GnRH binding polypeptides and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of GnRH antagonists and anti-GnRH ligands.

[0002] The present invention also relates to the fields of pharmacy and diagnostics. In particular, the present disclosure relates to the field of contraception and GnRH-related diseases. BACKGROUND

[0003] Various methods of contraception are practiced in the art, especially for livestock, including pigs.

[0004] One long-practiced method for pigs is to perform a live castration of piglets in the absence of any anesthesia. However, this long-practiced method has been deemed unethical as it causes unacceptable pain, suffering and distress to the piglets.

[0005] Animal contraception methods known in the art also include surgical contraception under anesthesia (e.g., tubal ligation or vasectomy), use of hormone-containing compositions, which include hormone-containing implants (e.g., GnRH analog-containing implants), insertion of an intrauterine device, direct injection of a chemical that destroys reproductive tissue into the testes, and methods for immunological contraception.

[0006] GnRH-binding ligands, in particular monoclonal antibodies, have been reported in the art. Known methods of immunological contraception include administration of a vaccine composition intended to immunize a subject against endogenous GnRH.

[0007] Specifically, GnRH vaccines stimulate antibody production to inactivate endogenous GnRH, which in turn causes a reduction in gonadotropin release, resulting in atrophy of the gonads in adult animals or underdevelopment in sexually immature animals.

[0008] This resulting atrophy or underdevelopment of the reproductive organs is known as immunological sterilization.

[0009] Due to their role as hormones, GnRH, GnRH analogs and GnRH-binding ligands have further been involved as therapeutic agents in a number of other medical conditions and clinical uses, either as GnRH agonists or as GnRH antagonists, including the treatment or prevention of diseases beyond infertility, especially sex hormone-dependent diseases, such as delayed puberty, ovarian hyperstimulation syndrome, precocious puberty, premenstrual syndrome, endometriosis, uterine leiomyoma, polycystic ovary syndrome, hirsutism, acne vulgaris, acute intermittent porphyria, breast cancer, uterine cancer, uterine fibrosis, endometrial cancer, prostate cancer, benign prostatic hyperplasia, pancreatic cancer, etc.

[0010] Silversides et al. ("Monoclonal antibodies against LHRH: development and immunoactivity in vivo and in vitro"; Journal of Reproductive Immunology, 1985) reported monoclonal antibodies from dog immunization trials, which were directed against GnRH / LHRH.

[0011] 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) reported an antibody preparation capable of inhibiting the progress of estrus in dogs.

[0012] Li et al. ("Vectored antibody gene delivery mediates long-term contraception"; Current Biology, 2015) and US 10,570,200 B2 teach antibody-mediated immunization for contraception, which includes directed AAV-dependent anti-GnRH antibody expression.

[0013] A purified monoclonal mouse IgGl kappa antibody specific for the C-terminal peptide of GnRH, known as clone number SMI41 (Absolute Antibody Ref. n° Ab00922-1.1) has been reported for use in immunocytochemistry and expressed in mice by injection of replication-incompetent recombinant adeno-associated virus to confer long-term infertility.

[0014] CEVA VALORA TM A GnRH / LHRH-based synthetic peptide vaccine for pigs is reported, which comprises three synthetic peptides as immunogens.

[0015] IMPROVAC TM A GnRH analogue-protein conjugate is reported, which is capable of stimulating the immune system of pigs to produce antibodies against GnRH for the control of sexual development.

[0016] Chang et al. (“Effects of a recombinant Gonadotropin-Releasing Hormone Vaccine on Reproductive Function in Adult Male ICR Mice”; Vaccines, 2021) report on a vaccine preparation comprising a multimeric fusion protein including multiple copies of a GnRH I decapeptide and a T-cell epitope.

[0017] Gonadotropin-releasing hormone (GnRH) (also known as LHRH) is a key, hypothalamic and highly conserved hormone in mammals, secreted by the pituitary, and acts as the master reproductive hormone by regulating the release of two major gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). GnRH has been extensively studied for the development of immunization vaccines for various mammalian species, including pigs, rodents, cats, dogs, and wild carnivores.

[0018] The amino acid sequence of GnRH is highly conserved among phylogenetically related species; it is typically 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 as a reference in mammals: (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 152).

[0019] The N-terminal region of native GnRH (pGlu-His-Trp) is characterized by a blocked amino-terminal pyroglutamic acid residue (pGlu), while the C-terminal region of native GnRH (Arg-Pro-Gly-NH2) is characterized by an amidated carboxy-terminal end.

[0020] In its folded conformation, both the N-terminal and C-terminal regions of GnRH are involved in receptor binding, although only the N-terminal seems to be involved in receptor activation.

[0021] The development of anti-GnRH ligands, especially in the context of immunization vaccines, is challenging for several reasons:

[0022] (i) GnRH is a small decapeptide with very low antigenicity;

[0023] (ii) Despite its small size, the folded conformation of mammalian GnRH has led to the identification of at least two key regions for receptor binding, with only the N-terminal region responsible for receptor activation;

[0024] (iii) It exists naturally in the body.

[0025] Therefore, it is recognized by the immune system as an "autoprotein," which elicits a weak and transient antibody response after administration to animals and includes numerous conformational epitopes.

[0026] Therefore, there is still a need in the art for novel ligands for GnRH, or alternatively, for the identification of ligands for novel epitopes of GnRH.

[0027] There is still a need in the field for ligands and formulations for GnRH that remain stable and compatible with large-scale production.

[0028] In particular, there is still a need in the art for ligands targeting GnRH that can be used as pharmaceuticals and / or for detection or diagnostic purposes.

[0029] In particular, there is still a need in the field for GnRH antagonists to treat diseases, especially sex hormone-related and / or GnRH-related conditions.

[0030] There is still a need for contraceptive methods in this field, especially for livestock.

[0031] In particular, there remains a need in the field for contraceptive methods that are easy to administer and simultaneously reversible and immediately effective.

[0032] The purpose of this disclosure is to meet these needs. Invention Overview

[0034] The first principal embodiment of the present invention relates to an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of natural GnRH.

[0035] A second principal embodiment of the present invention relates to an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of:

[0036] - CDR3 polypeptide sequence SEQ ID NO: 57 or 187;

[0037] - CDR3 polypeptide sequence SEQ ID NO: 33 or 188;

[0038] - CDR3 polypeptide sequence SEQ ID NO: 36;

[0039] - CDR3 polypeptide sequence SEQ ID NO: 96;

[0040] - CDR3 polypeptide sequence SEQ ID NO: 48;

[0041] - CDR3 polypeptide sequence SEQ ID NO: 42, 75, 81, 90, 93; or

[0042] - A variant of the CDR3 polypeptide sequence with one or two substitutions.

[0043] According to another main embodiment, the present invention relates to nucleic acids that encode antibodies or antigen-binding fragments thereof according to the present invention.

[0044] According to another main embodiment, the present invention relates to a vector comprising a nucleic acid according to the invention.

[0045] According to another main embodiment, the present invention relates to host cells containing nucleic acids or vectors according to the invention; in particular, eukaryotic cells containing nucleic acids or vectors according to the invention. Such host cells can be isolated by any method known in the art.

[0046] According to another principal embodiment, the present invention relates to a pharmaceutical composition comprising:

[0047] - An isolated antibody or its antigen-binding fragment, or nucleic acid, or vector, or host cell according to the invention; and

[0048] - Pharmaceutically acceptable excipients.

