated. In an embodiment, the VHH, antigen-binding polypeptide, composition or construct of the present invention may be used as a transient contraceptive in a subject in need thereof, and / or for the control of fertility in a subject in need thereof.

[0167] Because of their small size (around 15kDa), VHHs have a very short half-life in the body, due to renal glomerular filtration. Furthermore, the half-life of VHHs can be modulated by different strategies, such as fusion with a crystallizable fragment (Fc) of immunoglobulin, or fusion with albumin or a second albumin-binding VHH (Rotman et al. 2015; Chanier and Chames 2019). These strategies give the VHH a half-life similar to that of immunoglobulins, i.e., about 21 days (Liu 2018; Kang and Lee 2021; Mujic-Delic et al. 2014; De Pauw et al. 2023).

[0168] Therefore, in a particular embodiment, an antigen-binding polypeptide comprising the VHH of the invention, conjugated to a Ec fragment of IgG or IgA, or to a binding moiety that recognizes albumin, may be used as a transient contraceptive in a subject in need thereof and / or for the control of fertility in a subject in need thereof.

[0169] In an embodiment, the VHH, antigen-binding polypeptide, composition or construct of the present invention is for use for the manufacture of a medicament to be used as transient contraceptive in a subject in need thereof, and / or for the control of fertility in a subject in need thereof.

[0170] For instance, the VHH, antigen-binding polypeptide, composition or construct of the present invention may be used to prevent fertility. The subject may be a non-human animal, in particular a mammal such as dogs, cats, horses, bovines, pigs, sheep and non-human primates. Alternatively, the subject to treat may be a human, particularly a human of childbearing age, either a female or a male of childbearing age. The subject in need thereof can be administered with suitable amounts of the VHH, antigen-binding polypeptide, composition or construct according to the invention.

[0171] In a preferred embodiment of this invention, the contraceptive compositions of this invention are administered in oral dosage form, preferably in the form of pills or capsules. The pills or capsules can be packaged in any manner suitable for proper delivery and use. For instance, they are packaged in the form of a pharmaceutical kit or package in which the daily unit dosage forms are provided or arranged in a contiguous, sequential order which will enable the subject taking the pills to take the proper formulation at the appropriate time in the reproductive cycle. Alternatively, in another preferred embodiment, the contraceptive compositions are administered by systemic administration, preferably by injection, such as by intramuscular injection, or by mucosal route, such as vaginal administration for female.

[0172] The VHHs, antigen-binding polypeptides, compositions and constructs of the present invention find also application in the treatment or prevention of ovarian cancer. Indeed, FSHR is expressed by ovarian granulosa cells, and its role in ovarian cancers is well established. FSHR is expressed in most ovarian cancer subtypes, especially in 50%-70% of serous ovarian cancers. Furthermore, several polymorphisms of the receptor have been associated with cancer susceptibility, and studies have highlighted that FSHR activates oncogenic signaling pathways, facilitating an invasive phenotype (Perales-Puchalt et al. 2017; Feng et al. 2024).

[0173] The VHHs, antigen-binding polypeptides, compositions and constructs of the present invention find also application in the treatment or prevention of pre-menopausal and peri-menopausal hormone-dependent breast cancer, uterine fibroids or other menstrual-related disorders, or in the treatment or prevention of an estrogen dependent disease, since inhibition of FSH signaling pathway may allow to reduce production of estradiol in ovaries.

[0174] In one embodiment, the subject to treat is a non-human animal, in particular a mammal such as dogs, cats, horses, cows, pigs, sheep and non-human primates. Alternatively, the subject to treat may be a human, particularly a human, at any age, particularly a female at pre-menopausal and peri-menopausal stage.

[0175] Particularly, the subject is affected with a disease that involve pre-menopausal and peri- menopausal hormone(s). In one embodiment, the subject is suffering from cancer, in particular breast cancer or ovarian cancer (Bordoloi et al. 2022, JCI Insight. 2022;7(21):el62553) ; Bordoloi et al., 2023, Sci. Adv. 9, eadh4379). In another embodiment, the VHHs, antigen-binding polypeptides, compositions and constructs of the present invention find also application in the treatment or prevention of prostate cancer (Olayiwola O. Oduwole et al., 2021, The FASEB Journal / Volume 35, Issue 4).

[0176] Importantly, FSHR expression in the endothelium has been identified in a broad multitude of tumors, including (but not limited to): ovarian, breast, urothelial, thyroid, neuroendocrine, pancreatic, pituitary, soft tissue sarcomas, kidney, colon, lung, testicular, gastric, and hepatocellular cancers. The expression of FSHR in and around tumor blood vessels suggests that it is associated with metastasis and tumor neovascularization, and makes it an attractive target for affecting tumor microenvironment (Perales-Puchalt et al., 2017; Ghinea, 2018; Feng et al., 2024). In addition to its expression on the surface of tumor epithelial cells, whose proliferation, migration and invasion it promotes, FSHR is expressed in endothelial cells of tumor blood vessels, and then mediates endothelial FSH transport, tumor angiogenesis and vascular remodeling (Ghinea, 2018). Thus, targeting endothelial vascular FSHR may allow to inhibit the growth of FSHR-positive peritumoral blood vessels. Thus, the VHHs, antigenbinding polypeptides, compositions and constructs of the present invention find also application in the treatment or prevention of solid tumor expressing FSHR, by targeting the tumor microenvironment of the solid tumor.

[0177] A further object of the invention is a VHH, antigen-binding polypeptide, composition or construct of the present invention for use in the treatment of a disorder or disease involving a FSH receptor, such as cancer, and / or for use as a medicament.

[0178] Therefore, it is an object of the present invention to provide a method to prevent or treat a disease or disorder associated to FSH signalling pathway, such as breast cancer, ovarian cancer, endometriosis, prostate cancer, etc., in a subject in need thereof.

[0179] Accordingly, it is herein described methods for inhibiting the growth of a tumor or the spread of metastasis in a subject in need thereof and / or for treating a cancer in a patient in need thereof. The tumor may be a solid tumor or a liquid tumor, preferably a solid tumor. In some embodiments, the tumor or cancer expresses or overexpresses FSHR.

[0180] In certain embodiments, these methods comprise, or alternatively consist essentially of, or yet further consist of, administering to the subject or patient a therapeutically effective amount of VHH, antigen-binding polypeptide, composition or construct of the present invention. In a further aspect, the subject has been previously selected for the therapy by a diagnostic, preferably to evaluate if the tumor expresses or overexpresses FSHR.

[0181] Since FSHR is a relevant target for the treatment of disease or disorder, particularly such as cancer, the VHH of the present invention may be used as a drug, medicament or vaccine. The VHH, antigen-binding polypeptide, composition and construct of the present invention can be used as a medicament or vaccine or for the manufacture of a medicament or vaccine in the treatment of a disease, disorder, or condition in a subject. In some embodiments, such a medicament or vaccine can be used for treating cancer. In an embodiment, the VHH, antigenbinding polypeptide, composition or construct of the present invention is for use for the manufacture of a medicament to be used to prevent or treat a disease or disorder associated to FSH signalling pathway, such as breast cancer, ovarian cancer, endometriosis, etc., in a subject in need thereof.

[0182] The invention also relates to a method for treating a subject suffering from a disorder or disease involving a FSH receptor, wherein said method comprises administering to said subject a therapeutically effective amount of a VHH, antigen-binding polypeptide, composition or construct of the present invention.

[0183] In a particular embodiment, the disease or disorder is cancer, preferably solids tumors, even more preferably selected from the group consisting of endometrial cancer, ovarian cancer, breast cancer, testis cancer.

[0184] Accordingly, the present invention also relates to methods for inhibiting the growth of a tumor in a subject in need thereof and / or for inhibiting the growth and / or spread of metastasis. The tumor may be a solid tumor, or a liquid tumor. In some embodiments, the tumor or cancer expresses or overexpresses FSHR.

[0185] The VHH, antigen-binding polypeptide, composition and construct described herein may be administered with other therapeutics concomitantly or subsequently, including for example, small molecules, CAR-T or NKCE, radiation therapy, chemotherapy, surgery, particularly anticancer agents. An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions can be provided in a combined amount effective to kill or inhibit proliferation of the cell.

[0186] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the VHH, antigen-binding polypeptide, composition and construct to a subject, depending upon the type of diseases to be treated or the site of the disease. This composition can be administered via conventional routes, e.g., administered parenterally (e.g. by intravenous, subcutaneous, intradermal, or intramuscular route), via oral routes, or by mucosal routes (e.g. nasal, vaginal or rectal route). Use in diagnostic and prognostic

[0187] The VHH of the present invention may be used in cell immuno-staining, in in vivo or in vitro imaging and for diagnosis purposes. The invention also relates to a VHHs and antigen-binding polypeptides for use for diagnosing, imaging or treating cells expressing or over-expressing FSHR, such as cancer cells.

[0188] They may also be used as biological reagents in in vitro assays, e.g. as test compounds or competitive binders for the identification, the screening or the characterization of potential drugs targeting a FSH receptor.

[0189] The VHHs disclosed herein can be used diagnostically to monitor expression FSHR levels in tissue or cells as part of a clinical testing procedure in vitro or ex vivo as well as in vivo, e.g., to determine the efficacy of a given treatment regimen.

