Gnrh-binding polypeptides and uses thereof

Abstract

The invention relates to the field of GnRH antagonists and anti-GnRH ligands. The invention also relates to the pharmaceutical and diagnosis field. In particular, the present disclosure relates to the field of contraception and GnRH-related diseases.

Description

[0001] TITLE

[0002] GnRH-binding polypeptides and uses thereof

[0003] FIELD OF THE DISCLOSURE

[0004] The invention relates to the field of GnRH antagonists and anti-GnRH ligands .

[0005] The invention also relates to the pharmaceutical and diagnosis field. In particular, the present disclosure relates to the field of contraception and GnRH-related diseases.

[0006] BACKGROUND OF THE DISCLOSURE

[0007] Various contraceptive methods are practiced in the art, especially for livestock, including for porcine.

[0008] A long-practiced method for porcine consisted of live castration of piglets in the absence of any anesthesia. However, this long practiced method has been considered as being unethical because it causes unacceptable pain, anguish and suffering to the piglets.

[0009] Animal contraceptive methods known in the art also encompass surgical contraception under anesthesia (e.g. tubal ligation or vasectomy), the use of hormone- containing compositions, which include hormone-containing implants (e.g. GnRH analogcontaining implants), of insertion of intrauterine devices, injection of chemicals directly into the testes that destroy reproductive tissues, as well as methods for immunological contraception.

[0010] GnRH-binding ligands, in particular monoclonal antibodies, have been reported in the Art. Known methods of immunological contraception include the administration of vaccine compositions aimed at immunizing the subjects against endogenous GnRH.

[0011] In particular, GnRH vaccines stimulate antibody production to inactivate endogenous GnRH that in turn causes reduced release of gonadotrophic hormones leading to gonadal atrophy in adult animals or lack of development in sexually immature animals.

[0012] This resulting regression or lack of development of the reproductive organs is referred to as immunological sterilization.

[0013] Due to its role as an hormone, GnRH, GnRH analogues and GnRH-binding ligands have further been involved as therapeutics in a number of other medical conditions and clinical uses, either as GnRH agonists or GnRH antagonists, including the treatment or prevention of diseases beyond the treatment or prevention of infertility, especially sex-hormone dependent diseases, for example delay of puberty, ovarian hyperstimulation syndrome, precocious puberty, premenstrual syndrome, endometriosis, uterine leiomyoma, polycystic ovarian syndrome, hirsutism, acne vulgaris, acute intermittent porphyria, breast cancer, uterus cancer, uterine fibrosis, endometrial cancer, prostate cancer, benign prostatic hyperplasia, pancreas cancer, and the like.

[0014] Silversides et ai. (“Monoclonal antibodies against LHRH: development and immunoactivity in vivo and in vitro”; Journal of Reproductive Immunology, 1985) reports a monoclonal antibody from a dog immunization trial which is directed toward GnRH / LHRH.

[0015] Talwar et al. (“Bioeffective monoclonal antibody against the decapeptide gonadotropin-releasing hormone: Reacting determinant and action on ovulation and estrus suppression”; Proc. Natl. Acad. Sci. USA, 1985) reports an antibody preparation competent to suppress the progression of estrus in dogs.

[0016] Li et al. (“Vectored antibody gene delivery mediates long-term contraception”; Current Biology, 2015) and US 10,570,200 B2 teach antibody-mediated immunocontraception, including vectored, AAV-dependent, anti-GnRH antibody expression.

[0017] A purified monoclonal mouse IgGl, Kappa antibody referenced as clone number SMI41 (Absolute Antibody Ref. n° Ab00922-l.l), specific to a C-terminal peptide of GnRH, has also been reported for immunocytochemistry, and expressed in mice via injection of a replication-incompetent, recombinant adeno-associated virus, for conferring long-term infertility.

[0018] CEVA VALORA™ is reported as a GnRH / LHRH-based synthetic peptide vaccine for swine containing three synthetic peptides as the immunogen.

[0019] IMPROVAC™ is reported as a GnRH analogue-protein conjugate which is capable of stimulating a pig’s immune system to produce antibodies against GnRH, for controling sexual development.

[0020] Chang et al. (“Effects of a recombinant Gonadotropin-Releasing Hormone Vaccine on Reproductive Function in Adult Male ICR Mice”; Vaccines, 2021) reports a vaccine preparation comprising a multimeric fusion protein, including a plurality of copies a GnRH I decapeptide and T-cell epitopes.

[0021] Gonadotropin releasing hormone (GnRH), also known as LHRH, is a pivotal hypothalamic and well conserved hormone in mammals, secreted by the pituitary gland, and which acts as master reproductive hormone via regulation of the release of two major gonadotrophic hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH has been extensively studied for development of immunocontraceptive vaccines for various mammalian species, including pigs, rodents, cats, dogs, and wild carnivores.

[0022] The amino acid sequence of GnRH is highly conserved among phylogenetically closed species; it is generally reported as a decapeptide, characterized by a conserved N- terminal region, a central region and a conserved C-terminal region, with the following amino acid structure provided for reference in mammals: (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg- Pro-Gly-NH2(SEQ ID N°152).

[0023] The N-terminal region (pGlu-His-Trp) of native GnRH is characterized by a blocked amino-terminal pyroglutamic acid residue (pGlu), whereas the C-terminal region (Arg- Pro-Gly-NH2) of native GnRH is characterized by an amidated carboxy terminus.

[0024] The N-terminal and C-terminal region of GnRH, in its folded conformation, are both invoided in receptor binding, although only the N-terminus appears to be involved in receptor activation.

[0025] The development of anti-GnRH ligands, especially in the context of immunocontraceptive vaccines, is challenging for several reasons:

[0026] (i) GnRH is a small decapeptide with very low antigenicity;

[0027] (ii) In spite of its small size, the folded-conformation of mammalian GnRH has led to the identification of at least two critical regions for receptor binding, with the N- terminal region alone being responsible for receptor activation;

[0028] (iii) it is naturally present in the body.

[0029] Thus, it is recognized by the immune system as a " self protein” with a weak and transient antibody response following administration to an animal, and including a number of conformational epitopes.

[0030] Hence, there remains a need in the Art for novel ligands directed against GnRH, or alternatively for the identification of ligands directed against novel epitopes of GnRH.

[0031] There remains a need in the Art for ligands and formulations directed against GnRH, which remain stable and compatible with large-scale production.

[0032] In particular, there remains a need in the Art for ligands directed against GnRH, which can be of use as a medicament and / or for detection or diagnosis purposes. In particular, there remains a need in the Art for GnRH antagonists for use for treatment of disorders, especially sex hormone-related disorders and / or GnRH-related disorders.

[0033] There remains a need in the art for contraceptive methods, especially for livestock.

[0034] In particular, there remains a need in the art for contraceptive methods which can be easily administered, while being reversible and immediately efficient.

[0035] The present disclosure has for purpose to satisfy those needs.

[0036] SUMMARY OF THE DISCLOSURE

[0037] A first main embodiment of the invention relates to an isolated monoclonal anti- Gonadotropin-Releasing Hormone (anti-GnRH) single domain antibody (SdAb), or antigenbinding fragment thereof; wherein said antibody or antigen-binding fragment binds specifically to, both, a N-terminal region (pGlu-His-Trp) of native GnRH, and a C-terminal region (Pro- Gly-NH2) of native GnRH.

[0038] A second main embodiment, the invention relates to an isolated monoclonal anti- Gonadotropin-Releasing Hormone (anti-GnRH) antibody, or antigen-binding fragment thereof; wherein said antibody or antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of: a CDR3 polypeptide sequence SEQ ID NO 57 or 187; a CDR3 polypeptide sequence SEQ ID NO 33 or 188; a CDR3 polypeptide sequence SEQ ID NO 36; a CDR3 polypeptide sequence SEQ ID NO 96; a CDR3 polypeptide sequence SEQ ID NO 48; a CDR3 polypeptide sequence SEQ ID NO 42, 75, 81, 90, 93; or a variant of said CDR3 polypeptide sequence having 1 or 2 substitutions.

[0039] According to another main embodiment, the invention relates to a nucleic acid coding for an antibody or antigen-binding fragment thereof according to the invention.

[0040] According to another main embodiment, the invention relates to a vector comprising a nucleic acid according to the invention. According to another main embodiment, the invention relates to a host cell comprising the nucleic acid or the vector according to the invention; in particular an eukaryotic cell comprising the nucleic acid or the vector according to the invention. Such host cell may be isolated by any method known in the Art.

[0041] According to another main embodiment, the invention relates to a pharmaceutical composition comprising: the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell according to the invention; and a pharmaceutically acceptable excipient.

[0042] According to another main embodiment, the invention relates to an in vitro method for detecting Gonadotropin-Releasing Hormone (GnRH), comprising the steps of: a) providing a sample, in particular a biological sample or a fraction thereof; b) bringing in contact the sample with an isolated antibody or antigen-binding fragment thereof, according to the invention.

[0043] According to another main embodiment, the invention relates to a method for isolating an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody, or antigen-binding fragment thereof; comprising the steps of: a) providing a library of single domain antibodies (SdAb), or antigen-binding fragments thereof; b) subjecting the library to antigen- affinity selection, the selected SbAbs or fragments thereof being characterized for having specificity toward, both, the N-terminal region (pGlu-His-Trp) of native GnRH and the C-terminal region (Pro-Gly-NH2) of native GnRH.

[0044] DESCRIPTION OF THE FIGURES

[0045] Figure 1. Graphical representation of biotinylated biopeptide 1 (A) and biopeptide 2 (B) for ELISA and SPR. The ten highlighted amino acids are representative of the GnRH native peptide in mammals (SEQ ID N°152). Figure 2. Graphical representation of ELISA Quality Control result of directly coated or captured bio-peptides. Graphic illustrating the Optical Density (OD) at 450 nm vs. log concentrations of biopeptides 1 and 2, applying a nonlinear regression curve fit of a log (agonist) vs. response using four parameters. A) As a function of directly coated proteins in pg / ml in the x-axis. B) As a function of captured proteins in nM in the x-axis.

[0046] Figure 3. ELISA results from thirty SdAb (VHH) purified leads and the reference SMI41 antibody with Bio-peptide 1. The corresponding Optical Density (OD) at 450 nm is provided in the y-axis. The corresponding nM concentration of the VHH, or alternatively SMI41, is provided in the x-axis. For all panels, Bio-peptide 1 is coated.

[0047] Figure 4. SPR results from thirty SdAb (VHH) purified leads and the reference SMI41 antibody with Bio-peptide 1 and Bio-peptide 2. For each graph, the Relative Unit (RU) is provided in the y-axis over time (s). A. On the left panel: this represents the SPR signal from SMI41 (IgGl) antibody in the presence of coated Bio-peptide 1. On the right panel: this represents the SPR signal from SMI41 (IgGl) antibody in the presence of coated Bio-peptide 2. B. On the left panel: this represents the SPR signal from each VHH lead antibody in the presence of coated Bio-peptide 1. On the right panel: this represents the SPR signal from each VHH lead antibody in the presence of coated Bio-peptide 2.

[0048] Figure 5. Native GnRH binding of a selected VHH library by ELISA, with coated GnRH and antibody titration. Graphic illustrating the Optical Density (OD) at 450 nm vs. log concentrations (pM) of A. VHH antibody clones 1 to 12. B. VHH antibody clones 13 to 24. C. VHH antibody clones 25 to 36. D. Negative control experiment with polyclonal and monoclonal VHH VS. SMI41

[0049] Figure 6. Assessment of the capacity of five VHH purified leads to antagonize the human GnRH receptor when expressed in rat basophil leukemia cells. The % of inhibition is assessed in the y-axis, as a function of the concentration of each tested VHH, and of the reference SMI41 antibody. A) The concentration of each antibody is expressed in pg / mL. B) The concentration of each antibody is expressed in pM.

[0050] Figure 7. Assessment of antibody titer using reversed phase-high performance liquid chromatography (RP-HPLC). Samples of antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022 andpGnRH-024 were subjected to 5 Freeze / Thaw cycles, or stored for 2 weeks at room temperature (25 °C), or 40°C. TO represents the control condition and corresponds to the original sample before storage. A. Total area of the pic corresponding to the antibody from the clone in reference, as measured by RP-HPLC B. Main pic of the pic corresponding to the antibody from the clone in reference, as measured by RP- HPLC.