[0049] According to another main embodiment, the present invention relates to an in vitro method for detecting gonadotropin-releasing hormone (GnRH), comprising the following steps:

[0050] a) Provide samples, especially biological samples or fractions thereof;

[0051] b) Contact the sample with the isolated antibody or its antigen-binding fragment according to the invention.

[0052] According to another principal embodiment, the present invention relates to a method for isolating an anti-gonadotropin-releasing hormone (anti-GnRH) antibody or an antigen-binding fragment thereof, comprising the following steps:

[0053] a) Provide a library of single-domain antibodies (SdAbs) or their antigen-binding fragments;

[0054] b) Subject the library to antigen affinity selection, wherein the selected SdAb or fragment thereof is characterized as being specific for both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH. Attached Figure Description

[0055] Figure 1 Graphical representation of biotinylated biopeptide 1 (A) and biopeptide 2 (B) used in ELISA and SPR. Ten highlighted amino acids are representative of the native GnRH peptide in mammals (SEQ ID NO: 152).

[0056] Figure 2 Graphical representation of ELISA quality control results for directly coated or captured biopeptides. The figure illustrates the optical density (OD) vs. log concentrations of biopeptides 1 and 2 at 450 nm, where a nonlinear regression curve is fitted using four parameters: log (agonist) vs. response. A) as a function of directly coated protein in μg / ml on the x-axis. B) as a function of captured protein in nM on the x-axis.

[0057] Figure 3 Using bioactive peptide 1 derived from thirty SdAbs (V H H) ELISA results of the purified lead and reference SMI41 antibody. The corresponding optical density (OD) at 450 nm is provided on the y-axis. The corresponding nM concentration of VHH or alternatively SMI41 is provided on the x-axis. Biopeptide 1 is coated on all plates.

[0058] Figure 4 Using bioactive peptide 1 and bioactive peptide 2 from thirty SdAbs (V H H) SPR results for purified lead and reference SMI41 antibody. For each graph, relative units (RU) are provided on the y-axis over time (s). A. On the left plate: This depicts the SPR signal from the SMI41 (IgG1) antibody in the presence of coated biopeptide 1. On the right plate: This depicts the SPR signal from the SMI41 (IgG1) antibody in the presence of coated biopeptide 2. B. On the left plate: This depicts the SPR signal from each V in the presence of coated biopeptide 1. H SPR signaling of the H-lead antibody. On the right-hand plate: This depicts the SPR signaling from each V in the presence of the coated biopeptide 2. H SPR signaling of H-lead antibody.

[0059] Figure 5. Titration using coated GnRH and antibody via ELISA at the selected VH The H library binds to native GnRH. The figure illustrates the optical density (OD) at 450 nm versus the concentrations (μM) of the following at 450 nm: A, V. H H antibody clones 1 to 12; B.V H H antibody clones 13 to 24; C. V H H antibody clones 25 to 36. D. Using polyclonal and monoclonal V. H Negative control experiment of H vs. SMI41.

[0060] Figure 6 When expressed in rat basophilic leukemia cells, the five V... H The ability of the purified lead compound to antagonize the human GnRH receptor was evaluated. The percentage of inhibition was assessed on the y-axis as a percentage for each tested V. H H is a function of the concentration of the reference SMI41 antibody. A) The concentration of each antibody is expressed in μg / mL. B) The concentration of each antibody is expressed in μM.

[0061] Figure 7 Antibody titers were assessed using reversed-phase high-performance liquid chromatography (RP-HPLC). Samples of antibodies derived from clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 were subjected to five freeze-thaw cycles or stored at room temperature (25°C) or 40°C for two weeks. T0 represents the control conditions and corresponds to the original sample before storage. A. Total peak area corresponding to the antibody from the reference clone, as measured by RP-HPLC. B. Main peak corresponding to the antibody from the reference clone, as measured by RP-HPLC.

[0062] Figure 8. Sequences of Tm variants. A. Sequences of Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 (sequence variants) of cloned pGnRH-001, with arrows pointing to the permutations made in the sequence variants. B. Sequences of Tm variants 09Tm1 and 09Tm2 (sequence variants) of cloned pGnRH-009, with arrows pointing to the permutations made in the sequence variants.

[0063] Figure 9ELISA assays for bioactive peptides 1 and 2. The binding properties of antibodies against Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 (sequence variants) of clone pGnRH-001 were evaluated using ELISA assays for bioactive peptides 1 and 2. A. ELISA assays for bioactive peptide 1 using Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4. The corresponding optical density (OD) at 450 nm is provided on the y-axis. V is provided on the x-axis. H The corresponding nM concentration of H. B. ELISA assays for bioactive peptide 2 using Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4. The corresponding optical density (OD) at 450 nm is provided on the y-axis. V is provided on the x-axis. H The corresponding nM concentration of H.

[0064] Figure 10 ELISA assays for bioactive peptides 1 and 2. The binding properties of antibodies against Tm variants 09Tm1 and 09Tm2 (sequence variants) from clone pGnRH-009 were evaluated using ELISA assays for bioactive peptides 1 and 2. A. ELISA assays for bioactive peptide 1 using Tm variants 09Tm1 and 09Tm2. The corresponding optical density (OD) at 450 nm is provided on the y-axis. V is provided on the x-axis. H The corresponding nM concentration of H. B. ELISA assay for bioactive peptide 2 using Tm variants 09Tm1 and 09Tm2. The corresponding optical density (OD) at 450 nm is provided on the y-axis. V is provided on the x-axis. H The corresponding nM concentration of H.

[0065] Figure 11 Characterization of Tm variant stability. Samples containing antibodies generated from clones pGnRH-001, Tm variant 01Tm2 of clone pGnRH-001, clone pGnRH-009, and Tm variant 09Tm1 of clone pGnRH-009 were subjected to 5 freeze / thaw cycles (5x FT) or stored at room temperature (25°C) or 40°C for 2 weeks. T0 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 concentrations of the samples are provided in mg / mL on the y-axis for all samples and conditions specified on the x-axis. B. Turbidity measured at OD 500 nm is provided on the y-axis for all samples and conditions specified on the x-axis.

[0066] Figure 12Characterization of Tm variant stability. Samples of antibodies generated from clones pGnRH-001, the Tm variant 01Tm2 of clone pGnRH-001 (GnRH-001-Tm002), pGnRH-009, and the Tm variant 09Tm1 of clone pGnRH-009 (GnRH-009-Tm005) were subjected to 5 freeze / thaw cycles (5x FT) or stored at room temperature (25°C) or 40°C for 2 weeks. T0 represents the control condition and corresponds to the antibody titer of the original sample before storage. Samples were subjected to RP-HPLC. A. The total peak area corresponding to the antibody from the clone in the reference is indicated on the y-axis in milliabsorbance units per second (mAU*s), as measured by RP-HPLC, for all samples and conditions indicated on the x-axis. B. The y-axis indicates the percentage increase in high molecular weight species in samples from clones of the reference, as measured by RP-HPLC, for all samples and conditions indicated on the x-axis.

[0067] Figure 13 Antagonistic activity of Tm variants. Antagonistic activity against GnRH of antibodies derived from clones pGnRH-001, the Tm variant 01Tm2 (GnRH-001-Tm002) of pGnRH-001, pGnRH-009, and the Tm variant 09Tm1 (GnRH-009-Tm005) of pGnRH-009. A. Inhibition was assessed on the y-axis as per test V. H H is a function of the concentration of the reference SMI41 antibody. The concentration of each antibody is expressed in nM. B. The percentage of inhibition was assessed on the y-axis as a function of the concentration of each tested antibody. H H is a function of the concentration of the reference SMI41 antibody. The concentration of each antibody is expressed in μg / ml.