[0190] The detection method of the present disclosure can be used to detect levels of FSHR expression in a biological sample in vitro or ex vivo as well as in vivo, for example after a biopsy of an organ or tissue. In vitro or ex vivo techniques for detection of FSHR by the VHHs of the invention include, without limitation, enzyme linked immunosorbent assays (ELISAs), RIA, EIA and other "sandwich assays”, Western blots, flow cytometry, immunoprecipitations, radioimmunoassay, homogeneous time-resolved fluorescence (HTRF), Fluorescence resonance energy transfer technology with time-resolved measurement (TR-FRET), Bioluminescence Resonance Energy Transfer (BRET), protein complementation assay based on reconstitution of split fragments of a protein reporter (e.g., fluorescent or bioluminescent protein) and immunofluorescence (e.g., IHC). In in vivo techniques for detection of FSHR by the VHHs of the invention, the VHHs can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0191] The present invention also provides diagnostic, prognostic or predictive assays for determining whether a subject is at risk of developing a medical disease or condition associated with increased FSHR expression or activity (e.g., detection of a precancerous or cancerous cell that overexpress FSHR). Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a medical disease or condition characterized by or associated with FSHR expression or overexpression.

[0192] Kits

[0193] Any of the VHHs, antigen-binding polypeptides, compositions and constructs described herein may be included in a kit provided by the present invention. In certain embodiments the kit includes suitable container means, cells, buffers, cell media, vectors, primers, restriction enzymes, salts, and so forth, for example. The kits may also comprise means for containing a sterile, pharmaceutically acceptable buffer and / or other diluents.

[0194] In some embodiments, means of taking a sample from an individual and / or of assaying the sample may be provided in the kit.

[0195] In some embodiments, the kit further includes an additional agent for treating cancer and the additional agent may be combined with VHH, antigen-binding polypeptide, composition and construct, or other components of the kit of the present invention or may be provided separately in the kit.

[0196] In some cases of the invention, the kit also includes a second cancer therapy, such as chemotherapy and / or other immunotherapy, for example.

[0197] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. In an embodiment, the invention relates to a kit as defined above for a single-dose administration unit or for multiple-doses administration.

[0198] The kit of the invention may also contain a first recipient comprising a dried / lyophilized functional molecule (e.g., VHH) and a second recipient comprising an aqueous formulation. In certain embodiments of this invention, kits containing single-chambered and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided.

[0199] In a particular embodiment, the kit may comprise microneedle device or patch for transdermal delivery of the VHHs.

[0200] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.

[0201] The instructions related to the use of the VHH, antigen-binding polypeptide, composition and construct described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, or means for reconstituting or diluting such components. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual). In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of VHH, antigen-binding polypeptide, composition and construct described herein, particularly in the context of the treatment of a disease as described herein such as cancer. Alternatively, the included instructions can comprise a description of administration of VHH, antigen-binding polypeptide, composition and construct described herein, in the context of control of the fertility.

[0202] Other aspects and advantages of the present invention will become apparent upon consideration of the examples below, which are only illustrative in nature and which do not limit the scope of the present application.

[0203] EXAMPLES

[0204] MATERIALS ND METHODS

[0205] Recombinant FSH (GONAL-f®) was kindly provided by Merck (Darmstadt, Germany) and resuspended in mQ H2O. The chemical agoPAM B3 was previously characterised (De Pascali, F. et al., Int. J. Mol. Sci. 22, (2021); Sposini et al., Front. Pharmacol. 11, 1-14 (2020)). The Anti-Hen Egg Lysozyme (Anti-HEL) VHH (Akiba et al., Sci. Rep. 9, 4-6 (2019)) was chosen from the literature to serve as a non-relevant control VHH, and was produced either in bacteria (for the VHH format) or by Sino Biological (Diisseldorfer, Germany) (for the VHH-Hinge-Fc format).

[0206] Dyngo4a was purchased at MedChemExpress (South Bruunswick, NJ, USA). PitStop2 was from Sigma-Aldrich (St Louis, MO, USA).

[0207] Cell culture in adhesion

[0208] Human Embryonic Kidney 293 (HEK293A) (Thermo Fisher Scientific, Waltham, MA, USA), Human Embryonic Kidney 293 with P-arrestins 1 & 2 knock-out (HEK293AParrl / 2) (kindly donated by Dr Azuka Inoue, Tohuku University, Japan) and Chinese Hamster Ovary (CHO-K1) (ATCC) cells were respectively cultured in DMEM (Eurobio, Les Ulis, France) (for HEK293A and HEK293AParrl / 2) and F12K Nutri mix (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) (for CHO-K1) medium containing Glutabio and NAHCO3 and supplemented with 10% heat inactivated foetal bovine serum (Eurobio, Les Ulis, France), 100 lU / mL peniccillin and 0.1 mg / mL streptomycin (Eurobio, Les Ulis, France). Cells were kept at 37°C in a humidified 5% CO2 incubator. Cell culture in

[0209] ExpiCHO-S™ cells (kindly provided by Dr. Nicolas Aubrey, Tours, France) were cultured in ExpiCHO-S Expression Medium (Thermo Fisher Scientific, Waltham, MA, USA) and kept at 37 °C in a humidified 8% CO2 incubator under 12000g shaking (CellTron Infers HT, Massy, France). library

[0210] The synthetic phage library was designed in silico based on Next Generation Sequencing (NGS) analyses of different camelids immune repertoires. The VHH framework was previously described (McMahon et al. Nat. Struct. Mol. Biol. 25, 289-296 (2018)). A diversity of 2,O611xlO10was introduced within VHH complementarity-determining regions (CDRs) and framework (FR) sequences, with length variability introduced in CDR3 length. This resulted in 32 possible variants for CDR1, 8 possible variants for FR2, 36 possible variants for CDR2, 2 possible variants for FR3 and 4096, 65536 or 1048576 possible variants for CDR3 of 12, 16 or 20 amino acids length, respectively. The library was synthesised by Twist Bioscience (San Francisco, CA, USA) and inserted into pADE™-23c phagemid vector (Antibody Design Eabs, San Diego, CA, USA).

[0211] The anti-hFSHR phage display was performed on living cells expressing the receptor, starting from the synthetic phage library described above. To ensure the specificity of the selection, two other phage displays were conducted in parallel: an anti-hEH / CGR selection (the hEH / CGR being the receptor with the closer sequence identity to hFSHR), and a selection on nontransfected cells performed with a mix of phages recovered from rounds 1 of anti-hFSHR and anti-hEH / CGR phage displays (“control” selection). The first rounds were conducted on receptors only. In rounds 2 and 3, ligands were added at concentrations previously described as receptors’ internalisation EC50 (Ayoub, M, A. et al., Front. Endocrinol. (Lausanne). 6, 1-14 (2015)), in order to complete a selection on both active and inactive forms of the receptors: 3nM FSH + 3pM agoPAM B3 (De Pascali, F. et al., Int. J. Mol. Sci. 22, (2021), Sposini et al., Front. Pharmacol. 11, 1-14 (2020)) for the anti-hFSHR phage display, 5nM EH for the anti- hEH / CGR phage display. The two rounds of control phage display were performed in presence of these three ligands.

[0212] To perform VHH selection, lOpE of phage library at 1014phages / mE were diluted in lOOpL blocking buffer (Ca2+-Mg2+-free PBS with 2% dry milk), and depleted against 3xFlag peptide, non-transfected HEK293A and non-transfected CHO cells. For rounds 2 and 3, additional depletions were performed: on FSH and B3 for anti-hFSHR and control phage displays, and on LH for anti-hLH / CGR and control phage displays. 3 million of HEK293A (rounds 1 and 3) or CHO-K1 (round 2) cells were seeded in 100mm diameter disks 24h prior transfection, and transfected with lOpg / disk of DNA coding for either Flag-hFSHR (Tranchant et al., Mol. Cell. Endocrinol. 331, 109-118 (2016)) or Flag-hFH / CGR (kindly provided by Pr. Aylin Hanyaloglu, Imperial College Fondon, UK) using jetOPTIMUS® (Polyplus-transfection S.A, Illkirch, France) according to the manufacturer’s instructions. 24h (CHO-K1) or 48h (HEK293A) later, cells were detached with Ca2+-Mg2+-free PBS lOmM EDTA and washed with PBS before eliminating dead cells by magnetic sorting with Dead cell Removal MicroBeads (Miltenyi Biotec, Gladbach, Germany). Receptor expression levels were assessed by flow cytometry using Anti-Flag-PE antibody (Miltenyi Biotec, Gladbach, Germany) following the manufacturer’s instructions. Phages selection was performed by incubating one million of adequate transfected cells (or unstransfected cells for the control phage display) with the corresponding ligand(s) (rounds 2 and 3 only) and previously depleted phages during Ih on wheel (15rpm) at 4°C. Cells were then centrifuged (500g, 5min, 4°C), and supernatant containing unbound phages was removed. After a cold pH 7.4 PBS wash, two washes with cold pH 5.0 PBS were performed to remove phages with low binding specificity, followed by two cold pH 7.4 PBS washes. After centrifugation (500g, 5min, 4°C), cells were resuspended in IX PBS 0.5% BSA, and untransfected cells were eliminated by magnetic sorting using Anti-PE MicroBeads Ultra-Pure (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions. Recovered cells were centrifuged (500g, 5min, 4°C), and the pellet was resuspended in 50pg / mE trypsin, 50mM TrisHCl pH 8.0, ImM CaCh buffer to eluate phages. After 2min incubation at room temperature, this was added to E.coli TGI bacteria (Cambridge Bioscience, Cambridge, UK) culture in exponential growth phase and incubated Ih at 37°C under constant shaking (200rpm) for phages to infect bacteria. A fraction of the culture was collected to proceed with phages titration. The rest of the culture was centrifuged (3000g, lOmin, RT) and the pellet was resuspended in 2xTY, 2% glucose, lOOpg / mE ampicillin for overnight culture at 30°C under constant shaking (200rpm). A fraction of overnight culture was transferred into fresh 2xTY, 2% glucose, lOOpg / mE ampicillin and incubated Ih at 37°C under constant shaking (200rpm). Phages were rescued by adding 2xl09particles of helper phage (kindly provided by Dr. Pierre Martineau, IRCM, Montpellier, France), and Ih incubation at 37 °C without shaking was performed. The culture was centrifuged (3000g, lOmin, RT) and the pellet was resuspended in 2xTY, lOOpg / mE ampicillin, 25pg / mE kanamycin prior overnight incubation at 30°C under constant shaking (200rpm). Culture was centrifuged (3000g, lOmin, 4°C), and supernatant was recovered to precipitate phages by adding NaCl 2.5M, PEG6000 20% and conducting 40min incubation on ice. Centrifugations (10000g, 4°C) were performed to remove supernatant and remaining traces of PEG6000, and pellet was resuspended in PBS 15% glycerol. Centrifugation (lOmin, 10000g, 4°C) was conducted to eliminate cellular debris, and supernatant containing purified phages was stored at -20°C until further selection round. Two rounds of control phage display were performed from a mix of phages recovered from rounds 1 of anti-hFSHR and anti-hLH / CGR selections. Rounds 2 of anti-hFSHR and anti- hLH / CGR selections were performed with phages recovered from the corresponding round 1, and rounds 3 with phages recovered from the corresponding round 2. Alternating blocking buffers were used for the different rounds of selection: Ca2+-Mg2+-free PBS with 2% dry milk for rounds 1 and 3, and Ca2+-Mg2+-free PBS with 2% BSA for round 2. All incubations with phages were performed in low-binding plastic tubes.