[0051] Figure 8. Sequences of Tm variants. A. Sequences of Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4 (sequence variants) of clones pGnRH-001, arrows point to substitutions made in the sequence variants. B. Sequences of Tm variants 09Tml, 09Tm2, (sequence variants) of clone pGnRH-009, arrows point to substitutions made in the sequence variants.

[0052] Figure 9. ELISA assays against Bio-peptides 1 and 2. ELISA assays against Biopeptides 1 and 2 were used to assess the binding properties of antibodies from Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4 (sequence variants) of clone pGnRH-001. A. ELISA assays against Bio-peptide 1 of Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4. The corresponding Optical Density (OD) at 450 nm is provided in the y-axis. The corresponding nM concentration of the VHH, is provided in the x-axis. B. ELISA assays against Bio-peptide 2 of Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4. The corresponding Optical Density (OD) at 450 nm is provided in the y-axis. The corresponding nM concentration of the VHH, is provided in the x- axis.

[0053] Figure 10. ELISA assays against Bio-peptides 1 and 2. ELISA assays against Bio-peptides 1 and 2 were used to assess the binding properties of antibodies from Tm variants 09Tml, 09Tm2 (sequence variants) of clone pGnRH-009. A. ELISA assays against Bio-peptide 1 of Tm variants 09Tml, 09Tm2. The corresponding Optical Density (OD) at 450 nm is provided in the y-axis. The corresponding nM concentration of the VHH, is provided in the x- axis. B. ELISA assays against Bio-peptide 2 of Tm variants 09Tml, 09Tm2. The corresponding Optical Density (OD) at 450 nm is provided in the y-axis. The corresponding nM concentration of the VHH, is provided in the x-axis.

[0054] Figure 11. Characterization of Tm variants stability. Samples of antibodies produced by clone pGnRH-001, Tm variant 01Tm2 of clone pGnRH-001, clone pGnRH-009 and Tm variant 09Tml of clone pGnRH-009 were subjected to 5 Freeze / Thaw cycles (5x FT), or stored for 2 weeks at room temperature (25 °C), or 40°C. TO represents the control condition and corresponds to the antibody titer of the original sample before storage. Antibody concentration and turbidity were measured for each condition. A. Antibody concentration of samples is provided in mg / mL on the y-axis, for all samples and conditions indicated on the x- axis. B. Turbidity was measured at O.D. 500 nm is provided on the y-axis, for all samples and conditions indicated on the x-axis.

[0055] Figure 12. Characterization of Tm variants stability. Samples of antibodies produced by clone pGnRH-001, Tm variant 01Tm2 of clone pGnRH-001 (GnRH-001-Tm002), clone pGnRH-009 and Tm variant 09Tml of clone pGnRH-009(GnRH-009-Tm005) were subjected to 5 Freeze / Thaw cycles (5x FT), or stored for 2 weeks at room temperature (25°C), or 40°C. TO represents the control condition and corresponds to the antibody titer of the original sample before storage. The samples were submitted to RP-HPLC A. Total area of the pic corresponding to the antibody from the clone in reference, as measured by RP-HPLC is indicated in milli-Absorbance Units*seconds (mAU*s) on the y-axis, for all samples and conditions indicated on the x-axis B. Percent increase in high molecular weight species in sample from the clone in reference, as measured by RP-HPLC is indicated on the y-axis, for all samples and conditions indicated on the x-axis.

[0056] Figure 13. Antagonistic activity of Tm variants. Antagonist activity toward GnRH of antibodies produced by clone pGnRH-001, Tm variants 01Tm2 of clone pGnRH-001 (GnRH-001-Tm002), clone pGnRH-009 and Tm variant 09Tml of clone pGnRH-009 (GnRH- 009-Tm005). A. The % of inhibition is assessed in the y-axis, as a function of the concentration of each tested VHH, and of the reference SMI41 antibody. The concentration of each antibody is expressed in nM. B. The % of inhibition is assessed in the y-axis, as a function of the concentration of each tested VHH, and of the reference SMI41 antibody. The concentration of each antibody is expressed in pg / ml.

[0057] Figure 14 - Liabilities of top clones tested. Summary of the results obtained regarding physical and chemical stability of clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022 andpGnRH-024 as detailed in example 4.

[0058] Figure 15 - Liabilities of all Tm variants. Summary of the results obtained regarding physical and chemical stability of Tm variants , i.e. sequence variants of Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4 of clone pGnRH-001 and Tm variants 09Tml, 09Tm2 of clone pGnRH-009 as detailed in example 7. DETAILED DESCRIPTION

[0059] The inventors provide herein anti-GnRH antibodies, and antigen-binding fragments thereof. Surprisingly, the inventors disclose that such anti-GnRH antibodies have the capacity to target both the N-terminal and the C-terminal region of native GnRH, the two regions being involved in receptor binding.

[0060] This mechanism of binding is atypical in view of the already existing anti-GnRH antibodies reported in the Art, which do not share the same properties.

[0061] The inventors further provide in vitro and in vivo evidence that the selected single domain antibodies, which share the same binding mechanism, are sufficient for antagonizing human GnRH receptor activity in rat basophil leukemia cells.

[0062] Without wishing to be bound by the theory, the inventors are of the opinion that the mechanism of binding, which relies on the two terminal regions of native GnRH, is responsible for its biological effect.

[0063] The inventors further disclose methods for isolating monoclonal anti- Gonadotropin-Releasing Hormone (anti-GnRH) ligands, which are particularly selective toward other ligands having the same mechanism of binding.

[0064] Without wishing to be bound by the theory, the inventors are of the opinion that libraries of single domain antibodies (SdAb) are particularly convenient, as a novel class of ligands, for selecting antibodies with an atypical binding mechanism toward native GnRH.

[0065] The experimental data focus on the isolation of single domain antibodies (SdAb), in particular single variable domain on a heavy chain (VHH) antibodies, also known in the Art as nanobodies, although the provided hypervariable domains may also serve as a basis for the identification and / or engineering of non-VnH, conventional, bi-specific and tri-specific antibodies; as reported in Pekar et al. (“Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform”; Mabs, 12(1), 2020).

[0066] The following anti-GnRH antibodies, either as isolated (e.g. VHH) antibodies, antigen-binding fragments, or hypervariable regions thereof, are particularly considered.

[0067] Clone 1 corresponds to a VHH antibody of sequence SEQ ID N°l, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of sequences SEQ ID N° 31, 32 and 33.

[0068] Clone 2 corresponds to a VHH antibody of sequence SEQ ID N°2, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 34, 35 and 36. Clone 3 corresponds to a VHH antibody of sequence SEQ ID N°3, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 37, 38 and 39.

[0069] Clone 4 corresponds to a VHH antibody of sequence SEQ ID N°4, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 40, 41 and 42.

[0070] Clone 5 corresponds to a VHH antibody of sequence SEQ ID N°5, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 43, 44 and 45.

[0071] Clone 6 corresponds to a VHH antibody of sequence SEQ ID N°6, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 46, 47 and 48.

[0072] Clone 7 corresponds to a VHH antibody of sequence SEQ ID N°7, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 49, 50 and 51.

[0073] Clone 8 corresponds to a VHH antibody of sequence SEQ ID N°8, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 52, 53 and 54.

[0074] Clone 9 corresponds to a VHH antibody of sequence SEQ ID N°9, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 55, 56 and 57.

[0075] Clone 10 corresponds to a VHH antibody of sequence SEQ ID N°10, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 58, 59 and 60.

[0076] Clone 11 corresponds to a VHH antibody of sequence SEQ ID N°ll, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 61, 62 and 63.

[0077] Clone 12 corresponds to a VHH antibody of sequence SEQ ID N°12, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 64, 65 and 66.

[0078] Clone 13 corresponds to a VHH antibody of sequence SEQ ID N°13, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 67, 68 and 69.

[0079] Clone 14 corresponds to a VHH antibody of sequence SEQ ID N°14, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 70, 71 and 72.

[0080] Clone 15 corresponds to a VHH antibody of sequence SEQ ID N°15, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 73, 74 and 75.

[0081] Clone 16 corresponds to a VHH antibody of sequence SEQ ID N°16, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 76, 77 and 78.

[0082] Clone 17 corresponds to a VHH antibody of sequence SEQ ID N°17, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 79, 80 and 81.

[0083] Clone 18 corresponds to a VHH antibody of sequence SEQ ID N°18, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 82, 83 and 84. Clone 19 corresponds to a VHH antibody of sequence SEQ ID N°19, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 85, 86 and 87.

[0084] Clone 20 corresponds to a VHH antibody of sequence SEQ ID N°20, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 88, 89 and 90.

[0085] Clone 21 corresponds to a VHH antibody of sequence SEQ ID N°21, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 91, 92 and 93.

[0086] Clone 22 corresponds to a VHH antibody of sequence SEQ ID N°22, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 94, 95 and 96.

[0087] Clone 23 corresponds to a VHH antibody of sequence SEQ ID N°23, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 97, 98 and 99.

[0088] Clone 24 corresponds to a VHH antibody of sequence SEQ ID N°24, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 100, 101 and 102.

[0089] Clone 25 corresponds to a VHH antibody of sequence SEQ ID N°25, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 103, 104 and 105.

[0090] Clone 26 corresponds to a VHH antibody of sequence SEQ ID N°26, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 106, 107 and 108.

[0091] Clone 27 corresponds to a VHH antibody of sequence SEQ ID N°27, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 109, 110 and 111.

[0092] Clone 28 corresponds to a VHH antibody of sequence SEQ ID N°28, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 112, 113 and 114.

[0093] Clone 29 corresponds to a VHH antibody of sequence SEQ ID N°29, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 115, 116 and 117.

[0094] Clone 30 corresponds to a VHH antibody of sequence SEQ ID N°30, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 118, 119 and 120.

[0095] OlTml corresponds to a VHH antibody of sequence SEQ ID N°189, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 195, 196 and 197.

[0096] 01Tm2 corresponds to a VHH antibody of sequence SEQ ID N°190, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 31, 32 and 33.

[0097] 01Tm3 corresponds to a VHH antibody of sequence SEQ ID N°191, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 31, 32 and 33.

[0098] 01Tm4 corresponds to a VHH antibody of sequence SEQ ID N°192, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 31, 32 and 33. 09Tml corresponds to a VHH antibody of sequence SEQ ID N°193, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 55, 56 and 57.

[0099] 09Tm2 corresponds to a VHH antibody of sequence SEQ ID N°194, including CDR1, CDR2, and CDR3 hypervariable regions, respectively, of SEQ ID N° 198, 199 and 200.

[0100] In the context of the present invention, redundant sequences which are listed as part of distinct antibodies, or antigen-binding fragment, among SEQ ID N° 34 to 120 may be used interchangeably from one equivalent CDR to the other. This may, for example, be the case for CDR1 sequences SEQ ID N°31, 34 and 37 or CDR2 sequences SEQ ID N°32 and 35, which are shared among a plurality of the above-mentioned clones.

[0101] Accordingly, the present invention encompasses - but is not limited to - all the above-mentioned VHH clones, and / or combinations of their respective CDR1 , CDR2 and CDR3 hypervariable regions thereof; and uses thereof.

[0102] Consequently, a first main embodiment of the invention relates to an isolated monoclonal anti-Gonadotropin-Releasing Hormone (anti-GnRH) single domain antibody (SdAb), or antigen-binding fragment thereof; wherein said antibody or antigen-binding fragment binds specifically to, both, a N-terminal region (pGlu-His-Trp) of native GnRH, and a C-terminal region (Pro-Gly-NH2) of native GnRH.

[0103] According to particular embodiments, the isolated monoclonal anti-Gonadotropin- Releasing Hormone (anti-GnRH) single domain antibody (SdAb), or antigen-binding fragment thereof may be characterized in that it binds specifically to a first polypeptide sequence consisting of SEQ ID N°154 and to a second polypeptide sequence consisting of SEQ ID N°153.

[0104] According to a second main embodiment, the invention relates to an isolated monoclonal anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody, or antigen-binding fragment thereof; wherein said antibody or antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of: a CDR3 polypeptide sequence SEQ ID NO 57 or 187; a CDR3 polypeptide sequence SEQ ID NO 33 or 188; a CDR3 polypeptide sequence SEQ ID NO 36; a CDR3 polypeptide sequence SEQ ID NO 96; a CDR3 polypeptide sequence SEQ ID NO 48; a CDR3 polypeptide sequence SEQ ID NO 42, 75, 81, 90, 93; or a variant of said CDR3 polypeptide sequence having 1 or 2 substitutions.

[0105] According to a particular embodiment, when applicable, the variant of said CDR3 polypeptide sequence has 1 or 2 conversative substitutions.