[0068] Figure 14 Susceptibility of the tested top clones. A summary of the results obtained regarding the physical and chemical stability of clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 is provided in detail in Example 4.

[0069] Figure 15 Susceptibility to all Tm variants. A summary of the results obtained regarding the physical and chemical stability of the Tm variants, namely the Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 of clone pGnRH-001 and the Tm variants 09Tm1 and 09Tm2 of clone pGnRH-009, is provided in detail in Example 7. Invention Details

[0071] The inventors hereby provide anti-GnRH antibodies and their antigen-binding fragments. Surprisingly, the inventors disclose that such anti-GnRH antibodies have the ability to target both the N-terminal and C-terminal regions of natural GnRH, both of which are involved in receptor binding.

[0072] Given that existing anti-GnRH antibodies reported in the prior art do not share the same properties, this binding mechanism is atypical.

[0073] The inventors further provided in vitro and in vivo evidence showing that the selected single-domain antibodies (which share the same binding mechanism) were sufficient to antagonize human GnRH receptor activity in rat basophilic leukemia cells.

[0074] Not wanting to be bound by theory, the inventors believe that the binding mechanism, which depends on the two terminal regions of natural GnRH, is the reason for its biological effects.

[0075] The inventors further disclosed a method for isolating monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) ligands that is particularly selective for other ligands with the same binding mechanism.

[0076] Unwilling to be bound by theory, the inventors believe that, as a new class of ligands, libraries of single-domain antibodies (SdAbs) are particularly convenient for selecting antibodies with atypical binding mechanisms to natural GnRH.

[0077] Experimental data focus on single-domain antibodies (SdAbs) (especially in the heavy chain (V) H H) The separation of a single variable domain on an antibody (also known in the art as a nanobody), although the provided highly variable domain can also serve as a means of identifying and / or modifying non-V antibodies. H The basis of VHH, conventional, bispecific and trispecific 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).

[0078] The following anti-GnRH antibodies were specifically considered as isolates (e.g., V). H H) antibodies, their antigen-binding fragments, or their hypervariable regions.

[0079] Clone 1 corresponds to the V sequence of SEQ ID NO: 1. HThe H antibody contains CDR1, CDR2, and CDR3 hypervariable regions, respectively, of sequences SEQ ID NO: 31, 32, and 33.

[0080] Clone 2 corresponds to the V of sequence SEQ ID NO: 2. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 34, 35 and 36 respectively.

[0081] Clone 3 corresponds to the V of sequence SEQ ID NO: 3. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 37, 38 and 39 respectively.

[0082] Clone 4 corresponds to the V of sequence SEQ ID NO: 4. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 40, 41 and 42 respectively.

[0083] Clone 5 corresponds to the V of sequence SEQ ID NO: 5. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 43, 44 and 45 respectively.

[0084] Clone 6 corresponds to the V of sequence SEQ ID NO: 6. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 46, 47 and 48 respectively.

[0085] Clone 7 corresponds to the V of sequence SEQ ID NO: 7. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 49, 50 and 51 respectively.

[0086] Clone 8 corresponds to the V of sequence SEQ ID NO: 8. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 52, 53 and 54 respectively.

[0087] Clone 9 corresponds to the V of sequence SEQ ID NO: 9. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 55, 56 and 57 respectively.

[0088] Clone 10 corresponds to the V of sequence SEQ ID NO: 10. HH antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 58, 59 and 60 respectively.

[0089] Clone 11 corresponds to the V of sequence SEQ ID NO: 11. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 61, 62 and 63 respectively.

[0090] Clone 12 corresponds to the V of sequence SEQ ID NO: 12. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 64, 65 and 66 respectively.

[0091] Clone 13 corresponds to the V of sequence SEQ ID NO: 13. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 67, 68 and 69 respectively.

[0092] Clone 14 corresponds to the V of sequence SEQ ID NO: 14. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 70, 71 and 72 respectively.

[0093] Clone 15 corresponds to the V sequence of SEQ ID NO: 15. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 73, 74 and 75 respectively.

[0094] Clone 16 corresponds to the V of sequence SEQ ID NO: 16. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 76, 77 and 78 respectively.

[0095] Clone 17 corresponds to the V of sequence SEQ ID NO: 17. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 79, 80 and 81 respectively.

[0096] Clone 18 corresponds to the V of sequence SEQ ID NO: 18. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 82, 83 and 84 respectively.

[0097] Clone 19 corresponds to the V of sequence SEQ ID NO: 19. HH antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 85, 86 and 87 respectively.

[0098] Clone 20 corresponds to the V of sequence SEQ ID NO: 20. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 88, 89 and 90 respectively.

[0099] Clone 21 corresponds to the V of sequence SEQ ID NO: 21. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 91, 92 and 93 respectively.

[0100] Clone 22 corresponds to the V of sequence SEQ ID NO: 22. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 94, 95 and 96 respectively.

[0101] Clone 23 corresponds to the V sequence of SEQ ID NO: 23. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 97, 98 and 99 respectively.

[0102] Clone 24 corresponds to the V of sequence SEQ ID NO: 24. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 100, 101 and 102 respectively.

[0103] Clone 25 corresponds to the V sequence of SEQ ID NO: 25. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 103, 104 and 105 respectively.

[0104] Clone 26 corresponds to the V of sequence SEQ ID NO: 26. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 106, 107 and 108 respectively.

[0105] Clone 27 corresponds to the V of sequence SEQ ID NO: 27. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 109, 110 and 111 respectively.

[0106] Clone 28 corresponds to the V of sequence SEQ ID NO: 28.H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 112, 113 and 114 respectively.

[0107] Clone 29 corresponds to the V of sequence SEQ ID NO: 29. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 115, 116 and 117 respectively.

[0108] Clone 30 corresponds to the V of sequence SEQ ID NO: 30. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 118, 119 and 120 respectively.

[0109] 01Tm1 corresponds to the V sequence in SEQ ID NO: 189. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 195, 196 and 197 respectively.

[0110] 01Tm2 corresponds to the V sequence in SEQ ID NO: 190. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 31, 32 and 33 respectively.

[0111] 01Tm3 corresponds to the V sequence in SEQ ID NO: 191. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 31, 32 and 33 respectively.

[0112] 01Tm4 corresponds to the V sequence in SEQ ID NO: 192. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 31, 32 and 33 respectively.

[0113] 09Tm1 corresponds to the V sequence in SEQ ID NO: 193. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 55, 56 and 57 respectively.

[0114] 09Tm2 corresponds to the V sequence in SEQ ID NO: 194. H H antibody, which contains CDR1, CDR2 and CDR3 hypervariable regions of SEQ ID NO: 198, 199 and 200 respectively.

[0115] In the context of this invention, redundant sequences listed in SEQ ID NO: 34 to 120 as part of different antibody or antigen-binding fragments can be used interchangeably from one equivalent CDR to another. For example, this could be the case with respect to CDR1 sequences SEQ ID NO: 31, 34 and 37 or CDR2 sequences SEQ ID NO: 32 and 35 (which are shared among several of the clones mentioned above).

[0116] Therefore, this invention covers, but is not limited to, all of the V mentioned above. H H clones, and / or combinations of their respective CDR1, CDR2 and CDR3 hypervariable regions; and their uses.

[0117] Therefore, a first principal embodiment of the present invention relates to an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds to both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of natural GnRH.

[0118] According to a particular embodiment, the isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or its antigen-binding fragment is characterized in that it specifically binds to a first polypeptide sequence consisting of SEQ ID NO: 154 and a second polypeptide sequence consisting of SEQ ID NO: 153.