[0213] VHH and analysis Low-Throughput Sequencing: 96 clones Sanger sequencing

[0214] 96 E.coli TGI bacteria isolated colonies infected with phages selected from rounds 3 were randomly chosen and individually cultured in 2xTY medium overnight at 37°C under shaking. A Polymerase-Chain Reaction (PCR) was performed on each culture using P6S6 forward primer (5’-GTAAATGAATTTTCTGTATGAGG-3’ - SEQ ID NO: 17) and cpS4 reverse primer (5’-GCGGATAACAATTTGAATTCAAGGAGACAG-3’ - SEQ ID NO: 18) encompassing the multiple cloning site (MCS) of pADL™-23c phagemid vector (Antibody Design Labs, San Diego, CA, USA) with Platinium Taq DNA polymerase (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The obtained amplified VHH sequences were sent for Sanger sequencing to GENEWIZ™ (a service from Azenta Life Sciences, Burlington, MA, USA). Results were analysed by aligning sequences from each phage display to identify the number of repeated sequences (forming clusters) and their specificity for a given phage display.

[0215] VHH production and purification in E.coli bacteria

[0216] E.Coli BL21 (DE3) bacteria (NEB, Ipswich, USA) were transformed with a pET22b+ plasmid coding for a given 6xHis-tagged VHH. Bacteria cultures were amplified in 2xTY medium supplemented with 200pg / mL ampicillin and 0,1% glucose, and induction was performed by adding ImM IPTG (Invitrogen, Waltham, MA, USA) for overnight incubation at 28°C. Bacteria cultures were then centrifuged (4000g, 20min, 4°C). To extract periplasmic fraction, each pellet was resuspended in cold TES (lOOmM Tris-HCl pH 8.0, 0,5mM EDTA, 20% sucrose). After 30 minutes incubation on ice, cold distilled water (same volume as TES) was added before 15 minutes incubation on ice. After centrifugation (10000g, lOmin, 4°C), NaCl final concentration was adjusted to 0.5M, and SDS-PAGE followed by anti-6xHis Western Blot was performed to check VHH expression. The supernatant was used to extract leak fraction, by performing overnight dialysis against a pH 8.0 buffer (20mM Tris-HCl, 0.5M NaCl) at 4°C, using cellulose membrane tubing with a 6-8kDa molecular weight cut off (MWCO) (Thermo Fisher Scientific, Waltham, MA, USA). Depending on recovered amount, periplasm and leak fractions were either pooled or purified separately. VHH column purification was performed using Protino Ni- IDA 2000 kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer’s instructions. After 250mM imidazole (Merck, Darmstadt, Germany) elution, the eluate was dialysed (20mM Tris-HCl pH 7.4, 0.3M NaCl) to reduce imidazole concentration to less than ImM and concentrated with Centrifugal Filter Units 3.000 MWCO (Merck, Darmstadt, Germany). Purified VHHs were stored at -20°C.

[0217] VHH-Hinge-Fc production in ExpiCHO mammalian cells

[0218] ExpiCHO-S™ cells at 6xl06living cells / mL density were transfected with 0.8mg / mL pTwist CMV BG WPRE Neo plasmid vector (Twist Bioscience, San Francisco, CA, USA) coding for a given VHH fused to a FLAG-tagged, LALA-PG mutated (Lo, M. et al., J. Biol. Chem. 292, 3900-3908 (2017)) mouse IgG2a Hinge-Fc domain (VHH-Hinge-Fc), according to ExpiCHO- S™ Expression System (Thermo Fisher Scientific, Waltham, MA, USA) manufacturer’s instructions. Cells were cultured for 10- 12 days (37°C, 8% CO2) on a shaker platform (120rpm). The culture was centrifuged (4000g, lOmin, 4°C), and the collected supernatant was dialysed in cellulose membrane tubing with a 6-8kDa MWCO (ThermoFisher Scientific, Waltham, MA, USA) against a pH 8.0 buffer (50mM TrisHCl, lOOmM NaCl) overnight at 4°C under continuous stirring. VHH-Hinge-Fc purification was performed by Protein A affinity chromatography using UNOsphere SUPrA Affinity Chromatrography Media (Bio-Rad Laboratories, Hercules, CA, USA). Protein A resin was incubated with supernatant at room temperature for Ih, centrifuged (4000g, lOmin, 4°C), and transferred to a Poly-Prep® Chromatography Column (Bio-Rad Laboratories, Hercules, CA, USA). The resin was extensively washed (50mM TriHCl pH 8.0, lOOmM NaCl). Protein elution was performed with a lOOmM glycine pH 2.9 buffer, before neutralisation with a IM TrisHCl pH 8.0 solution. The eluates were dialysed against 50mM TrisHCl pH 8.0 and lOOmM NaCl, and purified VHH- Hinge-Fc were stored at -20°C until further use.

[0219] VHH stability assessment: Thermal Shift Assay (TSA)

[0220] Purified VHH and VHH-Hinge-Fc stability was assessed by Thermal Shift Assay (TSA). 5pg of purified VHH or VHH-Hinge-Fc were mixed with fluorescent dye SYPRO-Orange (Invitrogen, Waltham, MA, USA) (final reaction 5X) in Tris / NaCl buffer solution. The mix was exposed to a continuous temperature increase (from 25°C to 95°C, in a 10 seconds ramp increment of 0.5°C followed by a reading) in a BioRad CFX Opus Real-Time PCR Systems machine. A control was performed with the buffer solution only. Using CFX Maestro™ Software, fluorescence intensity at each temperature was collected to generate a melting curve and a plot showing negative regression of fluorescence versus temperature ((-dRFU) / dT). The greatest fluorescence change, represented as a visible peak, allowed determination of the protein melting temperature (Tm). Enzyme-linked immunoassay (ELISA)

[0221] Twenty thousand CHO-K1 cells per well were seeded in a 96-well plate 24h prior transfection. Adherent cells were transfected with 0.3pg / well of DNA coding for a given Flag-tagged receptor (hFSHR or non-relevant GPCR mouse oxytocin receptor (mOTR)) using jetOPTIMUS® (Polyplus-transfection S.l, Illkirch, France) transfection reagent. 24h after transfection, wells were blocked for Ih at 4°C with blocking buffer (PBS, 1.5% BSA). lOOpE of VHH crude periplasmic extract (previously prepared by performing an osmotic shock) 1 / 3 diluted in blocking buffer were added into wells containing cells transfected with either hFSHR or mOTR, and incubated Ih at 4°C. Wells were then washed (PBS, 0.5% BSA) and incubated Ih at 4°C with a 6xHis-HRP antibody (Miltenyi Biotec, Gladbach, Germany) diluted in blocking buffer. After final wash, ECE substrate (Thermo Scientific, Waltham, MA, USA) was added in each well. Signals were recorded with a Tristar plate reader (Berthold Technologies GmbH & Co., Wildbad, Germany).