[0106] According to particular and exemplified embodiments, the isolated antibody or antigen-binding fragment thereof is a single domain antibody (SdAb).

[0107] Hence, according to particular embodiments, the isolated monoclonal anti- Gonadotropin-Releasing Hormone (anti-GnRH) antibody or antigen-binding fragment thereof, is a single domain antibody (SdAb); said antibody or antigen-binding fragment comprising a variable domain having a complementarity-determining region 3 (CDR3) selected from the group as defined above.

[0108] According to particular embodiments, the isolated monoclonal anti-Gonadotropin- Releasing Hormone (anti-GnRH) antibody or antigen-binding fragment thereof binds to a first polypeptide of sequence SEQ ID NO 153, and to a second polypeptide of sequence SEQ ID NO 154.

[0109] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having a complementarity-determining region 3 (CDR3) comprising a sequence selected from: SEQ ID NO 57, 187, 33, 188, 36 and 96.

[0110] According to more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 57.

[0111] According to more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 187:

[0112] DX1X2X3X4GX5YYX6PDX7 wherein XI to X7 independently represent any amino acid, with XI being for example a R, with X2 being for example a G, with X3 being for example a P, with X4 being for example a S, with X6 being for example a Y, with X7 being for example a Y.

[0113] According to more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 187 as defined above: with XI being a R, with X2 being a G, with X3 being a P, with X4 being a S, with X6 being a Y, with X7 being a Y.

[0114] According to more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 33.

[0115] According to more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 188:

[0116] DX1X2YYX3X4X5X6X7PX8 wherein XI to X8 independently represent any amino acid, with XI being for example a D, with X2 being for example a A, with X3 being for example a P, with X4 being for example a G, with X8 being preferably a Q or an H.

[0117] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 188 as defined above: with XI being a D, with X2 being a A, with X3 being a P, with X4 being a G, with X8 being either a Q or an H.

[0118] According to other particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence selected from SEQ ID NO 182 to SEQ ID NO 186.

[0119] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0120] - a CDR1 comprising a sequence selected from: SEQ ID NO 55 and 82;

[0121] - a CDR2 comprising a sequence selected from: SEQ ID NO 56, 98, 116 and 199;

[0122] - a CDR3 comprising a sequence SEQ ID NO 187.

[0123] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0124] - a CDR1 comprising a sequence selected from: SEQ ID NO 55 and 82;

[0125] - a CDR2 comprising a sequence selected from: SEQ ID NO 56, 98, 116 and 199;

[0126] - a CDR3 comprising a sequence SEQ ID NO 57. According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0127] - a CDR1 comprising a sequence selected from: SEQ ID NO 31 and 195;

[0128] - a CDR2 comprising a sequence selected from: SEQ ID NO 32;

[0129] - a CDR3 comprising a sequence SEQ ID NO 188.

[0130] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0131] - a CDR1 comprising a sequence selected from: SEQ ID NO 31 and 195;

[0132] - a CDR2 comprising a sequence selected from: SEQ ID NO 32; a CDR3 comprising a sequence SEQ ID NO 33.

[0133] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0134] - a CDR1 comprising a sequence selected from: SEQ ID NO 34;

[0135] - a CDR2 comprising a sequence selected from: SEQ ID NO 35;

[0136] - a CDR3 comprising a sequence SEQ ID NO 36.

[0137] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0138] - a CDR1 comprising a sequence selected from: SEQ ID NO 94;

[0139] - a CDR2 comprising a sequence selected from: SEQ ID NO 95;

[0140] - a CDR3 comprising a sequence SEQ ID NO 96.

[0141] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having: a CDR1 comprising a sequence selected from: SEQ ID NO 46; a CDR2 comprising a sequence selected from: SEQ ID NO 47; a CDR3 comprising a sequence SEQ ID NO 48.

[0142] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0143] - a CDR1 comprising a sequence selected from: SEQ ID NO 195;

[0144] - a CDR2 comprising a sequence selected from: SEQ ID NO 196;

[0145] - a CDR3 comprising a sequence SEQ ID NO 197.

[0146] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a variable domain having:

[0147] - a CDR1 comprising a sequence selected from: SEQ ID NO 198;

[0148] - a CDR2 comprising a sequence selected from: SEQ ID NO 199;

[0149] - a CDR3 comprising a sequence SEQ ID NO 200.

[0150] According to more particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it comprises a framework region (FR) selected from the group consisting of: SEQ ID N° 121 to 151 , 201 to 204 or 211 to 214.

[0151] According to even more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a framework region 1 (FR1) selected from the group consisting of SEQ ID N° 121 to 137 , 201 to 204 or 211 to 214.

[0152] According to even more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a framework region 2 (FR2) selected from the group consisting of SEQ ID N° 138 to 141. According to even more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a framework region 3 (FR3) selected from the group consisting of SEQ ID N° 142 to 149.

[0153] According to even more particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it comprises a framework region 4 (FR4) selected from the group consisting of SEQ ID N° 150 or 151.

[0154] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it shares at least 80% of sequence identity with an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody of sequence SEQ ID N°1 to 30, 189 to 194, or 205 to 210.

[0155] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it shares at least 90% of sequence identity with an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody of sequence SEQ ID N°1 to 30 189 to 194, or 205 to 210.

[0156] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it shares at least 95% of sequence identity with an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody of sequence SEQ ID N°1 to 30 189 to 194, or 205 to 210.

[0157] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it shares at least 99% of sequence identity with an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody of sequence SEQ ID N°1 to 30 189 to 194, or 205 to 210.

[0158] According to particular embodiments, the isolated anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody or antigen-binding fragment thereof according to the invention is characterized in that: GnRH is selected in the group consisting of human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and feline GnRH.

[0159] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it is a recombinant protein.

[0160] According to particular embodiments, the isolated antibody, or antigen-binding fragment thereof, according to the invention binds specifically to more than one polypeptide, apart from native GnRH. According to said particular embodiment, the isolated antibody, or antigen-binding fragment thereof, may thus be plurispecific, for example bispecific or trispecific. In that regard, according to more particular embodiments, the isolated antibody, or antigen-binding fragment thereof, may consist of a bispecific single domain antibody (SdAb), especially a multispecific VHH antibody, or antigen-binding fragment thereof, characterized in that: it contains at least one antigen-binding fragment of the present invention, which binds specifically to a first polypeptide which is native GnRH, and comprising at least one of the hypervariable CDR3 regions, disclosed herein; it contains at least one antigen-binding fragment which binds specifically to a second polypeptide, for example a polypeptide selected from the group of albumin, a VHH antibody that binds specifically to albumin or a fragment thereof.

[0161] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention is characterized in that it is conjugated (i.e. linked covalently to another compound or polypeptide). For example, the isolated antibody or antigenbinding fragment thereof according to the invention may be N-terminally or C-terminally conjugated.

[0162] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention may be conjugated to a detectable agent such as a radioactive material (e.g., radionuclide), an enzyme, a fluorescent label, a cytotoxic drug label.

[0163] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof according to the invention may be conjugated to a polypeptide which is an albumin polypeptide or a fragment thereof.

[0164] According to other particular embodiments, the isolated antibody or antigenbinding fragment thereof according to the invention is characterized in that it is not conjugated.

[0165] According to more particular non-mutually exclusive, embodiments, the antibody or antigen-binding fragment thereof according to the invention is characterized in that it is modified in order to increase its biological half-life. Various approaches are possible.

[0166] When the antibody according to the invention is a single-domain antibody (SdAb), or an antigen-binding fragment thereof, more particularly a VHH antibody or antigen-binding fragment thereof, it may advantageously comprise or consist of a single domain antibody linked to an immunoglobulin domain, for example an Fc portion (such as a human Fc or a non-human Fc). Said Fc portion may be useful for increasing the half-life and even the production of the single domain antibody of the invention. For example the Fc portion can bind to serum proteins and thus increases the half-life on the single domain antibody.

[0167] In one embodiment, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 by Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al. Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the foetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitutions of Fc region residue 434 (US Patent No. 7,371,826).

[0168] Another modification of the antibodies or antigen-binding fragments herein that is contemplated is pegylation. An antibody or antigen-binding fragment can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0169] Another modification of the antibodies, or antigen-binding fragments, that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. Such approach is for example described in Ballance et al. EP0322094. Another possibility is a fusion of at least the antigenbinding region of the antibody of the invention to proteins capable of binding to serum proteins, such human serum albumin to increase half-life of the resulting molecule. Such approach is for example described in Nygren et al., EP 0486 525.

[0170] Polysialytion is another technology, which uses the natural polymer polysialic acid (PSA) to prolong the active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). Another technology includes the use of hydroxy ethyl starch ("HES") derivatives linked to antibodies.

[0171] A further aspect of the invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention.

[0172] Hence, according to another main embodiment, the invention relates to a nucleic acid coding for an antibody or antigen-binding fragment thereof according to the invention.

[0173] According to another main embodiment, the invention relates to a vector comprising a nucleic acid according to the invention.

[0174] According to another main embodiment, the invention relates to a host cell comprising the nucleic acid or the vector according to the invention; in particular an eukaryotic cell comprising the nucleic acid or the vector according to the invention. Such host cell may be isolated by any method known in the Art.

[0175] According to another main embodiment, the invention relates to a pharmaceutical composition comprising: the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell according to the invention; and a pharmaceutically acceptable excipient.

[0176] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell, or pharmaceutical composition, is for use a medicament, or for use in a method of diagnosis in vivo. In particular, the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell, or pharmaceutical composition, is for use in a method for modulating fertility in a human or non-human mammal.

[0177] According to some particular embodiments, the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell, or pharmaceutical composition, is for use in a method for decreasing, or suppressing, fertility in a human or non- human mammal.

[0178] According to some particular embodiments, the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell, or pharmaceutical composition, is for use as a medicament, directed toward non-castrated human or non-castrated non-human mammal.

[0179] According to some particular embodiments, the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell, or pharmaceutical composition, is for use as a medicament, directed toward castrated human or non-castrated non- human mammal.

[0180] Beyond sterilization, similar to surgery, the antibodies and antigen-binding fragments thereof, according to the invention, are also suitable for reducing the consequences (e.g. secondary effects) of surgical castration, such as those selected from urinary incontinence and lymphoma due to an overexpression of LH in spayed female (explained in Kutzler, 2020, Possible Relationship between Long-Term Adverse Health effects of Gonad Removing Surgical Sterilization and Luteinizing Hormone in Dogs, Animals).

[0181] Indeed, GnRH is physiologically regulated by negative feedback of oestrogene / progesterone. When a female dog is spayed (sterilized), no oestrogene / progesterone is produced, so GnRH production is not controlled and is overexpressed. This induces also an overexpression of LH, responsible for urinary incontinence and cancers, which can thus be prevented or attenuated.

[0182] According to particular embodiments, the isolated antibody or antigen-binding fragment thereof, or the nucleic acid, or the vector, or the host cell, or pharmaceutical composition, is for use for treating or preventing a sex-hormone related and / or GnRH-related disorder. Herein is further disclosed a use of an isolated antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or pharmaceutical composition according to the invention for manufacturing a medicament,

[0183] According to another main embodiment, the invention relates to an in vitro method for detecting Gonadotropin-Releasing Hormone (GnRH), comprising the steps of: a) providing a sample, in particular a biological sample or a fraction thereof; b) bringing in contact the sample with an isolated antibody or antigen-binding fragment thereof, according to the invention.

[0184] According to particular embodiments, the in vitro method for detecting Gonadotropin-Releasing Hormone (GnRH), according to the invention; which further comprises a step of detecting an interaction of said isolated antibody or antigen-binding fragment thereof, with GnRH in the sample, thereby detecting GnRH in the sample.

[0185] The sample may be any sample, for example any biological sample, which is susceptible to contain GnRH, and more particularly native GnRH, such as human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and feline GnRH.

[0186] A biological sample susceptible to contain GnRH may include blood, plasma, serum, urine, amniotic fluid, tissue extract, tissue fluid, in vitro cell culture supernatant, a cell lysate, or fractions thereof.

[0187] According to particular embodiments, the in vitro method for detecting Gonadotropin-Releasing Hormone (GnRH), according to the invention; which further comprises a step of comparing an amount of GnRH in the sample to a reference value.