[0119] According to a second principal embodiment, the present invention relates to isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibodies or antigen-binding fragments thereof, wherein the antibody or antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of:

[0120] - CDR3 polypeptide sequence SEQ ID NO: 57 or 187;

[0121] - CDR3 polypeptide sequence SEQ ID NO: 33 or 188;

[0122] - CDR3 polypeptide sequence SEQ ID NO: 36;

[0123] - CDR3 polypeptide sequence SEQ ID NO: 96;

[0124] - CDR3 polypeptide sequence SEQ ID NO: 48;

[0125] - CDR3 polypeptide sequence SEQ ID NO: 42, 75, 81, 90, 93; or

[0126] - A variant of the CDR3 polypeptide sequence with one or two substitutions.

[0127] According to a particular embodiment, when applicable, the variant of the CDR3 polypeptide sequence has one or two conserved substitutions.

[0128] According to specific and exemplary embodiments, the isolated antibody or its antigen-binding fragment is a single-domain antibody (SdAb).

[0129] Therefore, according to a particular embodiment, the isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment is a single-domain antibody (SdAb); the antibody or antigen-binding fragment contains a variable domain having a complementarity-determining region 3 (CDR3) selected from the group defined above.

[0130] According to a particular embodiment, the isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment is combined with a first polypeptide of sequence SEQ ID NO: 153 and a second polypeptide of sequence SEQ ID NO: 154.

[0131] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable domain having a complementarity determination region 3 (CDR3) comprising sequences selected from the following: SEQ ID NO: 57, 187, 33, 188, 36 and 96.

[0132] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment 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.

[0133] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment 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:

[0134] DX1 X2 X3 X4 G X5 Y Y X6 P D X7

[0135] Where X1 to X7 independently represent any amino acid,

[0136] For example, X1 is R.

[0137] X2 is, for example, G.

[0138] For example, X3 is P.

[0139] X4 is, for example, S.

[0140] X6 is, for example, Y.

[0141] X7 is, for example, Y.

[0142] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence as defined above (SEQ ID NO: 187).

[0143] Where X1 is R,

[0144] X2 is G,

[0145] X3 is P,

[0146] X4 is S.

[0147] X6 is Y,

[0148] X7 is Y.

[0149] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment 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.

[0150] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment 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:

[0151] DX1X2YYX3X4X5X6X7PX8

[0152] Where X1 to X8 independently represent any amino acid,

[0153] For example, X1 is D.

[0154] For example, X2 is A.

[0155] For example, X3 is P.

[0156] X4 is, for example, G.

[0157] X8 is preferably Q or H.

[0158] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence as defined above SEQ ID NO: 188:

[0159] Where X1 is D,

[0160] X2 is A.

[0161] X3 is P,

[0162] X4 is G.

[0163] X8 is either Q or H.

[0164] According to other particular embodiments, the isolated antibody or its antigen-binding fragment 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.

[0165] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0166] - CDR1, which contains sequences selected from SEQ ID NO: 55 and 82;

[0167] - CDR2, which contains sequences selected from SEQ ID NO: 56, 98, 116 and 199;

[0168] - CDR3, which contains the sequence SEQ ID NO: 187.

[0169] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0170] - CDR1, which contains sequences selected from SEQ ID NO: 55 and 82;

[0171] - CDR2, which contains sequences selected from SEQ ID NO: 56, 98, 116 and 199;

[0172] - CDR3, which contains the sequence SEQ ID NO: 57.

[0173] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0174] - CDR1, which contains sequences selected from SEQ ID NO: 31 and 195;

[0175] - CDR2, which contains a sequence selected from SEQ ID NO: 32;

[0176] - CDR3, which contains the sequence SEQ ID NO: 188.

[0177] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0178] - CDR1, which contains sequences selected from SEQ ID NO: 31 and 195;

[0179] - CDR2, which contains a sequence selected from SEQ ID NO: 32;

[0180] - CDR3, which contains the sequence SEQ ID NO: 33.

[0181] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0182] - CDR1, which contains a sequence selected from SEQ ID NO: 34;

[0183] - CDR2, which contains a sequence selected from SEQ ID NO: 35;

[0184] - CDR3, which contains the sequence SEQ ID NO: 36.

[0185] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0186] - CDR1, which contains a sequence selected from SEQ ID NO: 94;

[0187] - CDR2, which contains a sequence selected from SEQ ID NO: 95;

[0188] - CDR3, which contains the sequence SEQ ID NO: 96.

[0189] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0190] - CDR1, which contains a sequence selected from SEQ ID NO: 46;

[0191] - CDR2, which contains a sequence selected from SEQ ID NO: 47;

[0192] - CDR3, which contains the sequence SEQ ID NO: 48.

[0193] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0194] - CDR1, which contains a sequence selected from SEQ ID NO: 195;

[0195] - CDR2, which contains a sequence selected from SEQ ID NO: 196;

[0196] - CDR3, which contains the sequence SEQ ID NO: 197.

[0197] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a variable structural domain having:

[0198] - CDR1, which contains a sequence selected from SEQ ID NO: 198;

[0199] - CDR2, which contains a sequence selected from SEQ ID NO: 199;

[0200] - CDR3, which contains the sequence SEQ ID NO: 200.

[0201] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a framework region (FR) selected from the group consisting of: SEQ ID NO: 121 to 151, 201 to 204 or 211 to 214.

[0202] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a framework region 1 (FR1) selected from the group consisting of: SEQ ID NO: 121 to 137, 201 to 204 or 211 to 214.

[0203] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a framework region 2 (FR2) selected from the group consisting of: SEQ ID NO: 138 to 141.

[0204] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a framework region 3 (FR3) selected from the group consisting of: SEQ ID NO: 142 to 149.

[0205] According to a more particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it comprises a framework region 4 (FR4) selected from the group consisting of: SEQ ID NO: 150 or 151.

[0206] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it shares at least 80% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequences SEQ ID NO: 1 to 30, 189 to 194 or 205 to 210.

[0207] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it shares at least 90% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequences SEQ ID NO: 1 to 30, 189 to 194 or 205 to 210.

[0208] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it shares at least 95% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequences SEQ ID NO: 1 to 30, 189 to 194 or 205 to 210.

[0209] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it shares at least 99% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequences SEQ ID NO: 1 to 30, 189 to 194 or 205 to 210.

[0210] According to a particular embodiment, the isolated anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment according to the present invention is characterized in that the GnRH is selected from the group consisting of: human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and feline GnRH.

[0211] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it is a recombinant protein.

[0212] According to a particular embodiment, in addition to natural GnRH, the isolated antibody or its antigen-binding fragment according to the invention specifically binds to more than one polypeptide.

[0213] According to the specific embodiment described, the isolated antibody or its antigen-binding fragment can therefore be multispecific, such as bispecific or trispecific. In this regard, according to a more specific embodiment, the isolated antibody or its antigen-binding fragment can be composed of a bispecific single-domain antibody (SdAb), especially a multispecific V... H Composed of an H antibody or its antigen-binding fragment, characterized in that:

[0214] - It contains at least one antigen-binding fragment of the present invention, said antigen-binding fragment specifically binding to a first polypeptide as natural GnRH, and containing at least one of the hypervariable CDR3 regions disclosed herein;

[0215] - It contains at least one antigen-binding fragment that specifically binds to a second polypeptide (e.g., a polypeptide selected from albumin), and a V that specifically binds to albumin or a fragment thereof. H H antibody.