[0222] BioLayer Interferometry (BLI) hFSHR ectodomain coupled to Hinge-Fc portion (hFSHR ECD-Hinge-Fc with a 6xHis tag) was co-transfected with monocatenary FSH (kindly given by Yves Combarnous and Daniele Klett, CNRS, Nouzilly, France) in ExpiCHO-S™ cells. Culture in ExpiCHO-S™ and supernatants were processed as described previously. hFSHR ECD-Hinge-Fc was then purified using Protino® Ni-IDA packed columns (Macherey-Nagel, Hoerdt, France) and buffer exchanged using Desalting Prepacked Gravity Flow Columns (Bio-Rad Laboratories, Hercules, CA, USA). The hFSHR ECD-Hinge-Fc / FSH complex was then biotinylated using the EZ-link Sulfo-NHS-Biotinylation kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, with a 1:1 biotimectodomain ratio. To perform affinity measurements, all samples were diluted in home-made kinetics buffer consisting in IX Ca2+ / Mg2+-free PBS pH 7.4 with 0.002% BSA. Experiments were conducted with an Octet RED96 interferometer (Pall Forte Bio, Fremont, CA, USA) at 30°C under lOOOrpm shaking. Streptavidin coated sensors Octet® SAX Biosensors (Sartorius, Aubagne, France) were left for lOmin equilibration in kinetics buffer, before lOnM biotinylated hFSHR ECD-Hinge-Fc / FSH loading for 150sec. After two washing steps in kinetics buffer (respectively for 60sec and 30sec), sensors were incubated for lOOsec with different concentrations of VHH or VHH- Hinge-Fc, before assessing dissociation in kinetics buffer for lOOsec. For each VHH or VHH- Hinge-Fc tested, Anti-HEL (without or with Hinge-Fc, respectively) was used as a negative control for hFSHR binding at the same concentrations than the tested nanobody. Anti-HEL curves obtained were subtracted to the tested nanobody curves at the corresponding concentration for the analysis. For each nanobody, a second normalisation on the condition with no nanobody was done. The association / dissociation profiles were fitted with a 1:1 interaction model and the kinetics constants calculated using the Octet Data Analysis 9.0 software.

[0223] Determination of VHH binding EC50 by Homogeneous Time Resolved Fluorescence (HTRF)

[0224] Cell membranes were prepared as follows: three million CHO-K1 cells were seeded in a 100mm dish 24h prior transfection. Adherent cells were transfected with lOpg / dish of DNA coding for a Flag-tagged hFSHR using jetOPTIMUS® transfection reagent (Polyplus-transfection S.l, Illkirch, France). 24h after transfection, cells were detached, resuspended in PPI Terbium detection buffer (Revvity, Bussy-Saint-Martin, France) at the density of 20000 cclls / 5p L and frozen at -70°C for at least 24h. Cells were thawed at 37°C during 5 minutes, and frozen at - 70°C during at least one night. Then, cells were thawed a second time at 37 °C during 5 minutes and centrifuged (10000g, 10 minutes, 4°C). The pellet containing cell membranes was resuspended in PPI Terbium detection buffer at the same density as previously, for immediate use. HTRF was performed in white 384-wells plate. Prepared cell membranes were added and mixed with VHH diluted in PPI Terbium detection buffer (final VHH concentration from 0 to IpM). After Ih incubation at room temperature, an anti-Flag M2-Tb cryptate antibody and an anti-6xHis-d2 antibody (Revvity, Bussy-Saint-Martin, France) were added directly in each well according to the manufacturer’s instructions, and incubated overnight at 4°C away from light. Signals were recorded with a Tristar plate reader (Berthold Technologies GmbH & Co., Wildbad, Germany). Data were analysed and plotted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). For each VHH, values were expressed in percentage of maximal response. N=3, means ± SEM.

[0225] Flow cytometry

[0226] Four hundred thousand CHO-K1 or HEK293A cells per well were seeded in a 6-well plate 24h prior transfection. Adherent cells were transfected with 2pg / well of DNA coding for a given Flag-tagged receptor (except hTSHR with no tag) using jetOPTIMUS® (Polyplus-transfection S.l, Illkirch, France) transfection reagent. 24h (CHO-K1) or 48h (HEK293A) after transfection, cells were washed with Ca2+-Mg2+-free PBS, detached with lOmM EDTA Ca2+-Mg2+-free PBS, and resuspended in a home-made staining buffer (PBS, 0.5% BSA for CHO-K1; PBS, 1% SVF for HEK293A). Cells were incubated for Ih at 4°C with a determined concentration of VHH or VHH-Hinge-Fc, washed twice with staining buffer, and centrifuged (500g, 5min, 4°C). VHH being tagged with 6xHis and VHH-Hinge-Fc with Flag, cells were stained with different antibodies depending on the VHH format tested. Table 1: Cells staining

[0227] All stainings were conducted in staining buffer with 1 : 1000 diluted LIVE / DEAD Fixable Violet Dead Cell Stain (Invitrogen, Waltham, MA, USA). After 30min incubation at 4°C, cells were washed twice with staining buffer before being resuspended in PBS. Samples were analysed with a MACSQuant Analyser 10 flow cytometer (Miltenyi Biotec, Gladbach, Germany).

[0228] Bioluminescence Resonance Energy Transfer (BRET)

[0229] Fourty thousand HEK293Aor HEK293AParrl / 2 cells per well were seeded in previously 0.01% poly-lysine treated 96- well plates, and transiently transfected in suspension using Metafectene Pro transfection reagent (Biontex Laboratories, Miinchen, Germany) according to the manufacturer’s instructions, using the following DNA quantities.

[0230] Table 2: Quantity of DNA transfected / well

[0231] The Camyel sensor has been the first cAMP BRET sensor designed (L.I. jiang et al. Journal of Biological Chemistry vol. 282 10576-10584 2007). The cAMP BRET sensor NLuc- EpacD602A-VV-NES was designed from the cAMP BRET sensor NLuc-Epac-VV (Masuho et al. Physiol Behav. 176, 139-148, 2007) (kindly given by Prof. Kirill A. Martemyanov, The Scripps Research Institute Florida, FL, USA) by adding a nuclear exclusion signal (NES) sequence and a mutation in 602 position to improve the sensor’s dynamic range (Klarenbeek et al. PloS One 10, 1-11 2015).

[0232] 48 hours after transfection, BRET measurements were performed upon addition of 5pM coelenterazine-H (Interchim, Montlucon, France) diluted in Ca2+ / Mg2+-free PBS, containing no or different concentrations of FSH or agoPAM B3 (De Pascali et al. Int. J. Mol. Sci.22, 2021; Sposini et al. Front. Pharmacol, 11, 1-14 2020), with or without different concentrations of VHH or VHH-Hinge-Fc. For experiments performed in presence of Dyngo4a or PitStop2, cells were pre-incubated 35 minutes in presence of drug-containing buffers (or buffer with DMSO for control condition) before measurements and ligand(s) stimulation(s). Signals were recorded for at least 60 minutes with a Mithras LB 943 plate reader (Berthold Technologies GmbH & Co., Wildbad, Germany). BRET ratios were calculated as follows: 480nm / 540nm for cAMP experiments; 540nm / 480nm for mGs recruitment, P-arrestin2 recruitment, receptor internalisation and traffic to endosomes experiments. Data were analysed and plotted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Values were represented as means ± SEM. Statistical significance was determined by two- ways ANOVA performed on 0-60min area under the curve (AUC) values, and p-values considered significant if <0.05.

[0233] NanoBiT binding assays

[0234] To assess anti-FSHR VHH binding kinetics and determine whether these VHH competed with FSH for binding on hFSHR, two luminescence assays based on fusion of interest proteins with two different parts of a split luciferase were designed. a) VHH-LgBiT binding on SmBiT-FSHR

[0235] Fourty thousand HEK293A cells per well were seeded in previously 0.01% poly-lysine treated 96-well plates, and transiently transfected in suspension with 40ng / well DNA of hFSHR fused to luciferase SmBiT subunit (FSHR-SmBiT, synthesised by Twist Bioscience), using Metafectene Pro transfection reagent (Biontex Laboratories, Miinchen, Germany) according to the manufacturer’s instructions. VHH fused to luciferase LgBiT subunit (VHH-LgBiT, 6xHis- tagged) were produced in ExpiCHO-S™ cells and purified using Protino® Ni-IDA packed columns (Macherey-Nagel, Hoerdt, France). 48 hours after transfection, luminescence measurements were performed upon addition of lOpM 8-benzyl-2-(furan-2-ylmethyl)-6- phenylimidazo[l,2-a]pyrazin-3(7H)-one (AChemBlock, Hayward, CA, USA) diluted in Ca2+ / Mg2+-free PBS, containing OnM, 30nM or lOOnM of VHH-LgBiT. Signals were recorded for at least 60 minutes with a Mithras LB 943 plate reader (Berthold Technologies GmbH & Co., Wildbad, Germany). Data were represented as means ± SEM. b) FSH-LgBiT binding on SmBiT-FSHR

[0236] Fourty thousand HEK293A cells per well were seeded in previously 0.01% poly-lysine treated 96-well plates, and transiently transfected in suspension with 80ng / well DNA of hFSHR fused to luciferase SmBiT subunit (FSHR-SmBiT, synthesised by Twist Bioscience), using Metafectene Pro transfection reagent (Biontex Laboratories, Miinchen, Germany) according to the manufacturer’s instructions. FSH fused to luciferase LgBiT subunit (FSH-LgBiT, 6xHis- tagged) was produced in ExpiCHO-S™ cells and purified using Protino® Ni-IDA packed columns (Macherey-Nagel, Hoerdt, France). 48 hours after transfection, luminescence measurements were performed upon addition of lOpM 8-benzyl-2-(furan-2-ylmethyl)-6- phenylimidazo[l,2-a]pyrazin-3(7H)-one (AChemBlock, Hayward, CA, USA) diluted in Ca2+ / Mg2+-free PBS, containing no or different concentrations of FSH-LgBiT and lOOnM of VHH or lOOnM of VHH-Hinge-Fc. Signals were recorded for at least 60 minutes with a Mithras LB 943 plate reader (Berthold Technologies GmbH & Co., Wildbad, Germany). Data were analysed and plotted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Values were expressed in percentage of control condition’s maximal response and represented as means ± SEM. Statistical significance was determined by two- ways ANOVA performed on 0-60min area under the curve (AUC) values, and p-values considered significant if <0.05. assay