[0188] According to another main embodiment, the invention relates to a method for isolating an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody, or antigen-binding fragment thereof; comprising the steps of: a) providing a library of single domain antibodies (SdAb), or antigen-binding fragments thereof; b) subjecting the library to antigen- affinity selection, the selected SbAbs or fragments thereof being characterized for having specificity toward, both, the N-terminal region (pGlu-His-Trp) of native GnRH and the C-terminal region (Pro-Gly-NH2) of native GnRH. According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof may be characterized in that the library comprises, or consists of, VHH polypeptides of antigen-binding fragments thereof.

[0189] According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof may be characterized in that: the selected SbAbs are selected for binding to a first polypeptide of sequence SEQ ID NO 153, and to a second polypeptide of sequence SEQ ID NO 154.

[0190] According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that the GnRH is selected in the group consisting of human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and feline GnRH.

[0191] According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that it comprises a plurality of steps consisting of subjecting the library to antigen- affinity selection, the selected SbAbs or antigen-binding fragments thereof being characterized for having specificity toward, both, the N-terminal region (pGlu-His-Trp) of native GnRH and the C-terminal region (Pro- Gly-NH2) of native GnRH.

[0192] Advantageously, such one or more steps of subjecting the library to antigen-affinity selection may additionally include a further step of separating polypeptides which have become bound to single domain antibodies (SdAb) or antigen-binding fragments thereof, from unbound.

[0193] According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that step b) comprises the following sub-steps: bl) contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a N-terminal region of native GnRH (pGlu-His-Trp) and of a C-terminal region of native GnRH (Arg-Pro-Gly-NH2), or analogues thereof; b2) subjecting said polypeptide(s) to at least one washing step; b3) separating polypeptides which have become bound to single domain antibodies (SdAb) or antigen-binding fragments thereof, from unbound by separation through an organic phase, thereby separating candidate binding partners from other library members. According to some particular embodiments, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that it includes a first step of contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a N-terminal region of native GnRH (pGlu- His-Trp), or an analogue thereof; and a subsequent (e.g. second) step of contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a C-terminal region of native GnRH (Arg-Pro-Gly-NH2), or an analogue thereof.

[0194] According to some particular embodiments, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that it includes a first step of contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a C-terminal region of native GnRH (Arg- Pro-Gly-NH2), or an analogue thereof; and a subsequent (e.g. second) step of contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a N-terminal region of native GnRH (pGlu-His-Trp), or an analogue thereof.

[0195] According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that step b) comprises the following sub-steps: bl) contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a N-terminal region of native GnRH (pGlu-His-Trp), or analogues thereof; b2) subjecting said polypeptide(s) to at least one washing step; b3) separating polypeptides which have become bound to single domain antibodies (SdAb) or antigen-binding fragments thereof, from unbound by separation through an organic phase, thereby separating candidate binding partners from other library members; b4) contacting the candidate binding partners at the preceding step with one or more polypeptides consisting of a C-terminal region of native GnRH (Arg-Pro-Gly-NH2), or analogues thereof; b5) subjecting said polypeptide(s) to at least one washing step; b6) separating polypeptides which have become bound to single domain antibodies (SdAb) or antigen-binding fragments thereof, from unbound by separation through an organic phase, thereby separating candidate binding partners from other library members.

[0196] According to a particular embodiment, the method for isolating an anti-GnRH antibody, or antigen-binding fragment thereof, may be characterized in that step b) comprises the following sub-steps: bl) contacting the library of single domain antibodies (SdAb), or antigen-binding fragments thereof with one or more polypeptides consisting of a C-terminal region of native GnRH (Arg-Pro-Gly-Nth), or analogues thereof; b2) subjecting said polypeptide(s) to at least one washing step; b3) separating polypeptides which have become bound to single domain antibodies (SdAb) or antigen-binding fragments thereof, from unbound by separation through an organic phase, thereby separating candidate binding partners from other library members; b4) contacting the candidate binding partners at the preceding step with one or more polypeptides consisting of a N-terminal region of native GnRH (pGlu-His-Trp), or analogues thereof; b5) subjecting said polypeptide(s) to at least one washing step; b6) separating polypeptides which have become bound to single domain antibodies (SdAb) or antigen-binding fragments thereof, from unbound by separation through an organic phase, thereby separating candidate binding partners from other library members.

[0197] A library of SdAb or antigen-binding fragments thereof may comprise or consist of a library of virus particles comprising a plurality of virus particles, the virus particles displaying a plurality of different fusion proteins on the surface thereof, wherein each fusion protein comprises at least a portion of a SdAb or an antigen-binding fragment thereof conjugated to a filamentous phage coat protein.

[0198] Herein is further disclosed a method for modulating fertility in a non-human animal, preferably, a non-human mammal, such as companion animals, livestock, exotic, wild and zoo animals, comprising a step of administering an anti-GnRH antibody, or antigen-binding fragment thereof, according to the invention, to the said animal. In particular, the above-mentioned method is suitable for decreasing, or suppressing, fertility in the human and non-human animal.

[0199] Herein is further disclosed a method for modulating, preferably, decreasing or suppressing, boar taint in meat from an animal, comprising a step of administering an anti- GnRH antibody, or antigen-binding fragment thereof to the said animal; said animal being in particular a porcine, such as a non-castrated male porcine.

[0200] Herein is further disclosed a method for improving the quality of meat by reducing boar taint, comprising a step of administering an anti-GnRH antibody, or antigen-binding fragment thereof to the said animal; said animal being in particular a porcine, such as a noncastrated male porcine.

[0201] General definitions

[0202] As used herein the term “GnRH” or “Gonadotropin Releasing Hormone » or « LHRH » or « Luteinizing Hormone releasing Hormone » are used interchangeably. Although the main antigen considered by the present disclosure is mammalian GnRH, in particular human and non-human (e.g. livestock and / or porcine-related) GnRH, the term is also meant to include other non-mammalian GnRH in the absence of contrary instructions. In particular, GnRH may be selected in the group consisting of human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and feline GnRH. A polypeptide sequence of GnRH which may used as a reference is mammalian or porcine GnRH, which is characterized by the following structure :

[0203] (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID N°152).

[0204] As used herein the term « N-terminal region of native GnRH » refers to the blocked amino-terminal region of native GnRH, including a pyroglutamic acid residue (pGlu), which consists of the following reference sequence: pGlu-His-Trp.

[0205] As used herein the term « C-terminal region of native GnRH » refers to the C- terminal region of native GnRH, including a C-terminal amidated carboxyterminus, which consists of the following reference sequence: Arg-Pro-Gly-NH2.

[0206] As used herein the term "antibody" or "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. Unless specificed otherwise, this term thus encompasses both conventional antibodies and non-conventional antibodies, including single domain antibodies, bispecific antibodies, multispecific antibodies, and the like.

[0207] As used herein the term “non-conventional antibody”, in particular, may encompass engineered antibodies, such as bi-specific antibodies, and heavy-chain antibodies, or single domain antibodies such as those selected from the VHH-type, and those characterized as single heavy chain variable domains of antibodies of the VNAR-type.

[0208] As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found, for example, in Camelid mammals and sharks which are naturally devoid of light chains.

[0209] As used herein, single domain antibodies of the VHH type are also called VHH or VHH or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(ll):484-490; and WO 06 / 030220, WO 06 / 003388.

[0210] Camel immunoglobulins can be modified by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "nanobody" or “VHH”. See U.S. patent number 5,759,808 issued June 2, 1998; see also Stijlemans, B. et al. , 2004 J Biol Chem 279: 1256-1261 ; Dumoulin, M. et a / . , 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440- 448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in the camelid animal, i.e., is produced by the camelid following immunization with GnRH or a peptide fragment thereof, according to the present disclosure using techniques described herein for other antibodies. Alternatively, the Gn / ? / 7-binding camelid nanobody is engineered, i.e. , produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody proteins using panning procedures with GnRH as a target. As used herein the term “VNAR” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Sharks which are naturally devoid of light chains. IgNARs (Immunoglobulin New Antigen Receptors) heavy chain-only Ig-like molecules have been identified in all species of sharks studied so far. They are disulphide-bound homodimeric molecules composed of two polypeptide chains containing five constant domains and one variable region (VNAR) by which they bind antigens (Greenberg et al, Nature 1995 Mar 9; 374(6518): 168-73). VNARs are small (12kDa), stable, soluble, monomeric antigen-binding domains that can be configured into many different therapeutic modalities. The isolation of various VNAR based binding moieties has been described (see, e.g., W02003 / 014161 and W02005 / 118629). They have elongated CDR3 structures that potentially extend into antigen clefts and cavities.

[0211] The term “single domain antibody” may further encompass a non-conventional antibody with at least one single domain antibody of the invention, which binds specifically to GnRH, and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is typically also a single domain antibody. Such a polypeptide is referred to herein as ''multispecific'' polypeptide; in opposition to a polypeptide comprising the same single domain antibodies (“monospecific” polypeptide). Thus, in some embodiments, the polypeptide of the invention may also provide at least one further binding site directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope. Said binding site is directed against to the same protein, polypeptide, antigen, antigenic determinant or epitope for which the single domain antibody of the invention is directed against, or may be directed against a different protein, polypeptide, antigen, antigenic determinant or epitope) from the single domain antibody of the invention.

[0212] Typically, the one or more further binding site may comprise one or more parts, fragments or domains of conventional chain antibodies (and in particular human antibodies) and / or of heavy chain antibodies. For example, a single domain antibody of the invention may be linked to a conventional (typically human) VH or VL optionally via a linker sequence.

[0213] The term “single domain antibody” may further encompass, in some embodiments, the at least one single domain antibody linked to one or more (typically of mammalian origin, or even human) CHI, and / or CH2 and / or CH3 domains, optionally via a linker sequence.

[0214] For instance, a single domain antibody linked to a suitable CHI domain could for example be used - together with suitable light chains - to generate antibody fragments / structures analogous to conventional Fab fragments or F(ab')2 fragments, but in which one or (in case of an F(ab')2 fragment) one or both of the conventional VH domains have been replaced by a single domain antibody of the invention. In some embodiments, one or more single domain antibodies of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of / to form an Fc portion), to an Fc portion and / or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and / or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more CH2 and / or CH3 domains of an antibody, such as from a heavy chain antibody and more typically from a conventional human chain antibody; and / or may form and Fc region, for example from IgG (e.g. from IgGl , IgG2, IgG3 or IgG4), from IgE or from another human Ig such as IgA, IgD or IgM. For example, WO 94 / 04678 describes heavy chain antibodies comprising a Camelid VHH domain or a humanized derivative thereof (i.e. a single domain antibody), in which the Camelidae CH2 and / or CH3 domain have been replaced by human CH2 and CH3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a single domain antibody and human CH2 and CH3 domains (but no CHI domain), which immunoglobulin has the effector function provided by the CH2 and CH3 domains and which immunoglobulin can function without the presence of any light chains.

[0215] Typically, the polypeptide of the invention comprises a single domain antibody of the invention, which is optionally fused at its N terminal end, at its C terminal end, or both at its N terminal end and at its C terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein.

[0216] As used herein, the polypeptides that comprise a sole single domain antibody are referred to herein as "monovalent" SdAb polypeptides. Polypeptides that comprise or essentially consist of two or more single domain antibodies according to the invention are referred to herein as "multivalent" SdAb polypeptides.

[0217] As used herein, the term “antigen-binding fragment' refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to GnRH. The antigenbinding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain, or any fusion proteins comprising such antigen-binding fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.

[0218] As used herein, the term "dsFv" refers to a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. Such single chain antibodies may include one or more antigen binding portions or fragments of an antibody.

[0219] A unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies. Antigen binding fragments can be incorporated into single domain antibodies, SMIP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Antigen biding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) Which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870). These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0220] As used herein, the term “variable”, as in “variable domain”, refers to certain portions of the relevant binding protein which differ extensively in sequence between and among antibodies and are used in the specific recognition and binding of a particular antibody for its particular target. However, the variability is not evenly distributed throughout the entire variable domains of antibodies. The variability is concentrated segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariable regions, which in the context of conventional antibodies are present both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions or sequences.

[0221] As used herein, the term “VH domain”, or “VH domain” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin variable domain.

[0222] As used herein, the term “VL domain”, or “VL domain” can be used interchangeably and refer, when applicable, to the corresponding light chain immunoglobulin variable domain.

[0223] As used herein, the term ''hypervariable region'' when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. This term may be substituted by the terms “Complementarity Determining Regions” or “CDRs”.

[0224] Thus, as used herein “Complementarity Determining Regions” or “CDRs” refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of a conventional immunoglobulin each have three CDRs, designated CDR-L1, CDR-L2, CDR-L3 and CDR - Hl, CDR-H2, CDR-H3, respectively. A conventional antibody antigen-binding domain, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.