[0216] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it is conjugated (i.e., covalently linked to another compound or polypeptide). For example, the isolated antibody or its antigen-binding fragment according to the invention may be conjugated at the N-terminus or the C-terminus.

[0217] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the invention may be conjugated with a detectable reagent (e.g., a radioactive substance (e.g., a radionuclide), an enzyme, a fluorescent marker, or a cytotoxic drug marker).

[0218] According to a particular embodiment, the isolated antibody or its antigen-binding fragment according to the present invention can be conjugated with a polypeptide, said polypeptide being an albumin polypeptide or a fragment thereof.

[0219] According to other particular embodiments, the isolated antibody or its antigen-binding fragment according to the invention is characterized in that it is not conjugated.

[0220] According to a more specific, non-mutually exclusive embodiment, the antibody or its antigen-binding fragment according to the invention is characterized in that it is modified to increase its biological half-life. Various methods are possible.

[0221] When the antibody according to the invention is a single-domain antibody (SdAb) or its antigen-binding fragment (more particularly, V... HWhen H antibody (or its antigen-binding fragment) is used, it may advantageously comprise, or consist of, a single-domain antibody linked to, an immunoglobulin domain (e.g., an Fc portion (e.g., human Fc or non-human Fc)). The Fc portion may be useful for increasing the half-life and even generating the single-domain antibody of the present invention. For example, the Fc fragment may bind to serum proteins, thereby increasing the half-life of the single-domain antibody.

[0222] In one embodiment, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in Ward's U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody may be modified within the CH1 or CL region to include a salvage receptor-binding epitope of two loops of the CH2 domain derived from the Fc region of IgG, as described in Presta et al.'s U.S. Patent Nos. 5,869,046 and 6,121,022. Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for transferring maternal IgG to the fetus (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 contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following 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, for example, the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0223] Another modification of the antibody or antigen-binding fragment considered herein is PEGylation. Antibody or antigen-binding fragments can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, typically the antibody or a fragment thereof is reacted with polyethylene glycol (PEG) (e.g., a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups are attached to the antibody or antibody fragment. PEGylation can be performed by acylation or alkylation with a reactive PEG molecule (or a similar reactive water-soluble polymer). In some embodiments, the antibody to be PEGylated is an unglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this invention. See, for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0224] Another modification of the antibody or antigen-binding fragment contemplated by the present invention is a conjugate or protein fusion of at least the antigen-binding region of the antibody of the present invention with a serum protein (e.g., human serum albumin or a fragment thereof) to increase the half-life of the resulting molecule. Such a method is described, for example, in EP0322094 by Ballance et al. Another possibility is a fusion of at least the antigen-binding region of the antibody of the present invention with a protein capable of binding serum proteins (e.g., human serum albumin) to increase the half-life of the resulting molecule. Such a method is described, for example, in EP 0 486 525 by Nygren et al.

[0225] Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to extend the active lifespan and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). Another technique involves using hydroxyethyl starch (“HES”) derivatives linked to antibodies.

[0226] A further aspect of the invention relates to host cells that have been transfected, infected, or transformed with nucleic acids and / or vectors according to the invention.

[0227] Therefore, according to another main embodiment, the present invention relates to nucleic acids that encode antibodies or antigen-binding fragments thereof according to the present invention.

[0228] According to another main embodiment, the present invention relates to a vector comprising a nucleic acid according to the invention.

[0229] According to another main embodiment, the present invention relates to host cells containing nucleic acids or vectors according to the invention; in particular, eukaryotic cells containing nucleic acids or vectors according to the invention. Such host cells can be isolated by any method known in the art.

[0230] According to another principal embodiment, the present invention relates to a pharmaceutical composition comprising:

[0231] - An isolated antibody or its antigen-binding fragment, or nucleic acid, or vector, or host cell according to the invention; and

[0232] - Pharmaceutically acceptable excipients.

[0233] According to a particular embodiment, the isolated antibody or its antigen-binding fragment, or the nucleic acid, or the vector, or the host cell, or the pharmaceutical composition is used as a drug or for use in in vivo diagnostic methods.

[0234] Specifically, the isolated antibody or its antigen-binding fragment, or the nucleic acid, or the vector, or the host cell, or the pharmaceutical composition is used in a method for regulating fertility in humans or non-human mammals.

[0235] According to some specific embodiments, the isolated antibody or its antigen-binding fragment, or the nucleic acid, or the vector, or the host cell, or the pharmaceutical composition is used in a method for reducing or inhibiting fertility in humans or non-human mammals.

[0236] According to some specific embodiments, the isolated antibody or its antigen-binding fragment, or the nucleic acid, or the vector, or the host cell, or the pharmaceutical composition is used as a drug for uncastrated humans or uncastrated non-human mammals.

[0237] According to some specific embodiments, the isolated antibody or its antigen-binding fragment, or the nucleic acid, or the vector, or the host cell, or the pharmaceutical composition is used as a medicament for castrated humans or non-castrated non-human mammals.

[0238] In addition to sterilization (similar to surgical procedures), the antibodies and their antigen-binding fragments according to the invention are also suitable for mitigating the consequences of surgical castration (e.g., secondary effects), such as those selected from ovariectomized female animals with urinary incontinence and lymphoma due to LH overexpression (explained in Kutzler, 2020, Possible Relationship between Long-Term Adverse Health effects of Gonad Removing Surgical Sterilization and Luteinizing Hormone in Dogs, Animals).

[0239] In fact, GnRH is physiologically regulated by negative feedback from estrogen / progesterone. When female dogs are ovariectomized (sterilized), they do not produce estrogen / progesterone, so GnRH production becomes uncontrolled and overexpressed. This also induces LH overexpression, which is responsible for urinary incontinence and cancer, and can therefore be prevented or attenuated.

[0240] According to a particular embodiment, the isolated antibody or its antigen-binding fragment, or the nucleic acid, or the vector, or the host cell, or the pharmaceutical composition is used to treat or prevent hormone-related and / or GnRH-related conditions.

[0241] The use of isolated antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells or pharmaceutical compositions according to the present invention in the preparation of medicaments is further disclosed herein.

[0242] According to another main embodiment, the present invention relates to an in vitro method for detecting gonadotropin-releasing hormone (GnRH), comprising the following steps:

[0243] a) Provide samples, especially biological samples or fractions thereof;

[0244] b) Contact the sample with the isolated antibody or its antigen-binding fragment according to the invention.

[0245] According to a particular embodiment, the in vitro method for detecting gonadotropin-releasing hormone (GnRH) according to the present invention further includes the step of detecting the interaction between the isolated antibody or its antigen-binding fragment and GnRH in the sample, thereby detecting GnRH in the sample.

[0246] The sample can be any sample, such as any biological sample that readily contains GnRH, more particularly natural GnRH, such as human GnRH, swine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH and cat GnRH.

[0247] Biological samples that are likely to contain GnRH may include blood, plasma, serum, urine, amniotic fluid, tissue extracts, tissue fluid, in vitro cell culture supernatant, cell lysate or fractions thereof.

[0248] According to a particular embodiment, the in vitro method for detecting gonadotropin-releasing hormone (GnRH) according to the present invention further includes the step of comparing the amount of GnRH in the sample with a reference value.

[0249] According to another principal embodiment, the present invention relates to a method for isolating an anti-gonadotropin-releasing hormone (anti-GnRH) antibody or an antigen-binding fragment thereof, comprising the following steps:

[0250] a) Provide a library of single-domain antibodies (SdAbs) or their antigen-binding fragments;

[0251] b) Subject the library to antigen affinity selection, wherein the selected SdAb or fragment thereof is characterized as being specific for both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH.