[0237] Fourty thousand HEK293A cells per well were seeded in previously 0.01% poly-lysine treated 96-well plates, and then transiently transfected in suspension with lOng per well of hFSHR plasmid and 50ng per well of pSOM-Luc plasmid, using Metafectene Pro transfection reagent (Biontex Laboratories, Miinchen, Germany) according to the manufacturer’s instructions. The pSOM-Luc plasmid consists in the firefly luciferase reporter gene under the control of somatostatin promoter region cAMP responsive element (CRE), and allows to assess CRE- dependent gene transcription. After 48 hours transfection, cells were stimulated during 6 hours with FSH (concentrations from 0.03 to 30nM) diluted in serum-free DMEM (Eurobio), in presence of 300nM of VHH-Hinge-Fc. At the end of stimulation, supernatants were removed and cell lysis was induced by adding Bright-Glo Luciferase assay substrate (Promega, Madison, WI, USA). After 5 minutes incubation at room temperature and away from light, luminescence was quantified with a Mithras LB 943 plate reader (Berthold Technologies GmbH & Co.). Values were expressed in percentage of control condition’s maximal response and represented as means ± SEM. Statistical significance was determined by two-ways ANOVA, and p-values considered significant if <0.05.

[0238] Construction and expression of FSHR mutants

[0239] FSHR mutants were designed to determine the binding region of the anti-FSHR VHHs of the invention. Eleven FSHR mutants were designed, whose mutations were distributed along the loops linking the 11 leucin-reach repeats (ERR) of the receptor ectodomain. For each mutant, two to three mutations were introduced into wild-type FSHR sequence (as set in SEQ ID NO:21). Each mutant had mutations in one loop. Mutations were introduced in a way not to affect FSH binding, and to prevent major impact on receptor expression, structure and function. Sequences were sent to Twist Bioscience (San Francisco, CA, USA) for gene synthesis and inserted into pcDNA3.1 plasmid between EcoRI and Xhol restriction sites.

[0240] Softwares

[0241] Data collected in BRET experiments, NanoBiT binding assays and autologous reporter gene assays were analysed and plotted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Flow cytometry data were analysed and plotted using FlowJo 7.6.3 software (FlowJo, Ashland, OH, USA).

[0242] Example 1: Anti-FSHR VHH selection and candidates’ identification

[0243] A) Design of the VHH library and selection

[0244] In order to identify Variable Heavy domain of Heavy chain antibodies (VHHs) targeting the follicle-stimulating hormone receptor (FSHR), the inventors designed a synthetic phage library inspired from synthetic yeast library published by Kruse’s laboratory for G protein-coupled receptors (GPCRs) targeting (McMahon et al., 2018). The library was designed in silico based on Next Generation Sequencing (NGS) analyses of different camelids’ immune repertoires. It presented a diversity greater than IO10, with the majority of diversity introduced into the VHH complementarity-determining region 3 (CDR3), most commonly responsible for the antigenantibody recognition (Manglik, A. et al., Annu. Rev. Pharmacol. Toxicol. 19-37 (2017)). To select VHH targeting FSHR extracellular domain (ECD), phages from the synthetic library, each expressing a single VHH, were selected on living cells transiently over-expressing the receptor. A selection against the other gonadotropin receptor, the luteinizing hormone / choriogonadotropin receptor (LH / CGR), was also performed as a specificity control, this receptor having the closer sequence and structure identity to FSHR. After the first round of selection, recovered phages were used for two further selection rounds, as well as for a control selection on non-transfected cells mixing phages selected in anti-FSHR and anti-LH / CGR first selection rounds. To screen VHH on both inactive and active conformations of receptors, including conformations able to be internalised, second and third rounds of phage display were performed in presence of FSH or LH (or both for control selection) at internalisation EC50 concentrations (Ayoub, M. A. et al., Front. Endocrinol. (Lausanne). 6, 1-14 (2015)). Trafficking and intracellular signalling of gonadotropin receptors being important for their function (Sayers, N. & Hanyaloglu, A. C., Front. Endocrinol. (Lausanne). 9, 1-12 (2018); Sposini, S. et al., Cell Rep. 21, 2855-2867 (2017)), a small benzamide chemical molecule named B3, previously described as a super-agonist of P-arrestin2 recruitment and promoting FSHR internalisation and recycling (De Pascali, F. et al., Int. J. Mol. Sci. 22 (2021); Sposini, S. et al., Front. Pharmacol. 11, 1-14 (2020)), was also added in anti-FSHR selection.

[0245] B) Identification of anti-FSHR candidates

[0246] 96 clones from the third anti-FSHR selection round were randomly chosen and sequenced by Sanger method. Screening these 96 anti-FSHR clones for binding on their target receptor, the inventors identified four VHH showing a specific binding on FSHR by comparison to the non- relevant control GPCR oxytocin receptor (OTR): P52E8, P52E12, P52B7 and P52D6. Interestingly, these four VHH were not found in either anti-LH / CGR or control selection. The other clones did not show specific binding to FSHR. The 96 tested VHH sequences were extracted from NGS analysis to represent them separately (P52FSHR_binders, P52FSHR_non- binders) and improve the understanding of the whole selection. This allowed to see non-specific binders were mainly located in the same area, whereas the four anti-FSHR candidates were all found in a zone that was enriched only in the third round of anti-FSHR selection, and not in anti-LH / CGR third selection round or in control selection. In addition to the anti-FSHR selection specificity, these results reflected the candidates’ sequences similarity, their few differences lying only in CDR3. Table 3: List of VHH candidates

[0247] Table 4: List of VHH-Hinge-Fc candidates C) Anti-FSHR candidates are highly selective

[0248] P52E8, P52E12, P52B7 and P52D6 were produced and purified, and displayed excellent thermal stability (figure 1A). The inventors first confirmed the four candidates bound FSHR expressed at cell surface, observing a clear shift in flow cytometry compared to non-relevant anti-Hen Egg Lysozyme (HEL) VHH (previously characterised) (Akiba et al., Sci. Rep. 9, 4-6 (2019)) condition (figure 1C). Additionally, the VHH selectivity was explored regarding the two others glycoprotein hormones’ receptors, and revealed the four candidates had no visible binding on either LH / CGR or thyroid stimulating hormone receptor (TSHR) (figures 1C, ID), underlying a great selectivity of these VHH for FSHR. Furthermore, none of the candidates were able to bind mouse FSHR (mFSHR) (figure ID). Therefore, the inventors had selected four highly specific VHH, targeting FSHR with great selectivity and no cross-reactivity on mouse FSHR.

[0249] Example 2: Anti-FSHR VHH bind FSHR ectodomain on allosteric sites with high affinity

[0250] A) VHH binding affinity and kinetics constants

[0251] To improve VHH avidity, apparent affinity and in vivo half-life, the four anti-FSHR candidates were reformatted into “IgG-like” called VHH-Hinge-Fc, constituted of two mouse IgG2A Fc fragments, each one associated to a VHH, and linked together by a Hinge region. Within Fc fragments, LALA-PG mutation was introduced to inhibit recruitment of immune system effectors (Lo, M. et al., J. Biol. Chem. 292, 3900-3908 (2017)). VHH-Hinge-Fc were produced and purified, and as in VHH format, had excellent thermal stability (figure IB). Using flow cytometry, the inventors assessed VHH and VHH-Hinge-Fc binding at different concentrations on FSHR expressed at CHO-K1 cell surface, to determine binding EC50 (figures 2A, 2B, Figure 3 table A). P52E12 and P52E8 VHH had the lowest binding EC50, whereas P52B7 had the highest EC50 and P52D6 had an EC50 in-between those of the three first VHH. As expected, reformatting VHH into VHH-Hinge-Fc improved apparent affinity, as reflected by EC50 improvement. Interestingly, P52B7 had the best apparent affinity gain when reformatted into VHH-Hinge-Fc (Figure 3 table A). The VHH binding was also assessed using HTRF on HEK293A cell membranes expressing FSHR (figure 2F). Results were slightly different from what was obtained in flow cytometry, and suggested that P52B7 and P52D6 had a binding EC50 in the same range than that of P52E8 and P52E12. In spite of these differences, these results confirmed that the four VHHs had excellent binding EC50, and that P52E12 was the candidate with the lowest binding EC50, and therefore the best affinity. To evaluate the kinetic profile of VHH binding on FSHR, a luminescence assay based on fusion of two different parts of a split luciferase with the proteins of interest was designed: receptor was fused in its N-terminal region with luciferase SmBiT (SmBiT-FSHR), and VHH were fused to luciferase LgBiT (VHH- LgBiT). VHH-LgBiT binding on SmBiT-FSHR was assessed at two concentrations approximately corresponding to the four candidates’ mean EC50 previously determined (30nM and lOOnM) (figure 2C). The four anti-FSHR candidates showed specific association to SmBiT- FSHR compared to LgBiT alone and Anti-HEL- LgBiT (negative controls). P52E12-LgBiT had the fastest and strongest association to SmBiT-FSHR, with very little dissociation within 60 minutes. P52E8-LgBiT also had fast association to SmBiT-FSHR, but showed little more dissociation than P52E12-LgBiT. P52B7-LgBiT showed slower association but almost no dissociation within 60 minutes, whereas P52D6-LgBiT was the candidate with the highest dissociation from SmBiT-FSHR. Since the four candidates were able to bind FSHR expressed at cell surface with no cell permeability, it was postulated that they would likely bind the receptor ectodomain (ECD). Therefore, the inventors assessed VHH and VHH-Hinge-Fc binding on purified and biotinylated FSHR ECD bound to FSH, and calculated kinetic constants associated to this binding (figure 2G, figure 3 table B,). This revealed the four VHH did bind FSHR ECD and presented excellent binding affinity on it (approx. 10 to 18 nM), even more improved in VHH-Hinge-Fc format (approx. 4 to 7 nM). P52D6 and especially P52B7 seemed to have greater affinity on FSHR ECD bound to FSH than on FSHR without bound agonist (Figure 3), suggesting they could preferentially bind on the receptor when in active conformation. Notably, the four candidates were able to bind both FSHR alone (figures 2A, 2B, 2F) or associated to FSH (figure 2G), strongly suggesting their binding sites were allosteric.