[0225] A non-conventional antibody antigen-binding domain of the VHH type includes three CDRs, also reported herein as CDR1, CDR2 and CDR3.

[0226] A non-conventional antibody antigen-binding domain of the VNAR type includes two CDRs, also reported herein as CDR1 and CDR3, and CDR-like regions defined in the Art as hypervariable region 2 (HV2) and hypervariable region 4 (HV4). Also, as used herein, "Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin (i.e. light and heavy chain variable regions in a conventional antibody, or only heavy chain variable regions for single domain antibodies) that are relatively conserved among different immunoglobulins in a single species.

[0227] The light and heavy chains of an immunoglobulin each have four FRs, designated FR-L1, FR-L2, FR-L3, FR-L4, and FR-H1, FR-H2, FR-H3, FR-H4, respectively. Accordingly, in a conventional antibody antigen-binding domain, the light chain variable domain may thus be designated as (FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4) and the heavy chain variable domain may thus be designated as (FR-H1)-(CDR-H1)-(FR-H2)-(CDR- H2)-(FR-H3)-(CDR-H)-(FR4-H3).

[0228] The amino acid sequence and structure of a single domain antibody of the VHH type can be considered to be comprised of four framework regions or "FRs" 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; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http: / / imgt.cines.fr / ).

[0229] The VNAR protein scaffold consists of amino acid residues (aa) 1-25 of the framework 1 (FW1) region; aa 26-32 of the complementary determining region 1 (CDR1); aa 33-43 of FW2; aa 44-52 of the hypervariable 2 region (HV2); aa 53-85 of FW3; aa 61-65 of HV4; the CDR3 region (of variable length) and FW4 (11 residues starting at XGXG). Like all immunoglobulin family variable (V) domains, VNARs contain the two canonical cysteine residues that link FW1 and FW3 via a disulfide bond. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. For conventional antibodies, the CDRs of the heavy chain variable domain are located at residues 31-35B (H- CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. (http: / / www.bioinf.org.Uk / abs / #cdrdef).

[0230] The Kabat system may also be used to delineate the approximate boundaries of the CDRS within an antibody scaffold belong to non-conventional antibodies, such as a single domain antibody (SdAb) of the VHH type.

[0231] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as native GnRH, while having relatively little detectable reactivity with non-antigen proteins or structures. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules.

[0232] The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope, such as native GnRH. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

[0233] As used herein, the terms "monoclonal antibody" , "monoclonal Ab" , "monoclonal antibody composition" , "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0234] As used herein the term “humanized” refers to a conventional or non-conventional antibody, for example a single domain antibody of the VHH type according to the disclosure wherein an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring antibody (e.g. a conventional antibody or a VHH domain) has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring antibody sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional chain antibody from a human being. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be chosen such that the resulting humanized (e.g. conventional or single domain) antibodies still retain the favourable properties of anti-GnRH antibodies of the invention. The one skilled in the art is able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions.

[0235] As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl- CIO) alkoxy - or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.

[0236] As used herein, the terms “treat”, “treating” and “treatment” are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself. As used herein, the terms “prevent”, “preventing”, and “prevention” mean reducing the risk of onset or slowing the occurrence of a given phenomenon, namely in the present invention, a cancer and / or dysplasia and more particularly, a pre-cancerous condition, an early stage cancer or a non-metastatic cancer. The term “preventing” also encompasses “reducing the likelihood of occurrence” or “reducing the likelihood of reoccurrence”.

[0237] As used herein, the term “subject” or “patient” may refer indistinctively to all animals expressing GnRH or an anlog thereof, which includes in particular humans and nonhuman mammals. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals, including dogs and cats, zoo animals, as well as livestock such as horses, cattle, cows, horses pigs, sheep, and goats, or other mammalian species including, without limitation, rodents such as mice and rats, guinea pigs, rabbits, hamsters, and the like.

[0238] As used herein, the term “sex hormone related disorder” refers, both, to sex hormone related disorders in male and female, those which are specific to male and those which are specific to female. Such sex hormone related disorders may or may not be directly associated to a deficient GnRH expression or function. In a non-exhaustive manner, the following disorders are considered by the present disclosure: endometriosis, uterine fibroids, polycystic ovarian disease, hirsutism, precocious puberty, delay of puberty, ovarian hyperstimulation syndrome, premenstrual syndrome, endometriosis, sleep apnea, irritable bowel syndrome, gonadal steroid-dependent neoplasias such as cancers of the pancreas, prostate, breast, uterus, endometrium and ovary, gonadotrophe pituitary adenomas, benign prostatic hypertrophy, uterine leiomyoma, acne vulgaris, acute intermittent porphyria, uterine fibrosis, ebenign prostatic hyperplasia, and the like.

[0239] As used herein, a « GnRH related disorder » more specifically refers to a condition for which it is to prevent activation of the GnRH receptor. Exemplary GnRH- related disorders include, without limitation, sex hormone-related conditions, sex hormonedependent cancers, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, breast cancer, pituitary gonadotrophe adenomas, endometriosis, polycystic ovarian disease, uterine fibroids, primary hirsutism, luteinizing hormone surge, and precocious puberty.

[0240] As used herein, a "pharmaceutically acceptable carrier” is intended to include any and all carrier (such as any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like) which is compatible with pharmaceutical administration, in particular parenteral administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the present disclosure. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In a non-exhaustive manner, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM (e.g. 0.05M) phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and will in an embodiment be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In certain embodiments, isotonic agents are included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a pharmaceutically acceptable carrier” encompasses a plurality of pharmaceutically acceptable carriers, including mixtures thereof.

[0241] As used herein, « a plurality of » may thus include « two » or « two or more ».

[0242] As used herein, and unless instructed otherwise, the term “at least one” may encompass “one or more”, or even “two or more” (or “a plurality”). For instance, it may encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

[0243] 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,

[0244] 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,

[0245] 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more than 100.

[0246] As used herein, and unless instructed otherwise, the term “less than...” may encompass all values from 0 to the corresponding threshold, For instance, it may encompass less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

[0247] 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

[0248] 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,

[0249] 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or less than 100, when applicable.

[0250] As used herein, “comprise” or « comprising* may encompass “consist” or « consisting of ».

[0251] As used herein, the term “cell” may encompass any procaryotic cell or eukaryotic cell. Cell types which are particularly considered are those suitable for the production and / or engineering of recombinant antibodies, or fragments, or polypeptide chains thereof. In a non- exhaustive manner, such cells may be selected from the group consisting of: bacterial cells, yeast cells, mammalian cells, non-mammalian cells, insect cells, and plant cells.

[0252] The terms "host cell”, "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate binding proteins of the present disclosure. Host cells may thus include cultured cells, e.g. mammalian cultured cells, such as CHO cells, HEK cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, bacterial cells, yeast cells, insect cells, and plant cells, to name only a few.

[0253] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed". The nucleic acids of the invention may be used to produce an anti-GnRH antibody of the present invention in a suitable expression system.

[0254] As used herein, the term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cell"), and the like. The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present invention. Anti-GnRH antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0255] As used herein, the terms "vector”, "cloning vector" and "expression vector" mean the vehicle by which a nucleic acid sequence, for example a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Typically, the nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0256] So, a further aspect of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0257] In particular, the term “vector” may refer to those viral particules (e.g. AAV type) having a specific tissue tropism, such as a muscle tropism, which may for example include AAV8 and AAV9.

[0258] In particular, the term “retroviral” or “retrovirus” may refer to a lentivirus or lentiviral vector. As used herein, the terms "recombinant AAV”, “rAAV virion”, “AAV particle”, are defined herein as an infectious, replication-defective virus including an AAV protein shell, encapsidating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs. A rAAV virion is produced in a suitable host cell which has had sequences specifying an AAV plasmid, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV plasmid (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0259] By “recombinant virus” is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.

[0260] By “AAV virion” is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single- stranded AAV nucleic acid genome associated with an AAV capsid protein coat). In this regard, single- stranded AAV nucleic acid molecules of either complementary sense, e.g., “sense” or “antisense” strands, can be packaged into any one AAV virion and both strands are equally infectious.' Accordingly, this term may encompass, in the absence of other limitations, any AAV selected from a group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, AAV10, and rhesus macaque- cv vc serotypes including AAVrhlO, and mixtures thereof.

[0261] By "isolated nucleic acid molecule or polynucleotide” is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present disclosure, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator. As used herein, the term 'isolated” or “purified" refers to those molecules that have been altered by humans in their native state, that is, if such molecules exist in nature, that they have been changed and / or removed from their original environment.

[0262] As used herein, the “percentage identity” between two sequences of nucleic acids or proteins (e.g. GnRH binding antibodies) means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length. The comparison of sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an “alignment window”. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group,

[0263] 575 Science Dr., Madison, WI, or by the comparison software BLAST NR or BLAST P).

[0264] The percentage identity between two sequences is determined by comparing the two optimally-aligned sequences in which the sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the nucleotide or amino acid residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0265] As used herein, the term “amino acid” refers to natural or unnatural amino acids in their D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptidyl structure. Natural and unnatural amino acids are well known in the art. Common natural amino acids include, without limitation, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). Uncommon and unnatural amino acids include, without limitation, allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) and pentafluorophenylalanine.

[0266] As used herein, a 'modified form” of a given amino acid encompasses any equivalent, translationally or post-translationally modified amino acid or non-naturally occurring amino acid, including non standard amino acids, that result from a reaction at an amino group, carboxy group, side-chain functional group, or from the replacement of any hydrogen by a heteroatom. Hence, a modified amino acid may encompass an amino acid derivative which results from either one of modifications selected from: N-linked glycosylation, O-linked glycosylation, phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin-binding, cysteine oxidation, nitrosylation and ubiquity lation.

[0267] As used herein and above, an "equivalent amino acid" means an amino acid which may be substituted (i.e. through conservative substitution) for another amino acid in the peptide compounds according to the invention without any appreciable loss of function. Equivalent amino acids will be recognized by those of ordinary skill in the art. Substitution of like amino acids is made on the basis of relative similarity of side chain substituents, for example regarding size, charge, hydrophilicity and hydrophobicity as described herein. The phrase "or an equivalent amino acid thereof when used following a list of individual amino acids means an equivalent of one or more of the individual amino acids included in the list.

[0268] Illustratively, conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:

[0269] Aliphatic residues I, L, V, and M

[0270] Cycloalkenyl-associated residues F, H, W, and Y

[0271] Hydrophobic residues A, C, F, G, H, I, E, M, R, T, V, W, and Y

[0272] Negatively charged residues D and E

[0273] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0274] Positively charged residues H, K, and R

[0275] Small residues A, C, D, G, N, P, S, T, and V

[0276] Very small residues A, G, and S

[0277] Residues involved in turn A, C, D, E, G, H, K, N, Q, R, S, P, and formation T

[0278] Flexible residues Q, T, K, S, G, P, D, E, and R As used herein, the terms “phage display” and “bacteriophage display” are considered as synonymous, and refer to a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. In the absence of other indications, the term is not limited to one specific type of bacteriophage particle nor one specific type of fusion protein and / or strategy of enrichment, and / or particular phage library.

[0279] EXAMPLES

[0280] A. MATERIALS AND METHODS

[0281] Immunization

[0282] Single domain antibodies were obtained from llamas immunized with 6 injections at twoweekly intervals. Eight days after the last boost, sera were collected to define antibody titers against GnRH peptides by ELISA. In this ELISA, 96-well plates (Maxisorp; Nunc) were coated with the two Biotinylated GnRH peptides. After blocking and adding diluted sera samples, the presence of anti GnRH antibodies was demonstrated by using Mouse anti-llama IgG (FJB, Cat. nr. FJ1203MAB01B09) followed by Donkey anti-mouse IgG-HRP antibody (JIR, Cat. nr. 715- 035-150).

[0283] Library Construction

[0284] RNA was extracted from PBMC of 2 immunized llamas (400 ml each). 40 pg of RNA was used for cDNA synthesis using random primers. The cDNA was used in a primary PCR amplification using non-tagged primers annealing at the Leader sequence and Hinge CHI regions, followed by a secondary PCR amplification introducing restriction endonuclease sites for cloning of VHH genes in pDCLl phagemid vector. The libraries were electroporated into TGI E. coli cells and bacterial glycerol stock of the immune libraries were stored at - 80 °C.