[0252] According to a particular embodiment, the method for isolating anti-GnRH antibodies or their antigen-binding fragments is characterized in that the library contains V H H polypeptide or its antigen-binding fragment, or components thereof.

[0253] According to a particular embodiment, the method for separating anti-GnRH antibody or its antigen-binding fragment may be characterized in that the selected SbAb is selected for binding to a first polypeptide of sequence SEQ ID NO: 153 and a second polypeptide of sequence SEQ ID NO: 154.

[0254] According to a particular embodiment, the method for isolating anti-GnRH antibodies or antigen-binding fragments thereof is characterized in that the GnRH is selected from the group consisting of: human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH, and feline GnRH.

[0255] According to a particular embodiment, the method for isolating anti-GnRH antibodies or antigen-binding fragments thereof may be characterized in that it includes a plurality of steps consisting of subjecting the library to antigen affinity selection, wherein the selected SdAb or antigen-binding fragment is characterized as being specific for both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH.

[0256] Advantageously, one or more steps that subject the library to antigen affinity selection may additionally include a further step of separating peptides that have bound to a single-domain antibody (SdAb) or its antigen-binding fragment from unbound peptides.

[0257] According to a particular embodiment, the method for separating anti-GnRH antibodies or their antigen-binding fragments is characterized in that step b) includes the following sub-steps:

[0258] b1) Contact the single-domain antibody (SdAb) or a library of its antigen-binding fragment with one or more polypeptides composed of the N-terminal region (pGlu-His-Trp) of natural GnRH and the C-terminal region (Arg-Pro-Gly-NH2) of natural GnRH or analogues thereof.

[0259] b2) Subject the polypeptide to at least one washing step;

[0260] b3) By separating peptides bound to single-domain antibodies (SdAbs) or their antigen-binding fragments from unbound peptides via organic phase separation, candidate binding partners are separated from other library members.

[0261] According to some specific embodiments, the method for isolating anti-GnRH antibodies or their antigen-binding fragments may be characterized by comprising a first step of contacting a library of the single-domain antibody (SdAb) or its antigen-binding fragment with one or more polypeptides composed of the N-terminal region (pGlu-His-Trp) of natural GnRH or its analogues; and a subsequent (e.g., second) step of contacting the library of the single-domain antibody (SdAb) or its antigen-binding fragment with one or more polypeptides composed of the C-terminal region (Arg-Pro-Gly-NH2) of natural GnRH or its analogues.

[0262] According to some specific embodiments, the method for isolating anti-GnRH antibodies or their antigen-binding fragments may be characterized by comprising a first step of contacting a library of the single-domain antibody (SdAb) or its antigen-binding fragment with one or more polypeptides composed of the C-terminal region (Arg-Pro-Gly-NH2) of natural GnRH or its analogues; and a subsequent (e.g., second) step of contacting a library of the single-domain antibody (SdAb) or its antigen-binding fragment with one or more polypeptides composed of the N-terminal region (pGlu-His-Trp) of natural GnRH or its analogues.

[0263] According to a particular embodiment, the method for separating anti-GnRH antibodies or their antigen-binding fragments is characterized in that step b) includes the following sub-steps:

[0264] b1) Contact the single-domain antibody (SdAb) or a library of its antigen-binding fragment with one or more polypeptides composed of the N-terminal region (pGlu-His-Trp) of natural GnRH or its analogues.

[0265] b2) Subject the polypeptide to at least one washing step;

[0266] b3) By separating peptides bound to single-domain antibodies (SdAbs) or their antigen-binding fragments from unbound peptides via organic phase separation, candidate binding partners are separated from other library members.

[0267] b4) The candidate binding partner from the previous step is contacted with one or more peptides consisting of the C-terminal region (Arg-Pro-Gly-NH2) of natural GnRH or its analogues;

[0268] b5) subject the polypeptide to at least one washing step;

[0269] b6) By separating peptides bound to single-domain antibodies (SdAbs) or their antigen-binding fragments from unbound peptides via organic phase separation, candidate binding partners are separated from other library members.

[0270] According to a particular embodiment, the method for separating anti-GnRH antibodies or their antigen-binding fragments is characterized in that step b) includes the following sub-steps:

[0271] b1) Contact the single-domain antibody (SdAb) or a library of its antigen-binding fragment with one or more polypeptides composed of the C-terminal region (Arg-Pro-Gly-NH2) of natural GnRH or its analogues.

[0272] b2) Subject the polypeptide to at least one washing step;

[0273] b3) By separating peptides bound to single-domain antibodies (SdAbs) or their antigen-binding fragments from unbound peptides via organic phase separation, candidate binding partners are separated from other library members.

[0274] b4) The candidate binding partner from the previous step is contacted with one or more peptides consisting of the N-terminal region (pGlu-His-Trp) of natural GnRH or its analogues;

[0275] b5) subject the polypeptide to at least one washing step;

[0276] b6) By separating peptides bound to single-domain antibodies (SdAbs) or their antigen-binding fragments from unbound peptides via organic phase separation, candidate binding partners are separated from other library members.

[0277] A library of SdAb or its antigen-binding fragment may comprise or consist of a viral particle library containing multiple viral particles exhibiting multiple different fusion proteins on their surface, wherein each fusion protein contains at least a portion of an SdAb or its antigen-binding fragment conjugated to a filamentous phage capsid protein.

[0278] This document further discloses a method for regulating fertility in non-human animals (preferably non-human mammals, such as companion animals, livestock, exotic animals, wild animals, and zoo animals), comprising the step of administering an anti-GnRH antibody or an antigen-binding fragment thereof according to the invention to said animal.

[0279] In particular, the methods mentioned above are suitable for reducing or suppressing fertility in humans and non-human animals.

[0280] This document further discloses a method for modulating (preferably reducing or suppressing) the muttony odor in meat from an animal, comprising the step of administering an anti-GnRH antibody or an antigen-binding fragment thereof to the animal; the animal being, in particular, a pig, such as an uncastrated male pig.

[0281] This document further discloses a method for improving meat quality by reducing the gamey odor of boars, comprising the step of administering an anti-GnRH antibody or an antigen-binding fragment thereof to the animal; the animal being, in particular, a pig, such as an uncastrated male pig.

[0282] General definition

[0283] As used herein, the terms “GnRH” or “gonadotropin-releasing hormone” or “LHRH” or “luteinizing hormone-releasing hormone” are used interchangeably. While the primary antigen considered in this disclosure is mammalian GnRH, particularly human and non-human (e.g., livestock and / or porcine) GnRH, the term is also intended to include other non-mammal GnRH unless otherwise indicated. In particular, GnRH can be selected from the group consisting of: human GnRH, porcine GnRH, bovine GnRH, equine GnRH, sheep GnRH, canine GnRH, and feline GnRH. A polypeptide sequence of mammalian or porcine GnRH that can be used as a reference is characterized by the following structure:

[0284] (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 152).

[0285] As used herein, the term “N-terminal region of natural GnRH” refers to the closed amino-terminal region of natural GnRH, which includes pyroglutamic acid residues (pGlu) consisting of the following reference sequence: pGlu-His-Trp.

[0286] As used herein, the term “C-terminal region of natural GnRH” refers to the C-terminal region of natural GnRH, including the C-terminal amidated carboxyl terminus, which consists of the following reference sequence: Arg-Pro-Gly-NH2.