[0252] B) Anti-FSHR candidates do not compete with FSH for FSHR binding

[0253] To explore the allosteric nature of VHH binding, the inventors assessed whether their binding on FSHR could affect that of FSH. To do so, the binding of FSH-LgBiT on SmBiT-FSHR in the presence of either non-relevant Anti-HEL or anti-FSHR VHH was measured (figure 2D). Results showed the four VHH did not have a negative impact on FSH-LgBiT binding on SmBiT-FSHR. This indicated the absence of competition between VHH and the hormone for binding on the receptor, and strengthened the hypothesis of an allosteric binding of the VHH. With the VHH-Hinge-Fc format, a partial decrease of FSH-LgBiT binding compared to control condition was observed at some concentrations (figure 2E). VHH-Hinge-Fc being larger molecules than VHH, and FSH-LgBiT being larger than the native hormone, it is likely that a steric hindrance could occur with VHH-Hinge-Fc bound to SmBiT-FSHR and FSH-LgBiT, partially affecting its binding.

[0254] Example 3: Anti-FSHR VHH act as negative allosteric modulators of signalling

[0255] A) Negative modulation of cAMP production

[0256] The binding characteristics of the four anti-FSHR VHH determined, the inventors asked about the ability of these candidates to modulate receptor signalling, first and foremost regarding cAMP production. Cytosolic cAMP kinetics measurements were conducted by Bioluminescence Resonance Energy Transfer (BRET). In the conditions of this assay and for the concentrations tested, FSH-induced cAMP production was reduced in presence of P52E8, P52E12 and P52D6, but not in presence of P52B7 or negative control Anti-HEL in VHH format (figure 4A). The inventors proceeded to a complete characterisation of cAMP response modulation with VHH-Hinge-Fc, measuring cytosolic cAMP production and degradation kinetics at different concentrations of both FSH and VHH-Hinge-Fc (figure 4B, 4C). Regardless of the FSH or VHH-Hinge-Fc concentration, the cAMP response was not found to be modulated in presence of Anti-HEL-Hinge-Fc. Notably, a moderate inhibition of cAMP response was observed in the presence of P52B7-Hinge-Fc. It is likely that reformatting into VHH-Hinge-Fc, by bivalence and avidity improvement, allowed to obtain a negative modulation effect of cAMP response that was not observed with the VHH format of P52B7, probably due to the sensitivity of the assay. With the four anti-FSHR VHH-Hinge-Fc, the inventors observed a dose-dependent shift to the right of cAMP response EC50, demonstrating a dose-dependent inhibition of FSH- induced cAMP response by VHH-Hinge-Fc. VHH-Hinge-Fc had no significant effect on cAMP response Emax. These results demonstrated the four candidates acted as negative allosteric modulators (NAM) of FSHR cAMP response. Despite their comparable effects on cAMP response, the four anti-FSHR VHH-Hinge-Fc displayed inhibitions of different intensities, P52E12-Hinge-Fc and P52D6-Hinge-Fc having the strongest NAM effect. The amplitude of the effect did not seem to depend on VHH-Hinge-Fc affinity, since P52E8-Hinge-Fc had a similar or better affinity on FSHR ECD fused to FSH than P52D6-Hinge-Fc and P52E12- Hinge-Fc, respectively (Figure 3, table B).

[0257] To estimate these differences of inhibition intensities, mathematical modelling of the biological data was conducted, according to a previously described method (Leach et al. 2007, Trends in Pharmacological Sciences, doi.org / 10.1016 / j.tips.2007.06.004). This operational model of allosteric modulation is mainly characterised by a parameter a describing allosteric effect on the binding affinity, and a parameter P describing allosteric effect on the efficacy. Fitting of the data revealed the model is over-parametrized and lead to ambiguous parameter values. In agreement with the binding competition results (figures 1G, S7), the allosteric parameter on the binding affinity was fixed to a=l (no competition or synergy between FSH and the VHH-Hinge- Fc). Fitting of the data with a=l allowed to determine P and therefore to calculate allosteric coefficient for each VHH-Hinge-Fc. The allosteric coefficient P was to be interpreted as follows: i) P<1: negative allosteric effect of the VHH-Hinge-Fc, ii) P>1: positive allosteric effect of the VHH-Hinge-Fc, iii) P=l: neutral allosteric effect of the VHH-Hinge-Fc. Anti-HEL- Hinge-Fc had P close to 1, indicating it had no or very little effect on FSH-induced cAMP production, and thus validating this VHH as a negative control for FSHR signalling modulation. On the other hand, the four anti-FSHR VHH-Hinge-Fc had P significantly lower than 1 (P<0.2 for all candidates), clearly demonstrating their negative modulation effect on FSH-induced cAMP. Mathematical modelling confirmed that P52B7-Hinge-Fc, that displayed P considerably higher than the three other candidates (P=0.19, as opposed to P<0.043), had a less strong inhibitory effect on cAMP production. Table 5: Allosteric coefficient P of VHH-Hinge-Fc

[0258] B) Negative modulation of Gs protein recruitment

[0259] Because of the candidates’ NAM effect on cAMP production, the inventors were interested in their effect on FSHR coupling to Gas protein. The fluorescent biosensor mini Gs protein (mGs), corresponding to GTPase domain of Gas protein, was employed to allow the measurement of coupling events (Wan, Q. et al., J. Biol. Chem. 293, 7466-7473 (2018)). FSH-induced recruitment of mGs at FSHR was not affected by addition of non-relevant Anti-HEL in VHH or VHH-Hinge-Fc format (figures 4D,4E). All four anti-FSHR VHH-Hinge-Fc had a significant negative effect on mGs recruitment (figure 4E, 4F). P52E12-Hinge-Fc showed the greater inhibition, that was full inhibition at the highest VHH-Hinge-Fc concentration. P52B7-Hinge- Fc was the candidate with the still significant but least inhibitory effect, although it was as efficient as P52E12 in VHH format (figure 4D). P52E8-Hinge-Fc and P52D6-Hinge-Fc, as in VHH format (figure 4D), had similar efficacies, ranging in between P52E12-Hinge-Fc and P52B7-Hinge-Fc inhibitory effects (figure 4E). Though having similar effects on mGs recruitment at the receptor, the four anti-FSHR candidates displayed different inhibition efficacies, highlighting different pharmacological profiles.

[0260] C) Negative modulation of fi-arrestin recruitment

[0261] The inventors then assessed the impact of anti-FSHR candidates on FSHR coupling to P- arrestins, involved in both receptor internalisation and signalling. FSH-induced P-arrestin2 recruitment at FSHR was not affected by Anti-HEL in VHH or VHH-Hinge-Fc format (figures 4G, 4H). All four anti-FSHR VHH-Hinge-Fc had a significant negative effect on P-arrestin2 recruitment (figures 4H, 41). P52E12-Hinge-Fc was again the candidate inducing the strongest inhibition, whereas P52E8-Hinge-Fc and P52B7-Hinge-Fc were a bit less efficient in inhibiting P-arrestin2 recruitment at FSHR. Interestingly, reformatting P52D6 into VHH-Hinge-Fc allowed a great gain in its inhibiting capacity, shifting from a very slight inhibition of P-arrestin2 recruitment in VHH format (figure 4G) to inhibition comparable to that caused by P52E12- Hinge-Fc (figure 4H). D) Negative modulation of CRE-dependent gene transcription

[0262] Given the NAM effect of the four anti-FSHR candidates observed on cAMP signalling, Gs protein and P-arrestin recruitment, the inventors studied their impact on an integrative functional response by determining whether VHH-Hinge-Fc affected downstream cAMP responsive element (CRE) -dependent gene transcription (figure 4J). The results demonstrated that P52E8-Hinge-Fc, and especially P52E12-Hinge-Fc, acted as partial but strong negative modulators of FSH-induced CRE-dependent gene expression. P52D6-Hinge-Fc showed a less pronounced but still noteworthy inhibitory effect, whereas P52B7-Hinge-Fc had an effect on CRE-dependent gene expression at high FSH concentration. P52B7-Hinge-Fc being the candidate with the weakest NAM effect on mGs recruitment, P-arrestin recruitment, and especially cAMP signalling, these results suggested a stronger inhibition of FSHR signalling was necessary to impact downstream functional responses as CRE-dependent gene expression.