[0285] Phage Display Selection Phage production from the llama VHH library pool were used in three consecutive rounds of phage display selection using Biotinylated GnRH peptide 1 (also referred herein as “Biopeptide 1”). The first round of selection (Round I) was carried out on Biotinylated GnRH peptide 1, at 5 nM, pre-captured on a neutravidin (5 pg / mL, ThermoFisher; Cat. nr. 31000) coated plate. Non-specific phages were washed of (PBS buffer pH7.4), followed by specific phage elution with trypsin (total elution). The second round (Round II) of selections was performed with a lower number of phages (1:5 of the volume of phage used on the first round).

[0286] Overall, Round II of selection was achieved following two different strategies.

[0287] According to one strategy, the Biotinylated GnRH peptide 1 was pre-captured on a neutravidin plate, at 5 and 0.5 nM.

[0288] According to a second strategy, the same peptide was directly coated, at 1 pg / mL.

[0289] Non-specific phage were washed (PBS buffer pH7.4), followed by specific phage elution with trypsin (total elution).

[0290] Phages from both Round II strategies were then selected under a third round (Round III), where the number of phages was further reduced (1 : 10 of the volume of phage used on the first round), and Biotinylated GnRH peptide 1 was pre-captured on a neutravidin plate, at 0.5 and 0.05 nM. Non-specific phages were washed (PBS buffer pH7.4), followed by specific phage elution with trypsin (total elution).

[0291] Serial dilutions of the eluted phages were performed and used to infect exponentially growing TGI. Infected TGI was plated on LBCarbl00Glu2% plates and enrichment values calculated over the background (without antigen for selection).

[0292] ELISA screening in the context of VHH library pool selection

[0293] Individual clones from the second and third round of selection conditions outputs were picked into 96-well Master Plates and tested as Periplasmic Extract (P.E.) for binding to Biotinylated GnRH peptide 1 and 2 (also referred herein as “Biopeptide 2”), via binding ELISA.

[0294] For P.E. binding ELISA, MaxiSorp™ high protein-binding capacity 96 well ELISA plates, were coated with 5 pg / ml of neutravidin (ThermoFisher; Cat. nr. 31000), diluted in PBS, overnight at 4°C. The next day, plates were washed 3X with PBS Tween 0.05% (pH 7.4) and blocked for 1 hour at room temperature with 250 pL / well with 1% Casefn / PBS. After blocking, plates were washed 3X with PBS Tween 0.05% (pH7.4) and incubated for 1 hour at room temperature with 100 pL / well of the Biotinylated GnRH peptides 1 or 2, at 5 nM, diluted in 0.1% Casefn / PBS (pH7.4). After capturing, plates were washed 3X with PBS Tween 0.05% (pH7.4) and incubated per well with 20 pl of P.E. + 80 pl in in 0.1% Casefn / PBS (pH7.4), for 1 hour at RT with shaking. Plates were washed 3X with PBS Tween 0.05% (pH7.4) and incubated with 100 pl of anti-c-Myc antibody (Roche; Cat. nr. 11667203001) followed by secondary antibody donkey anti-mouse-HRP (JIR; Cat. nr. 715-035-150) in 0.1% Casefn / PBS (pH7.4), for 1 hour at RT with shaking. Plates were washed 3X with PBS Tween 0.05% (pH7.4) and the substrate solution (TMB solution) was added to the plates. Reaction was stopped with H2SO4 and plates read in the plate reader at 450 nm.

[0295] Off-rate screening by Surface Plasmon Resonance (SPR)

[0296] Individual clones from the second and third round of selection conditions outputs were picked into 96-well Master Plates and the off-rate (kd) of the clones, tested as Periplasmic Extract (P.E.), on Biotinylated GnRH peptide 1 captured on a StrepAvidin (SA) sensor chip (Cytiva®, Cat. nr. BR100531), was determined via Surface plasmon resonance (SPR) (Biacore 8K, GE Healthcare). Approximately 1140 to 1270 RUs of Biotinylated GnRH peptide 1, were immobilized onto a SA chip via standard streptavidin coupling method.

[0297] QC of the immobilization was done using a commercial antibody anti-GnRH (SMI41, Absolute Antibody, Cat. nr. Ab00922-l.l) at 5 nM diluted in HBS-EP pH 7.4 buffer, lx HBS-EP pH 7.4, was utilized as a running buffer during binding kinetic measurements. 20 pl of P.E. + 80 pl lx HBS-EP pH 7.4 were injected, at 30 pl / min for 2 minutes, followed by an off-rate wash for 1 min between injections. Off-rate wash was 300s after the last concentration injection in each cycle. RU levels were restored to base levels after regeneration with two injections of 15 pL of 10 Glycine pH 1.5 between samples.

[0298] Data was analysed using the multi cycle kinetics predefined evaluation method of the Biacore Insight Evaluation Software. Sensorgams were referenced blank / non-immobilized reference flow cell. Dissociation constants were calculated using the 1:1 dissociation Langmuir binding model.

[0299] Sequencing

[0300] The positive binders were sent to be sequenced. Clones were classified by families according to their different HCDR3 sequences.

[0301] Expression and Purification of VHH Candidate Antibodies The synthetic genes codifying to the VHH variable domains with FLAG and His tags were purchased cloned into pET15b bacterial expression vector with a periplasmic secretion leader sequence. E. coli strain BL21 DE3 were transformed and grown in ZYP-5052 autoindution media for 68 hours at 18°C. Produced VHH antibodies were captured from clarified supernatants using Ni-NTA beads (Qiagen) on gravity fed columns. Eluted antibodies were buffer exchanged to lx PBS pH 7.4 and concentrated using 10k cut-off spin concentrators (Amicon, Cat. nr. UFC801096D). Purified VHH protein was analysed by SDS-PAGE for the presence of correct chains.

[0302] Dose-response ELISA with purified VHH

[0303] MaxiSorp™ high protein-binding capacity 96 well ELISA plates, coated with 5 pg / ml of neutravidin (ThermoFisher; Cat. nr. 31000), diluted in PBS, overnight at 4°C. The next day, plates were washed 3X with PBS Tween 0.05% (pH 7.4) and blocked for 1 hour at room temperature with 250 pL / well with 1% Casefn / PBS. After blocking, plates were washed 3X with PBS Tween 0.05% (pH7.4) and incubated for 1 hour at room temperature with 100 pL / well of the Biotinylated GnRH peptide 1, at 5 nM, diluted in 0.1% Casefn / PBS (pH7.4). After capturing, plates were washed 3X with PBS Tween 0.05% (pH7.4) and incubated with VHH in 0.1% Casefn / PBS (pH7.4). (pH7.4) were diluted from 1000 to 0.01 nM in 5-fold, 7 steps dilutions and added to the Biotinylated GnRH peptide 1 captured and blocked ELISA wells for 1 hr at RT.

[0304] Plates were washed 3X with PBS Tween 0.05% (pH7.4) and incubated with 100 pl of anti-c- Myc antibody (Roche; Cat. nr. 11667203001) followed by secondary antibody donkey anti- mouse-HRP (JIR; Cat. nr. 715-035-150) in 0.1% Casefn / PBS (pH7.4), for 1 hour at RT with shaking. Plates were washed 3X with PBS Tween 0.05% (pH7.4) and the substrate solution (TMB solution) was added to the plates. Reaction was stopped with H2SO4 and plates read in the plate reader at 450 nm. O.D. 450 nm values vs. log concentrations of VHH were plotted using GraphPad Prism 7, applying a nonlinear regression (curve fit) of a log (agonist) vs. response - variable slope (four parameters), and EC50 was determined.

[0305] Affinity determination of purified VHH by Biacore

[0306] To assess the affinity of selected purified clones to Biotinylated GnRH peptide 1 and 2, captured on a StrepAvidin (SA) sensor chip (Cytiva®, Cat. nr. BR100531) by standard streptavidin coupling method. Surface plasmon resonance (SPR) (Biacore T200, GE Healthcare) was used to determine the binding kinetics of selected single domain antibodies at pH7.4. Approximately 1140 to 1270 RUs of Biotinylated GnRH peptide 1 and 2, were immobilized onto a SA chip. QC of the immobilization was done using a commercial antibody anti-GnRH (SMI41, Absolute Antibody, Cat. nr. Ab00922-l.l) at 5 nM diluted in HBS-EP pH 7.4 buffer, lx HBS-EP pH 7.4, was utilized as a running buffer during binding kinetic measurements. Purified VHH were injected 100 nM in HBS-EP pH 7.4, at 10 pl / min for 5 minutes, followed by an off-rate wash for 1 min between injections. Off-rate wash was 20 min after the last concentration injection in each cycle. RU levels were restored to base levels after regeneration with 15 pl of 1 M of 10 Glycine pH 1.5 between samples. Data was analysed using the binding kinetics predefined evaluation method of the Biacore Insight Evaluation Software Fitting. Sensorgrams were referenced blank / non-immobilized reference flow cell 1. Dissociation constants were calculated using the 1:1 dissociation Langmuir binding model to calculate the kinetic constants of the antibodyantigen interactions including association rate (ka), dissociation rate (kd) and affinity (KD).

[0307] Native protein binding by ELISA

[0308] (Gly-OHIO)-Luteinizing hormone releasing hormone (Sigma-Aldrich, Cat nr. L8OO8) was coated in a Maxisorp plate at, 1 pg / ml in IxPBS, 4°C, O / N. Coated plate was blocked with 4% milk in lx PBS. The GnRH VHH antibody panel was titrated by performing 7-steps, five-fold serial dilutions, starting at a 30 pM in 1% milk in lx PBS.

[0309] VHH were detected with an anti-c-myc HRP antibody (Roche, Cat. nr. 11 667 203 001) at 1:200. SMI41 monoclonal (Absolute Antibody, Ab00922-l.l and GnRHl Polyclonal Antibody (Invitrogen, PAI-121) titrations were included. This was followed by anti-mouse and antirabbit IgG-HRP at 1:10,000 (JIR, 715-035-150 and 111-035-144).

[0310] Plates were developed using TMB solution (Invitrogen, 00-4201-56) followed by H2SO4 (Fisher Chemical, J / 8430 / 15) .

[0311] Binding to native porcine GnRH was assessed by measuring O.D. at 450 nm

[0312] GnRH cell-based functional assay

[0313] To evaluate the antagonist activity of the top 5 VHH clones, on a GnRH cell based functional assay, using rat basophil leukemia cells stably expressing human GnRH-receptor. The inhibitory function was determined by measuring the VHH effect on a GnRH-dependent cytosolic Ca2+ion mobilization, using a fluorimetric detection method. For that, the cells were suspended in HBSS buffer (Invitrogen) complemented with 20 mM Hepes, and distributed in microplates at a density of 1.186E+04 cells / well. The fluorescent probe (Fluo8 Direct, AAT Bioquest) was mixed with probenicid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen, pH 7.4), added into each well and equilibrated with the cells for 60 min at 30°C. The 5 purified VHH were then titrated, 5-steps, 5-fold serial dilution starting at 100 pg / mL, added to the plate and incubated for 5 min. 8nM of GnRH (1:1 to 1:800 ratio GnRH / VHH) was subsequently added to the plate, and changes in fluorescence intensity, which varied proportionally to the free cytosolic Ca2+ion concentration, were measured using a microplate reader (FlipR Tetra, Molecular Device). The standard reference antagonist, Cetrorelix acetate, was tested in each experiment at several concentrations and an IC50 value was calculated. The commercial antibody anti-GnRH (SMI41, Absolute Antibody, Cat. nr. Ab00922-l.l) was also included, at two different concentrations at 100 and 20 pg / mL, as a comparator and a positive control.

[0314] 1: Binding of VHH to GnRH and comparison with a reference antibody

[0315] 1.1. Binding of the SMI41 reference antibody to GnRH-derived peptides

[0316] The SMI41 reference antibody corresponds to a mouse IgGl monoclonal anti-human LHRH (anti-huLHRH) antibody sold by Absolute Antibody (at. Nr. PAI-121) with an expected molecular weight of 155 kDa.

[0317] The second reference polyclonal antibody preparation is a rabbit polyclonal anti- amphibian / mouse GnRHl antibody (anti-GnRHl) sold by ThermoFisher (Cat. Nr. PAI-121), with an expected molecular weight of 150 kDa.

[0318] The aim of this experiment is to perform an ELISA QC on the two biotinylated GnRH peptides (Bio-peptide 1 and Bio-peptide 2), by either having them directly coated or captured by neutravidin. The monoclonal antibody, SMI41, and a polyclonal antibody are used for peptide detection, and the presence of Biotin is confirmed by ExtrAvidin-HRP.