[0287] As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and will be used equivalently in this invention. The term "antibody" as used herein refers to immunoglobulin molecules and the immunologically active portion of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to antigens. Thus, the term "antibody" covers not only whole antibody molecules but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments. Therefore, unless otherwise stated, the term covers both conventional and unconventional antibodies, including single-domain antibodies, bispecific antibodies, multispecific antibodies, etc.

[0288] As used herein, the term “unconventional antibody” may specifically cover engineered antibodies, such as bispecific antibodies, and heavy chain antibodies, or single-domain antibodies, such as those selected from the VHH-type and those with single heavy chain variable domains characterized as VNAR-type antibodies.

[0289] 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 the type of antibody (which naturally lacks a light chain) that can be found, for example, in camel mammals and sharks.

[0290] As used in this article, VHH type single-domain antibodies are also referred to as VHH or V. H H or “nanobody®”. For a general description of (single) domain antibodies, see also the prior art cited above, as well as EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490, and WO 06 / 030220, WO 06 / 003388.

[0291] Camel immunoglobulins can be genetically engineered to produce small proteins with high affinity for targets, resulting in low molecular weight antibody-derived proteins known as "nanobodies" or "VHHs". See U.S. Patent No. 5,759,808, issued June 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279:1256-1261; Dumoulin, M. et al., 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 camel antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. In some embodiments herein, the camel antibodies or nanobodies are naturally generated in camels, i.e., produced by camels after immunization with GnRH or its peptide fragments, according to this disclosure, using the techniques described herein for other antibodies. Alternatively, the GnRH-binding camel nanobodies are engineered, i.e., generated using a GnRH-targeted panning procedure, for example, by selection from a phage library exhibiting appropriately mutagenic camel nanobody proteins.

[0292] As used herein, the term “VNAR” has its general meaning in the art and refers to the single-heavy variable domain of the type of antibody (which naturally lacks a light chain) that can be found in sharks. Ig-like molecules consisting only of IgNARs (immunoglobulin neoantigen receptors) heavy chains have been identified in all shark species studied to date. These are disulfide-bound homodimer molecules composed of two polypeptide chains containing five constant domains and a variable region (VNAR) through which they bind antigens (Greenberg et al., Nature 1995 Mar 9; 374(6518): 168-73). The VNAR is a small (12 kDa), stable, soluble monomeric antigen-binding domain that can be configured into many different therapeutic modalities. Various VNAR-based binding moieties have been isolated (see, for example, WO2003 / 014161 and WO2005 / 118629). They have elongated CDR3 structures that may extend into antigen cleavages and cavities.

[0293] The term "single-domain antibody" can further encompass unconventional antibodies having at least one of the single-domain antibodies of this invention (which specifically binds to GnRH) and at least one other binding unit (i.e., targeting another epitope, antigen, target, protein, or peptide) (which is also typically a single-domain antibody). Such peptides are referred to herein as "multispecific" peptides, in contrast to peptides containing the same single-domain antibody ("single-specific" peptides). Thus, in some embodiments, the peptides of this invention may also provide at least one further binding site targeting any desired protein, peptide, antigen, antigenic determinant, or epitope. This binding site may target the same protein, peptide, antigen, antigenic determinant, or epitope targeted by the single-domain antibody of this invention, or it may target a protein, peptide, antigen, antigenic determinant, or epitope different from the single-domain antibody of this invention.

[0294] Typically, the one or more further binding sites may comprise one or more portions, fragments, or domains of conventional chain antibodies (particularly human antibodies) and / or heavy chain antibodies. For example, the single-domain antibody of the present invention may be linked to a conventional (typically human) VH or VL, optionally via a linker sequence.

[0295] In some implementations, the term "single-domain antibody" may further encompass at least one single-domain antibody linked (optionally via a linker sequence) to one or more (typically mammalian-derived, or even human) CH1 and / or CH2 and / or CH3 domains.

[0296] For example, a single-domain antibody linked to a suitable CH1 domain can be used, for instance, with a suitable light chain to generate an antibody fragment / structure similar to a conventional Fab fragment or F(ab')2 fragment, but wherein one or both of the conventional VH domains (in the case of the F(ab')2 fragment) have been replaced by the single-domain antibody of the present invention. In some embodiments, one or more single-domain antibodies of the present invention can be linked (optionally via a suitable linker or hinge region) to one or more constant domains (e.g., two or three constant domains that can be used as part of an Fc moiety / for forming an Fc moiety), linked to an Fc moiety, and / or linked to one or more antibody moieties, fragments, or domains that confer one or more effector functions to the polypeptide of the present invention, and / or can confer the ability to bind to one or more Fc receptors. For example, and not 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 those from heavy chain antibodies, and more typically from conventional human chain antibodies; and / or may form an Fc region, such as those from IgG (e.g., from IgG1, IgG2, IgG3, or IgG4), from IgE, or from another human Ig, such as IgA, IgD, or IgM. For example, WO 94 / 04678 describes a heavy chain antibody (i.e., a single-domain antibody) comprising a camelid VHH domain or a humanized derivative thereof, wherein the camelid CH2 and / or CH3 domains have been replaced by human CH2 and CH3 domains to provide an immunoglobulin consisting of two heavy chains, each heavy chain comprising a single-domain antibody and human CH2 and CH3 domains (but without a CHI domain), the immunoglobulin having effector functions provided by the CH2 and CH3 domains, and the immunoglobulin being able to function in the absence of any light chains.

[0297] Typically, the polypeptide of the present invention comprises a single-domain antibody of the present invention, which is optionally fused to at least one further amino acid sequence at its N-terminus, its C-terminus, or both its N-terminus and its C-terminus, so as to provide a fusion protein.

[0298] As used herein, a polypeptide comprising a single single-domain antibody is referred to herein as a “monovalent” SdAb polypeptide. A polypeptide comprising two or more single-domain antibodies according to the invention, or substantially composed of them, is referred to herein as a “multivalent” SdAb polypeptide.

[0299] As used herein, the term "antigen-binding fragment" refers to a full-length antibody or one or more fragments of an antibody that retain the ability to bind specifically to GnRH. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered in the term "antigen-binding fragment" of an antibody include: Fab fragments, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; F(ab')2 fragments, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of the antibody; dAb fragments (Ward et al., 1989 Nature 341:544-546), consisting of a VH domain; or any fusion protein containing such antigen-binding fragments. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked together using recombination methods via synthetic linkers, resulting in single-chain proteins in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, 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" in the term antibody.

[0300] As used herein, the term "dsFv" refers to a VH::VL heterodimer stabilized by disulfide bonds. Divalent and multivalent antibody fragments can be spontaneously formed by association with monovalent scFvs, or generated by coupling monovalent scFvs, such as divalent sc(Fv)2, via peptide linkers. Such single-chain antibodies may contain one or more antigen-binding portions or fragments of the antibody.

[0301] Unibody antibodies are another type of antibody fragment that lacks the hinge region of IgG4 antibodies. The absence of the hinge region results in molecules that are essentially half the size of conventional IgG4 antibodies and have a monovalent binding region instead of the bivalent binding region of IgG4 antibodies. Antigen-binding fragments can be incorporated into single-domain antibodies, SMIPs, macrobodies, minibodies, intrabody antibodies, diabody antibodies, triabody antibodies, and tetrabody antibodies (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The terms "diabody," "triabody," or "tetrabody" refer to small antibody fragments with multiple antigen-binding sites (2, 3, or 4), contained within a single polypeptide chain (VH-VL) linked to a heavy chain variable domain (VH) connected to a light chain variable domain (VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of another chain, creating two antigen-binding sites. The antigen-binding fragment can be incorporated into a single-chain molecule containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062; and U.S. Patent No. 5,641,870).