[0263] Altogether, these results showed the four anti-FSHR VHH and VHH-Hinge-Fc acted as NAMs of FSH-induced signalling and transcriptional response. These parameters controlling downstream physiological responses (i.e., steroidogenesis, ovulation in female and spermatogenesis in male), this suggested these candidates could act as NAMs of steroidogenesis and / or fertility.

[0264] Example 4: Anti-FSHR VHH prevent FSHR internalisation but do not inhibit signalling through this mechanism

[0265] A) Anti-FSHR VHH affect receptor trafficking

[0266] In an effort to explain anti-FSHR VHH NAM action mode, the inventors first wondered whether they affected receptor cellular trafficking. Enhanced bystander BRET (ebBRET) experiments (Namkung, Y. et al., Nat. Commun. 7 (2016)) were conducted to monitor FSHR location at plasma membrane after FSH stimulation, reflecting agonist-induced receptor internalisation (figure 5A). Results showed that in presence of P52E8-Hinge-Fc, P52E12-Hinge-Fc and P52D6-Hinge-Fc, FSHR remained at plasma membrane after FSH stimulation, and that the first two VHH-Hinge-Fc led to a full inhibition of receptor internalisation. P52B7-Hinge-Fc also had a negative effect on receptor internalisation, but this was more partial than with the three other anti-FSHR VHH-Hinge-Fc. FSHR internalisation has a great importance in its function, and internalised receptors have a significant contribution in agonist-induced cAMP signalling (Sayers, N. & Hanyaloglu, A. C., Front. Endocrinol. (Lausanne). 9, 1-12 (2018)). Therefore, the NAM effect of all four candidates on cAMP signalling could be due to receptor internalisation inhibition. However, P52E8, P52E12, P52B7 and P52D6 blocked Gas and P- arrestin recruitment as well, phenomena known to occur at least in part at plasma membrane. Thus, the inventors wondered whether the inhibition of FSHR internalisation observed with VHH-Hinge-Fc was the cause of downstream signalling inhibition, or was a consequence of an inhibition of receptor activation by the anti-FSHR candidates.

[0267] B) Anti-FSHR VHH still exert their NAM effect on signalling of receptors blocked at plasma membrane

[0268] To explore these questions, a study was conducted to assess the effect of the anti-FSHR VHH- Hinge-Fc on cAMP signalling of FSHR whose internalisation was inhibited. Firstly, a chemical approach was used by treating cells with either the dynamin inhibitor Dyngo4a (Mccluskey, A. et al., Traffic 14, 1272-1289 (2013)) or the clathrin inhibitor PitStop2 (Dutta, D. et al., PLoS One 7, 1-9 (2012)), and cAMP dynamics were monitored (figure 5B1). The four anti-FSHR VHH-Hinge-Fc still had a NAM effect on cAMP signalling downstream FSHR blocked at plasma membrane, suggesting their previously characterised NAM effect (figure 4B, 4C) was not a consequence of FSHR internalisation inhibition. To confirm the results and analyse further the VHH-Hinge-Fc mode of action, cAMP dynamics measurements were performed in HEK293AParrl / 2 cells, in which FSHR could not be internalised (De Pascali, F., Int. J. Mol. Sci. 22 (2021)). VHH-Hinge-Fc were added either 5 or 15 minutes after FSH stimulation, to assess their ability to reverse the FSH-induced receptor activation and signalling (figure 5B2). Interestingly, addition of anti-FSHR VHH-Hinge-Fc led to almost immediate decrease of cAMP response, until reaching strong inhibition level with P52B7-Hinge-Fc, and complete inhibition with P52E12-Hinge-Fc, P52E8-Hinge-Fc and P52D6-Hinge-Fc. This kind of profile was not observed when adding Anti-HEL-Hinge-Fc, with which cAMP response remained sustained. These results confirmed that anti-FSHR candidates’ NAM effect was not a consequence of receptor internalisation inhibition, and showed these VHH-Hinge-Fc were able to reverse FSH- induced receptor activation. Using P52E12-Hinge-Fc as model, the inventors demonstrated this effect was not specific to FSH-induced cAMP response, but that the VHH-Hinge-Fc also inhibited cAMP response induced by the agoPAM B3 (De Pascali, F. et al., Int. J. Mol. Sci. 22 (2021); Sposini et al., Front. Pharmacol. 11, 1-14 (2020)) (figure 5C1, 5C2). Though not reaching full inhibition as on FSH-induced response, P52E12-Hinge-Fc strongly inhibited B3- induced cAMP response when added before or with the agonist. Interestingly, when added after agonist stimulation, P52E12-Hinge-Fc seemed to reverse FSHR activation even more rapidly when it was stimulated with B3. cAMP production being dependent on Gs protein coupling with the activated receptor, the inventors assessed whether VHH-Hinge-Fc were also able to inhibit Gs recruitment when added after FSH stimulation, and if this effect was dependent on FSHR cellular location. mGs recruitment at FSHR in control (DMSO) and internalisation inhibition (Dyngo4a) conditions was monitored, and VHH-Hinge-Fc were added 10 minutes after FSH stimulation (figure 5D). Addition of any anti-FSHR VHH-Hinge-Fc led to immediate blocking of mGs recruitment, whereas Anti-HEL-Hinge-Fc had no effect on mGs recruitment. Interestingly, no decrease of mGs recruitment after anti-FSHR VHH-Hinge-Fc addition was observed, but the response immediately reached a plateau, suggesting mGs already recruited at FSHR did not dissociate. This was the case in both control and Dyngo4a conditions, showing VHH-Hinge-Fc exerted the same effect when FSHR was blocked at plasma membrane. Using a non-chemical approach to inhibit receptor internalisation, the inventors confirmed in HEK293AParrl / 2 cells, with P52E12-Hinge-Fc as a model, that the effect of anti-FSHR VHH-Hinge-Fc on mGs recruitment was not due to their negative impact on FSHR internalisation (figure 5E).

[0269] Based on these data, the complete decrease in cAMP response observed when adding VHH- Hinge-Fc on already activated FSHR may be due to anti-FSHR candidates blocking receptors in a conformation in which they could not produce cAMP anymore, even if pre-associated with Gs protein.

[0270] Example 5: Anti-FSHR VHH epitope determination

[0271] A) Determination of anti-FSHR VHH-Hinge-Fc cross -reactivity

[0272] The inventors previously demonstrated that none of the anti-FSHR VHH displayed binding on mouse receptor (figure ID). To confirm the absence of cross-reactivity, the inventors monitored cAMP production and degradation kinetics induced by FSH binding on human and mouse FSHR (hFSHR and rnFSHR, respectively) (figure 6A). As expected, all four anti-FSHR VHH- Hinge-Fc had a NAM effect on hFSHR cAMP signalling, and did not modulate rnFSHR cAMP response.

[0273] B ) Determination of VHH binding region on FSHR ectodomain

[0274] To determine the binding region of the anti-FSHR VHHs of the invention, 11 FSHR mutants were designed, whose mutations were distributed along the loops linking the 11 leucin-reach repeats (LRR) of the ectodomain. Mutations were introduced in a way not to affect FSH binding, and to prevent major impact on receptor expression, structure and function (Table 5). The positions disclosed in Table 5 are numbered by reference to the amino acid sequence of the hFSHR, as set forth in SEQ ID NO:21.

[0275] Table 6: Design of FSHR mutants

[0276] Interestingly, these mutants, covering possible binding areas in the whole FSHR ectodomain without affecting FSH binding or dramatically impacting receptor expression, could be useful to map epitopes of any FSHR-targeting drug.

[0277] Flow cytometry experiments were first conducted to assess anti-FSHR VHH binding on the 11 FSHR mutants (figure 6B). As expected, Anti-HEL VHH did not bind to any of the FSHR mutants or to wild-type (WT) FSHR. All the four anti-FSHR VHH bound to each mutant except mutants 5 and 6. To confirm these results, cAMP response downstream each FSHR mutant was monitored, and showed the four anti-FSHR VHH-Hinge-Fc maintained their NAM effect on all mutants’ signalling, except for mutants 5 and 6 (figure 6C).

[0278] Altogether, these data indicated the four anti-FSHR candidates bound human FSHR ectodomain in region located between LRR 3 and LRR 5, in particular between residues H98 and D153, more particularly, between residues KI 04 and DI 53.

[0279] Conclusions

[0280] The four anti-FSHR VHH studied, P52E8, P52E12, P52B7 and P52D6, are highly selective for the human FSHR. They all bind FSHR ectodomain (ECD) on allosteric sites, without competing with FSH, and with great affinity and kinetics constants. Interestingly, the four VHH have very similar sequences, which translate into similar pharmacological effects.