[0319] Bio-peptide 1 is detected with both anti-GnRH antibodies (SMI41 & the polyclonal antbiodies), either when directly coated or captured. A complete titration curve was further obtained for anti-GnRH, SMI41, monoclonal antibody Bio-peptide 1 was directly coated, allowing the determination of the EC50 value (0.037 |ag / ml). No background signal was observed, and no specific binding was detected for the secondary control.

[0320] Bio-peptide 2 is only detected by the anti-GnRH polyclonal antibody. It displayed a complete titration curve, both when the peptide was directly coated or captured, allowing for an EC50 the determination.

[0321] Given that SMI41 antibody is the current reference antibody, this experiment shows that Bio- peptide 1 is the peptide with the most relevant epitopes

[0322] ExtrAvidin-HRP confirmed the presence of biotin, on both peptides. No background signal was observed, and no binding was detected for the secondary control only.

[0323] As a conclusion, it is found that the monoclonal antibody labeled as SMI41, cannot bind specifically to bio-peptide 2 which mimics the N-terminal region of native GnRH.

[0324] On the other hand, the polyclonal antibody binds, both, to an analogue of the N-terminal region (biopeptide 2) and of the C-terminal region (biopeptide 1).

[0325] 1.2. Binding of VHH to GnRH-derived peptides and selection of 30 relevant VHH clones

[0326] A plurality of VHH clones were identified through four outputs of phage display selection, which were binding to capture GnRH bio-peptide 1 with a high hit rate.

[0327] P.E. binding ELISA was performed on GnRH Bio-peptide 1 and Bio-peptide-2 captured in a neutravidin-coated plate, using clones from three rounds of selections on GnRH Bio-peptide 1. Very high hit-rates (88-100%) were obtained on all four outputs of the phage display protocol, meaning that the selected VHH clones binding to Bio-peptide 1 also bind Bio-peptide 2.

[0328] The aim of the experiment was then to determine the dissociation rate constant (kd, off-rate) of P.E. containing soluble VHH antibodies to GnRH Bio-peptide 1 captured on a SA sensor chip. From the 368 clones tested, 346 showed specific association to GnRH bio-peptide- 1 (RU>10) and kd values ranged from 3.47E-02 to 6.61E-04 (1 / s).

[0329] 22 clones did not present specific association to GnRH bio-peptide- 1 or have RU levels below 10, therefore the kd values were not determined. No binding of the irrelevant or blank controls was observed, and no RU response was detected when buffer was used as sample (lxHBS-EP+ pH 7.4).

[0330] From the 347 sequenced clones, a total of 339 valid VHH sequences (97.7 %) were obtained. 103 unique VHH sequences were identified and 8 of these were considered enriched sequences with 10 or more representative clones.

[0331] 24 unique HCDR3 sequences were identified and 8 of these were considered enriched HCDR3 sequences with 10 or more representative clones. The enriched HCDR3 sequences were represented in all the four outputs, independently of the selection round.

[0332] A total of 30 VHH clones were thus selected, produced, purified, and further characterized, based on the following critera:

[0333] Binding to both Bio-peptide 1 and Bio-peptide 2, in P.E. binding ELISA;

[0334] Dissociation rate constant (kd, off-rate, < IE-02 1 / s) on Bio-peptide 1;

[0335] Enriched HCDR3 sequence;

[0336] Enriched VHH sequence;

[0337] HCDR3 Liabilitie.

[0338] 1.3. Binding of the selected individual VHH to GnRH through ELISA and SPR method

[0339] The aim of the experiment was to determine the dissociation rate constant (kd, off-rate) of P.E. containing soluble VHH antibodies to GnRH Bio-peptide 1 captured on a SA sensor chip

[0340] The VHH antibody panel exhibited a wide range of EC50 to GnRH Bio-peptide 1, ranging from 415 pM to 14 nM. Clone pGnRH-001 presented the lowest EC50 of the panel and clone pGnRH-019 presented the highest.

[0341] In order to determine the affinity of the GnRH antibody panel and the SMI41 reference antibody, the GnRH bio-peptide 1 and bio-peptide 2 were immobilized on a SA chip.

[0342] SMI41, on SPR, only binds GnRH Bio-peptide 1 confirming what was previously seen in ELISA. The following kinetic parameters were obtained:

[0343] - ka (l / Ms) : 4.76E +06

[0344] - kd (l / s) : 2.81E-03 KD (nM) : 0.59

[0345] On the other hand, all clones from the antibody panel were able to bind to both GnRH Biopeptide 1 and GnRH Bio-peptide 2, when captured on a SA-chip

[0346] The VHH antibody panel exhibited a wide range of affinities to GnRH Bio-peptide 1 and GnRH Bio-peptide 2, ranging from ~7 nM to 125 nM.

[0347] Clone 1 presented the highest affinity of the panel to both GnRH Bio-peptide 1 (13.5 nM) and GnRH Bio-peptide 2 (7.1 nM)

[0348] 1.4. Binding of the selected individual VHH to native porcine GnRH

[0349] The aim of the experiment was to determine the ability of the GnRH VHH antibody panel binding to native GnRH protein, by ELISA.

[0350] A plurality of the lead VHH clones previous mentioned were tested, and the corresponding titration graphs are disclosed in figure 5.

[0351] All the VHH panel successfully bound the native porcine GnRH protein at the highest concentrations (30 pM), when determined as 3 times the GFP VHH OD value at the highest concentration. On the other hand, SMI41 monoclonal antibody does not readily bind the native porcine GnRH protein. The polyclonal antibody correctly binds to native GnRH (as a positive control). This demonstrates a particular mechanism of binding in the selected ligands, which is not present in the SMI41 monoclonal antibody reported in the Art.

[0352] Example 2 : Antagonist activity of VHH against GnRH

[0353] A cell-based GnRH functional assay was established, based on five selected VHH clones, including clone 1, clone 2, clone 9, clone 22 and clone 24. The selection critera included:

[0354] Lowest EC50;

[0355] Higher affinity (lower KD) and slowest off-rate (lower kd)

[0356] Fast on-rate (Higher ka)

[0357] Production yields. The antagonist activity of those five purified VHH was verified by expressing the human GnRH receptor in rat basophil leukemia cells. Their effect on agonist-induced cytosolic Ca2+ ion mobilization was measured using a fluorimetric detection method

[0358] The results were expressed as a percent inhibition of the control response to 8 nM GnRH.

[0359] Cetrorelix acetate was used as the standard reference antagonist, tested at several concentrations to generate a concentration-response curve from which its IC50 value was calculated, and then reported in figure 6.

[0360] All top five VHH clones were able to inhibit GnRH on the cell-based functional assay. Clones 9 and 1 presented the best profile, with clone 9 exhibiting a better profile than the reference antibody, SMI41.

[0361] Example 3: Alanine scanning screening of selected VHH clones

[0362] VHH clones 1 and 9 (SEQ ID N°1 and 9, respectively) were further screened by Alanine scanning (ELISA) in order to determine key residues involved in GnRH binding.

[0363] Accordingly, neutravidin (Thermo Fisher Scientific, Cat nr. 31000) was coated in a Maxisorp plate at, 5 pg / ml in IxPBS, 4°C, overnight. The coated plate plate was blocked with 1% casein in lx PBS. The GnRH Bio-Peptide 1 (SB-Peptide, Lot nr. 10869) of sequence SEQ ID N° 155 was captured at 5 nM in 0.1% casein in lx PBS. The clone 1 and clone 9, and their corresponding variants wherein the CDR3 hypervariable region is substituted with a sequence SEQ ID N°159 to 186, were titrated by performing 7-steps, five-fold serial dilutions, starting at a 1 pM in 0.1% casein in lx PBS. VHH were detected with an anti-c-myc antibody (Roche, Cat. nr. 11 667 203 001) at 1:200 dilution in 0.1% casein in lx PBS, followed by anti-mouse IgG-HRP (JIR, Cat. nr. 715-035-150) at 0.16 pg / mL 0.1% casein in lx PBS. Binding to GnRH Bio-Peptide 1 was assessed by measuring optical density at 450 nm.

[0364] The corresponding results were obtained with clone 1 and its tested CDR3 variants.

[0365]

[0366] Table 1 - Alanine scanning on clone 1 and CDR3 variants.

[0367] The corresponding results were obtained with clone 9 and its tested CDR3 variants.

[0368] Table 2 - Alanine scanning on clone 9 and CDR3 variants. Mutations were further introduced, distinct from alanines, in selected positions within the CDR3 of clone 1. Surprisingly, substitution of the last “D” residue with a histidine (H) led to improved affinity toward the antigen. Table 3 - tested mutations in Clone 1.

[0369] In view of the above, the two following CDR3 consensus regions, were obtained, of sequence

[0370] SEQ ID N° 187 and 188:

[0371] Example 4: Liabilities of several clones of interest

[0372] In order to assess their liability, antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022 andpGnRH-024 were tested more particularly for their physical and chemical stability after storage at different temperatures.

[0373] Each antibody composition was subjected to temperature variations to assess its stability, in particular:

[0374] - in one test condition, the samples were submitted to 5 rounds of Freeze / Thaw cycles (5x FT); - in other test conditions, the samples were stored at room temperature (25 °C) for 2 weeks, and 40°C for 2 weeks.

[0375] Physical stability was evaluated by measuring concentration, turbidity (OD500) and SE-HPLC before and after stress testing. Chemical stability was evaluated by analysis of the samples on RP-HPLC before and after stress testing. In addition, HIC (Hydrophobic Interaction Chromatography) analysis was performed on the non-stressed samples to rank the molecules based on their hydrophobicity.

[0376] Concentration and turbidity

[0377] Were measured using

[0378] Instrument: UV / Vis spectrophotometer (Unchained Labs Lunatic)

[0379] Plate: Lunatic plate (2 pL, 0.5 mm path, measuring range 0.03-40 OD; 10 mm

[0380] SE-HPLC

[0381] The samples were centrifuged for 5 min at 14000 g before injection. Samples were analyzed on an Agilent 1100 HPLC system. Instrument control and data analysis was performed with Chemstation for LC systems (rev. B.04.03-SP1).

[0382] RP-HPLC

[0383] Samples were analyzed on an Agilent 1200 HPLC system. Instrument control and data analysis was performed with Chemstation for LC systems (rev. B.04.03-SP1).

[0384] HIC

[0385] Samples were analyzed on an Agilent 1100 HPLC system. Instrument control and data analysis was performed with Chemstation for LC systems (rev. B.04.03-SP1).

[0386] Results are presented in Figure 7 A (representing the area of the HPLC pic corresponding to the antibody analysed), and Figure 7AB (representing the % of the main HPLC pic corresponding to the antibody analysed). The results of the analysis of the stability of the antibodies are summarized in the table illustrated in Figure 14.

[0387] Based on the data obtained, it was concluded that: - all antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022 andpGnRH-024 are stable when submitted to 5 Freeze / Thaw cycles;

[0388] - all antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022 andpGnRH-024 are stable when stored for 2 weeks at 25 °C;

[0389] - when stored for 2 weeks at 40°C

[0390] - antibody samples from pGnRH-009 and pGnRH-024 showed some protein loss;

[0391] - antibody samples from pGnRH-001, pGnRH-002 and pGnRH-022 showed an increase in HMW (High Molecular Weight species) typically a hallmark of antibody degradation;

[0392] - antibody samples from pGnRH-001, pGnRH-002 and pGnRH-022 showed an increase of single variant of more than 5% on RP-HPLC. of some clones of interest

[0393] It was proceeded with sequence optimization on antibodies produced by clones pGnRH-001 and pGnRH-009, which were considered as of interest.

[0394] With respect to clone pGnRH-001, 4 different sequence variants, namely OlTml (which sequence corresponds to SEQ ID NO: 205), 01Tm2 (which sequence corresponds to SEQ ID NO: 206), 01Tm3 (which sequence corresponds to SEQ ID NO: 207), 01Tm4 (which sequence corresponds to SEQ ID NO: 208). were produced. The sequences of these variants are illustrated in Figure 8 A, where the amino-acid residues which were mutated, compared to the sequence of original clone pGnRH-001, are pointed to by arrows.

[0395] With respect to clone pGnRH-009, 2 different sequence variants, namely 09Tml (which sequence corresponds to SEQ ID NO: 209), 09Tm2 (which sequence corresponds to SEQ ID NO: 210), were produced. The sequences of these variants are illustrated in Figure 8 B, where the amino-acid residues which were mutated, compared to the sequence of original clone pGnRH-009, are pointed to by arrows.