[0281] All four VHH act as negative allosteric modulators (NAM) of cAMP signalling, mGs and P- arrestin2 recruitment, as well as CRE-dependent gene transcription, though with some diversity in their pharmacological profiles. These four anti-FSHR VHH inhibit FSHR internalisation, while it is not the cause of their NAM effect. Indeed, the four anti-FSHR VHH can exert the same NAM effect on receptors blocked at plasma membrane’s signalling, showing FSHR internalisation inhibition is only a consequence of receptor inactivation by VHH. In addition, these VHH are able to inhibit signalling of already activated FSHR, leading to an arrest in mGs recruitment and a complete reduction of cAMP response. Interestingly, the VHH also exert their NAM effect on cAMP signalling when FSHR is activated by B3 agoPAM, suggesting their action mechanism would be conserved whatever the agonist activating the receptor. Altogether, and given that these VHH had no competition with FSH for binding to the receptor, these data strongly suggested the candidates’ powerful NAM effect was due to induction of FSHR conformation change, stabilising an inactive or partially active conformation of the receptor, thus leading to inactivation or weak activation of downstream signalling.

[0282] Cryo-EM structure of FSHR has been recently solved, revealing a “push-pull” activation mechanism (Duan, J. et al. Nat. Commun. 14, 1-12 (2023)). When the receptor is in an inactive state, the ECD is tilted towards the plasma membrane. Binding of FSH to the concave upper part of the ECD, at LRRs level, engenders a 48° upward rotation (“push”). Binding of FSH also induces a conformation change of the hormone, allowing FSH residues located between its two a and P subunits to form a binding pocket for sulphated Y335 of receptor’s Hinge region. This causes a conformation change of the Hinge region, stabilising FSH-FSHR interaction and removing inverse agonist effect exerted by the FSHR ECD on its own activity (Landomiel, F. et al. Front. Endocrinol. (Lausanne). 10, 0-15 (2019)). The ECD is then pulled nearby the transmembrane domains (TMDs) by the Hinge region (“pull”), allowing a conformation change of TMDs. In particular, binding of FSH to FSHR engenders an outward movement of TM6. Conformation change of TMDs allows recruitment and coupling of signal transducer molecules to cytosolic parts of the receptor, and thus the activation of intracellular signalling cascade. Considering this activation mechanism together with the present data, it is assumed that the four anti-FSHR VHH would bind to FSHR ectodomain and stabilise a receptor conformation in which the ECD could not be fully pulled to the TMDs and / or the TM6 outward movement could not occur, even in the presence of FSH or another agonist. Therefore, the present data demonstrate that targeting FSHR with anti-FSHR VHHs according to the present invention, that bind to a region comprised between LRR3-LRR5, allows to strongly inhibit FSHR’s activation, providing valuable information for design of new pharmacological agents targeting FSHR. In particular, regarding their in vitro efficacy on signalling and gene transcription, it is likely that these VHH can act as non-hormonal negative modulators of reproductive functions.

Claims

56CLAIMS1. An anti-FSH receptor (FSHR) VHH, wherein said VHH(i) is directed against and / or specifically binds to FSHR ectodomain in a region located between leucine-rich repeat 3 (LRR 3, H98) and leucine-rich repeat 5 (LRR 5, DI 53);(ii) is a negative allosteric modulator of FSHR;(iii) does not block the binding between FSH and FSHR; and(iv) does not bind to LH / CG receptor and TSH receptor.

2. The anti-FSHR VHH according to claim 1, comprising complementary determining regions whereinCDR1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, andCDR2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6, with optionally 1 or 2 amino acid modifications, selected from addition, deletion and / or substitution, andCDR3 comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 7, 8, 9, and 10, with optionally 1, 2, 3 or 4 amino acid modifications, selected from addition, deletion and / or substitution, wherein the CDRs are determined by IMGT.

3. The anti-FSHR VHH according to claim 1 or 2, comprising complementary determining regions wherein the amino acid sequence of CDR1 consists of SEQ ID NO: 5 or a variant thereof having at least 80% amino acid identity to SEQ ID NO: 5 over the entire length thereof, the amino acid sequence of CDR2 consists of SEQ ID NO: 6 or a variant thereof having at least 80% amino acid identity to SEQ ID NO: 6 over the entire length thereof,57 the amino acid sequence of CDR3 is selected from SEQ ID NOs: 7, 8, 9 and 10 or a variant thereof having at least 80% amino acid identity to any one of said sequences over the entire length thereof.

4. The anti-FSH receptor (FSHR) VHH according to any one of claims 1 to 3, comprising an amino acid sequence comprising or consisting of 4 framework regions (FR1 to FR4) and 3 complementary determining regions (CDR1 to CDR3) according to the formula (1):FR 1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 ( 1 ) whereinFR1 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 11, with optionally 1, 2, 3, 4 or 5 amino acid modifications, selected from addition, deletion and / or substitution, and / orFR2 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 15, with optionally 1, 2, 3, 4 or 5 amino acid modifications, selected from addition, deletion and / or substitution, and / orFR3 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 13, with optionally 1, 2, 3, 4, 5, 6, 7 or 8 amino acid modifications, selected from addition, deletion and / or substitution, and / orFR4 comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 14, with optionally 1, 2, 3 or 4 amino acid modifications, selected from addition, deletion and / or substitution wherein the FRs are determined by IM GT.

5. The anti-FSHR VHH according to any one of the previous claims, wherein said VHH comprises or consists of- an amino acid sequence of SEQ ID NO:1 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:1, or- an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:2, or58- an amino acid sequence of SEQ ID NO:3 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:3, or- an amino acid sequence of SEQ ID NO:4 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid identity with the amino acid sequence of SEQ ID NO:4.

6. The anti-FSHR VHH according to any one of the previous claims, wherein said VHH specifically binds to human FSHR and / or wherein said VHH binds FSHR with a Kd comprised between 8 nM and 20 nM, preferably between 10 nM and 18 nM.

7. An antigen-binding polypeptide comprising a VHH according to any one of claims 1 to 6 conjugated to at least one stabilizing group, preferably selected from an antibody or a fragment thereof such as a Fc fragment of IgG or IgA.

8. The antigen-binding polypeptide according to claim 7, wherein said antigen-binding polypeptide binds FSHR with a Kd comprised between 2 nM and 10 nM, preferably between 3.5 nM and 7.5 nM.

9. A pharmaceutical composition comprising a VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, and a pharmaceutically acceptable carrier.

10. A polynucleotide, wherein said polynucleotide encodes the protein consisting of the VHH according to any one of claims 1 to 6, or the protein consisting of the antigen-binding polypeptide according to claims 7 or 8.

11. An expression vector, wherein said vector contains the polynucleotide according to claim 10.

12. A host cell, wherein said host cell contains the expression vector according to claim 11, or the polynucleotide according to claim 10 integrated within the genome of the host cell.

13. An anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9, for use as a medicament.

14. Use of an anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9, for contraception or fertility regulation.5915. An anti-FSHR VHH, or an antigen-binding polypeptide, or a pharmaceutical composition for use according to claim 13, in the treatment or prevention of ovarian cancer.

16. An anti-FSHR VHH, or an antigen-binding polypeptide, or a pharmaceutical composition for use according to claim 13, in the treatment or prevention of pre-menopausal and peri- menopausal hormone-dependent breast cancer.

17. An anti-FSHR VHH, or an antigen-binding polypeptide, or a pharmaceutical composition for use according to claim 13, in the treatment or prevention of uterine fibroids or other menstrual-related disorders.

18. An anti-FSHR VHH, or an antigen-binding polypeptide, or a pharmaceutical composition for use according to claim 13, in the treatment or prevention of an estrogen dependent disease.

19. An anti-FSHR VHH, or an antigen-binding polypeptide, or a pharmaceutical composition for use according to claim 13, in the treatment or prevention of prostate cancer.

20. An anti-FSHR VHH, or an antigen-binding polypeptide, or a pharmaceutical composition for use according to claim 13, in the treatment or prevention of solid tumor, in particular ovarian, breast, urothelial, thyroid, neuroendocrine, pancreatic, pituitary, soft tissue sarcomas, kidney, colon, lung, testicular, gastric, and hepatocellular cancers.

21. An anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9, for use in the manufacture of a medicament for the treatment or prevention of ovarian cancer, in the treatment or prevention of pre-menopausal and peri-menopausal hormone-dependent breast cancer, in the treatment or prevention of uterine fibroids or other menstrual-related disorders, or in the treatment or prevention of an estrogen dependent disease.

22. An anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9, for use in the manufacture of a medicament for the treatment or prevention of prostate cancer.

23. An anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9, for use in the manufacture of a medicament for the treatment or prevention of a solid tumor, in particular ovarian, breast, urothelial, thyroid, neuroendocrine, pancreatic, pituitary, soft tissue sarcomas, kidney, colon, lung, testicular, gastric, and hepatocellular cancers.6024. Method of treatment or prevention of ovarian cancer of a subject in need thereof, wherein an anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9 is administered to said subject.

25. Method of treatment or prevention of pre-menopausal and peri-menopausal hormonedependent breast cancer of a subject in need thereof, wherein an anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9 is administered to said subject.

26. Method of treatment or prevention of uterine fibroids or other menstrual-related disorders of a subject in need thereof, wherein an anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9 is administered to said subject.

27. Method of treatment or prevention of an estrogen dependent disease in a subject in need thereof, wherein an anti-FSHR VHH according to any one of claims 1 to 6, or an antigenbinding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9 is administered to said subject.

28. Method of treatment or prevention of a prostate cancer in a subject in need thereof, wherein an anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9 is administered to said subject.

29. Method of treatment or prevention of a solid tumor in a subject in need thereof, wherein an anti-FSHR VHH according to any one of claims 1 to 6, or an antigen-binding polypeptide according to claim 7 or 8, or a pharmaceutical composition according to claim 9 is administered to said subject

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