[0396] Example 6: Binding to GnRH (Biopeptides 1 and 2) of sequences variants from clones pGnRH-001 and pGnRH-009.

[0397] ELISA assays against Bio-peptides 1 and 2, as described in the material and methods section, were used to assess the binding properties of antibodies from Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4, 09Tml, 09Tm2, (sequence variants) of clones pGnRH-001 and pGnRH-009 respectively, compare them with that of the antibodies from the original clones, with the aim to identify if the sequence mutations (substitutions) that were included to remove physical and chemical liabilities have any impact on clone binding ability to GnRH peptides.

[0398] The set up of the ELISA assays was as follows:

[0399] Neutravidin (Thermo Fisher Scientific, Cat nr. 31000) was coated in a Maxisorp plate at, 5 pg / ml in IxPBS, 4°C, O / N

[0400] Coated plate was blocked with 1% casein in lx PBS

[0401] GnRH Bio-Peptide 1 (SB-Peptide, Lot nr. 10869) and Bio-Peptide 2 (SB-Peptide, Lot nr. 10870) were captured at 5 nM in 0.1% casein in lx PBS

[0402] The pGnRH_001 and pGnRH_009 VHH Tm variants, were titrated by performing 7- steps, five-fold serial dilutions, starting at a 1 pM in 0.1% casein in lx PBS

[0403] VHH were detected with an anti- VHH antibody (JIR, Cat. nr. 128-005-230) at 1:1000 dilution in 0.1% casein in lx PBS, followed by anti-goat IgG-HRP (JIR, Cat. nr. 705-035-147) at 1:10000 dilution 0.1% casein in lx PBS

[0404] Coating control was performed with Anti-GnRH polyclonal antibody at 1 pg / mL in 0.1% casein in lx PBS, followed by an anti-rabbit IgG-HRP (JIR, Cat. nr. 111-035-144) at 1:10000 dilution in 0.1% casein in lx PBS

[0405] Binding to GnRH Bio-Peptide 1 was assessed by measuring O.D. at 450 nm.

[0406] The results are presented in Figure 9 (sequence variants OlTml, 01Tm2, 01Tm3, 01Tm4 of clone pGnRH-001), Table 4, Figure 10 (sequence variants 09Tml, 09Tm2 of clone pGnRH- 009) and Table 5.

[0407] Table 4 - Binding properties to Bio-peptide 1 and 2 of OlTml, 01Tm2, 01Tm3, 01Tm4.

[0408] Table 5 - Binding properties to Bio-peptide 1 and 2 of 09Tm 1, 09Tm2.

[0409] As shown in Figure 9 and Table 4, sequence optimization leading to sequence variants OlTml, 01Tm2, 01Tm3, 01Tm4 of clone pGnRH-001 allowed for an improvement of the binding to all of these variants for Bio-peptide 1.

[0410] As shown in Figure 10 and Table 5, sequence optimization leading to sequence variants 09Tml, 09Tm2 of clone pGnRH-009 allowed for an improvement of the affinity of all of these variants for Bio-peptide 1.

[0411] Example 7: Liabilities and stability of sequences variants from clones pGnRH-001 and pGnRH-009.

[0412] The Tm variants OlTml, 01Tm2, 01Tm3, 01Tm4 of clone pGnRH-001 and Tm variants 09Tml, 09Tm2 of clone pGnRH-009 were expressed in E. Coli, purified via Ni-NTA affinity chromatography, and formulated in PBS.

[0413] The physical and chemical stability was tested based on the same protocols as detailed in Example 4. In particular, each antibody composition was subjected to temperature variations to assess its stability, more precisely:

[0414] - in one test condition, the samples were submitted to 5 rounds of freeze / thawn steps (5x FT);

[0415] - in other test conditions, the samples were stored at room temperature (here 25°C) for 2 weeks, and 40°C for 2 weeks. For all these conditions, the antibody concentration was measured (results are illustrated in Figure 11A), to assess possible protein loss. In parallel, the turbidity was measured at O.D. 500 nm for all samples and conditions (results are illustrated in Figure 11B).

[0416] The samples were submitted to RP-HPLC, the total area for each pic was measured, the increase in high molecular weight species was measured. The results are illustrated in Figure 12.

[0417] For the purpose of these experiments, the antibodies were renamed as follows in Table 6.

[0418] Table 6 - Table of correspondence for references and names used in the example part with respect to the variants herein disclosed. The results of the analysis of the stability of the antibodies are summarized in the table illustrated in Figure 15.

[0419] Based on the data obtained, it was concluded that:

[0420] - all antibodies produced by clones pGnRHTm-001, pGnRHTm-002, pGnRHTm-003, pGnRHTm-00, pGnRHTm-005 and pGnRHTm-006 are stable when submitted to 5 Freeze / Thaw cycles; - all antibodies produced by clones pGnRHTm-001, pGnRHTm-002, pGnRHTm-003, pGnRHTm-00, pGnRHTm-005 and pGnRHTm-006 are stable when stored for 2 weeks at 25°C;

[0421] - when stored for 2 weeks at 40°C

[0422] - antibody samples from pGnRHTm-002, pGnRHTm-003, pGnRHTm-004 and pGnRHTm-005 showed some increase in High Molecular Weight species, however this increase was less important than that observed in similar conditions for the original clones 1 and 9;

[0423] - antibody sample from pGnRHTm-001 and pGnRHTm-003 showed an increase of single variant of more than 4% and 5% respectively on RP-HPLC.

[0424] Example 8: Antagonist activity of variants GnRH 001 Tm002 and GnRH 009 Tm005

[0425] The antagonist activity of antibodies produced by clones GnRH_001_Tm002 and GnRH_009_Tm005 at the human GnRH receptor expressed in rat basophil leukemia cells, was determined by measuring their effect on agonist-induced cytosolic Ca2+ion mobilization using a fluorimetric detection method.

[0426] The set up for this experiment was as follows:

[0427] - rat basophil leukemia cells, resuspended in HBSS buffer (Invitrogen), were distributed in a microplate at a density of 1.186E+04 cells / well;

[0428] - the fluorescent probe (Fluo8 Direct, AAT Bioquest) mixed with probenicid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Invitrogen, pH 7.4) was then added into each well and equilibrated with the cells for 60 min at 30°C;

[0429] - the most promising mutated versions of GnRH_001 and GnRH_009 monovalent VHH, GnRH-001-Tm002 and GnRH-009-Tm005 were, added to the cells and incubated for 5 min

[0430] The SMI41 positive control was tested at 100, 20, 4, 0.8 and 0.16 pg / mL;

[0431] E. coli productions of GnRH_001 and GnRH_009 monovalent VHH were also included as controls;

[0432] - 8nM of GnRH was then added to the cells and the measurements of changes in fluorescence intensity which varies proportionally to the free cytosolic Ca2+ion concentration;

[0433] - the results were expressed as a percent inhibition of the control response to 8 nM GnRH; - cetrorelix acetate was used as the standard reference antagonist, tested at several concentrations to generate a concentration-response curve from which its IC50 value was calculated.

[0434] The results are illustrated in Figure 13 and in Table 7 below.

[0435] Table 7 - IC50 of Tm variants and original clones.

[0436] Based on these results, it could be concluded that:

[0437] GnRH_001_Tm002 candidate was shown to have a similar profile to GnRH_001 suggesting that introduced mutations were able to ameliorate the chemical liabilities and stability of the parental clone, without affecting its functional activity;

[0438] GnRH_009 candidate produced for this very experiment exhibited a different profile and IC50 than the antibody obtained during Lead Identification stage ;

[0439] GnRH_009_Tm005 candidate was shown to have a similar profile to GnRH_009 suggesting that introduced mutations were able to ameliorate the chemical liabilities and stability of the parental clone, without affecting its functional activity.

[0440] Overall, the data confirms the benefit of the mutations implemented in the Tm variants, in particular in the sequences of GnRH_001_Tm002 and GnRH_009_Tm005, in terms of physical and chemical stability, while keeping their capabilities on GnRH binding and antagonistic activity of the antibodies.

[0441]

Claims

CLAIMS1. An isolated monoclonal anti-Gonadotropin-Releasing Hormone (anti-GnRH) single domain antibody (SdAb), or antigen-binding fragment thereof; wherein said antibody or antigen-binding fragment binds specifically to, both, a N-terminal region (pGlu-His-Trp) of native GnRH, and a C-terminal region (Pro-Gly-NH2) of native GnRH.

2. An isolated monoclonal anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody, or antigen-binding fragment thereof; wherein said antibody or antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of: a CDR3 polypeptide sequence SEQ ID NO 33 or 188; a CDR3 polypeptide sequence SEQ ID NO 57 or 187; a CDR3 polypeptide sequence SEQ ID NO 36; a CDR3 polypeptide sequence SEQ ID NO 96; a CDR3 polypeptide sequence SEQ ID NO 48; a CDR3 polypeptide sequence SEQ ID NO 42, 75, 81, 90, 93; or a variant of said CDR3 polypeptide sequence having 1 or 2 substitutions.

3. The isolated antibody or antigen-binding fragment thereof according to claim 2, wherein said antibody is a single domain antibody (SdAb).

4. The isolated antibody or antigen-binding fragment thereof according to any of the preceding claims; characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence SEQ ID NO 57 or 187.

5. The isolated antibody or antigen-binding fragment thereof according to any of claims 1 or 2; characterized in that it comprises a variable domain having:- a CDR1 comprising a sequence selected from: SEQ ID NO 55 and 82;- a CDR2 comprising a sequence selected from: SEQ ID NO 56, 98, 116 and 199;- a CDR3 comprising a sequence SEQ ID NO 57.

6. The isolated antibody or antigen-binding fragment thereof according to any of claims 1 or 2; characterized in that it comprises a variable domain having:- a CDR1 comprising a sequence selected from: SEQ ID NO 31 and 195;a CDR2 comprising a sequence selected from: SEQ ID NO 32; a CDR3 comprising a sequence SEQ ID NO 33.

7. The isolated antibody or antigen-binding fragment thereof according to any of claims 1 or 2; characterized in that it comprises a variable domain having:- a CDR1 comprising a sequence selected from: SEQ ID NO 34, a CDR2 comprising a sequence selected from: SEQ ID NO 35, a CDR3 comprising a sequence SEQ ID NO 36; or- a CDR1 comprising a sequence selected from: SEQ ID NO 94, a CDR2 comprising a sequence selected from: SEQ ID NO 95, a CDR3 comprising a sequence SEQ ID NO 96; or- a CDR1 comprising a sequence selected from: SEQ ID NO 46, a CDR2 comprising a sequence selected from: SEQ ID NO 47, a CDR3 comprising a sequence SEQ ID NO 48.

8. The isolated antibody or antigen-binding fragment thereof according to any of the preceding claims; wherein GnRH is selected in the group consisting of human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and feline GnRH.

9. A nucleic acid coding for an antibody or antigen-binding fragment thereof according to any of the preceding claims.

10. A vector comprising a nucleic acid according to the preceding claim.

11. A host cell comprising the nucleic acid according to claim 9, or the vector according to claim 10.

12. A pharmaceutical composition comprising: the isolated antibody or antigen-binding fragment thereof according to any of claims 1 to 8, or the nucleic acid according to claim 9, or the vector according to claim 10, or the host cell according to claim 11; and a pharmaceutically acceptable excipient.

13. The isolated antibody or antigen-binding fragment thereof according to any of claims 1 to 8, or the nucleic acid according to claim 9, or the vector according to claim 10, or the host cell according to claim 11, or pharmaceutical composition according to claim 12; for use a medicament, or for use in a method of diagnosis in vivo.

14. An in vitro method for detecting Gonadotropin-Releasing Hormone (GnRH), comprising the steps of: a) providing a sample, in particular a biological sample or a fraction thereof;b) bringing in contact the sample with an isolated antibody or antigen-binding fragment thereof, according to any of claims 1 to 8.

15. A method for isolating an anti-Gonadotropin-Releasing Hormone (anti-GnRH) antibody, or antigen-binding fragment thereof; comprising the steps of: a) providing a library of single domain antibodies (SdAb), or fragments thereof; b) subjecting the library to antigen- affinity selection, the selected SbAbs or fragments thereof being characterized for having specificity toward, both, the N-terminal region (pGlu-His-Trp) of native GnRH and the C-terminal region (Pro-Gly-NH2) of native GnRH. c)

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