[0302] These antibody fragments are obtained using conventional techniques known to those skilled in the art, and are used to screen the fragments in the same manner as intact antibodies.

[0303] As used herein, the term "variable" (as in "variable domain") refers to certain portions of a related binding protein that differ significantly in sequence between and within antibodies and are used in the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated in regions called complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) (also known as hypervariable regions), which are present in both the light and heavy chain variable domains in the context of conventional antibodies. More highly conserved portions of the variable domain are called framework (FR) regions or sequences.

[0304] As used herein, the terms "VH domain" or "V" are used in conjunction with other terms. H The term "domain" can be used interchangeably and refers to the corresponding variable domain of a heavy chain immunoglobulin.

[0305] As used herein, the term "VL domain" or "V" refers to a domain structure. L The term "domain" can be used interchangeably, and when applicable, it refers to the corresponding light chain immunoglobulin variable domain.

[0306] As used herein, the term "hypervariant region" refers to the amino acid residue of the antibody responsible for antigen binding. This term may be replaced by the terms "complementarity-determining region" or "CDR".

[0307] Therefore, as used herein, the "complementarity-determining region" or "CDR" refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of conventional immunoglobulins each have three CDRs, named CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively. Thus, the conventional antibody-antigen-binding domain comprises six CDRs, including a set of CDRs from each of the variable regions of the heavy and light chains.

[0308] The unconventional antibody antigen-binding domain of the VHH type contains three CDRs, which are also reported in this paper as CDR1, CDR2 and CDR3.

[0309] The unconventional antibody antigen-binding domain of the VNAR type contains two CDRs, which are also reported in this paper as CDR1 and CDR3, as well as CDR-like regions defined in the art as hypervariable region 2 (HV2) and hypervariable region 4 (HV4).

[0310] Furthermore, as used herein, “framework region” (FR) refers to the amino acid sequence inserted between CDRs, that is, those portions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species (i.e., the light chain and heavy chain variable regions in conventional antibodies, or only the heavy chain variable region for single-domain antibodies).

[0311] Immunoglobulins each have four FRs in their light and heavy chains, named FR-L1, FR-L2, FR-L3, FR-L4 and FR-H1, FR-H2, FR-H3, FR-H4, respectively. Therefore, in the conventional antibody-antigen binding domain, the variable domain of the light chain can be named (FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), and the variable domain of the heavy chain can be named (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H)-(FR4-H3).

[0312] VH The amino acid sequence and structure of a type H single-domain antibody can be considered to contain four framework regions, or "FRs," referred to in the art and herein as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4," respectively. These framework regions are interrupted by three complementarity-determining regions, or "CDRs" (referred to in the art as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively). Therefore, the single-domain antibody can be defined as having an amino acid sequence with the following general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3. In the context of this invention, the amino acid residues of the single-domain antibody are numbered according to the general numbering for the VH domain given by the international ImMunoGeneTics information system (http: / / imgt.cines.fr / ).

[0313] The VNAR protein scaffold consists of the following: amino acid residues (aa) 1-25 in the framework 1 (FW1) region; aa 26-32 in complementarity-determining region 1 (CDR1); aa 33-43 in FW2; aa 44-52 in the hypervariable 2 region (HV2); aa 53-85 in FW3; aa 61-65 in HV4; the CDR3 region (with variable length); and FW4 (11 residues starting at XGXG). Like all immunoglobulin family variable (V) domains, VNAR contains two canonical cysteine ​​residues linked to FW1 and FW3 via disulfide bonds.

[0314] Residues in the antibody variable domain are routinely numbered according to a system devised by Kabat et al. This system is described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used in this specification. Kabat residue naming does not always correspond directly to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than in a strict Kabat number, corresponding to shortening or insertion of structural components of the basic variable domain structure, whether framework regions or complementarity-determining regions (CDRs). For a given antibody, the correct Kabat number of the residues can be determined by comparing homologous residues in the antibody sequence with a "standard" Kabat-numbered sequence. For conventional antibodies, the CDR of the heavy chain variable domain is located at residues 31-35B (H-CDR1), 50-65 (H-CDR2), and 95-102 (H-CDR3) according to the Kabat numbering system. The CDR of the light chain variable domain is located at residues 24-34 (L-CDR1), 50-56 (L-CDR2), and 89-97 (L-CDR3) according to the Kabat numbering system. (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0315] The Kabat system can also be used to map antibodies that belong to unconventional antibody groups (e.g., V). H Approximate boundaries of CDRs within the antibody scaffold of H-type single-domain antibodies (SdAbs).

[0316] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen (e.g., native GnRH) while simultaneously exhibiting a relatively small detectable reactivity with non-antigen proteins or structures. Specificity can be determined relatively by binding or competitive binding assays using, for example, Biacore instruments, as described elsewhere herein. Specificity can be demonstrated by, for example, an affinity / affinity ratio of approximately 10:1, approximately 20:1, approximately 50:1, approximately 100:1, 10000:1, or greater, to binding to a specific antigen relative to non-specific binding to other unrelated molecules.

[0317] As used herein, the term "affinity" refers to the strength of an antibody's binding to an epitope (e.g., native GnRH). The affinity of an antibody is given by a dissociation constant Kd, defined as [Ab] × [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in the following references: Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entirety. A preferred and standard method well known in the art for determining the affinity of mAbs is the use of a Biacore instrument.

[0318] As used herein, the terms “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” “mAb,” etc., refer to antibody molecular preparations having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0319] As used herein, the term "humanized" refers to conventional or unconventional antibodies, such as VHH-type single-domain antibodies according to this disclosure, wherein the amino acid sequence corresponding to 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 the naturally occurring antibody sequence (particularly, in the framework sequence) with one or more amino acid residues appearing at corresponding positions in the VH domain of a conventional chain antibody derived from humans. Methods for humanizing single-domain antibodies are well known in the art. Typically, humanization substitutions should be selected so that the resulting humanized (e.g., conventional or single-domain) antibody still retains the advantageous properties of the anti-GnRH antibodies of the present invention. Those skilled in the art can identify and select suitable humanization substitutions or suitable combinations of humanization substitutions.

[0320] As used herein, the term “polyethylene glycol” is intended to cover any of the PEG forms used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.

[0321] As used herein, the term “treatment” is intended to include relief or elimination of a symptom, disease, or condition, or one or more symptoms associated with said symptom, disease, or condition; or relief or elimination of the cause of said symptom, disease, or condition itself.

[0322] As used herein, the term “prevention” means reducing the risk of or slowing the occurrence of a given phenomenon (i.e., in this invention, cancer and / or dysplasia, and more particularly, precancerous conditions, early-stage cancer, or non-metastatic cancer). The term “prevention” also encompasses “reducing the likelihood of occurrence” or “reducing the likelihood of recurrence.”

[0323] As used herein, the terms “subject” or “patient” may refer indiscriminately to all animals expressing GnRH or similar substances, including, in particular, humans and non-human mammals. Mammal species that may benefit from the disclosed treatments include, but are not limited to: humans, non-human primates (e.g., apes, chimpanzees, monkeys, and orangutans), domesticated animals (including dogs and cats), zoo animals, and livestock (e.g., horses, cattle, cows, pigs, sheep, and goats) or other mammal species (including, but not limited to, rodents such as mice and rats, guinea pigs, rabbits, hamsters, etc.).

[0324] As used herein, the term "sex hormone-related disorders" refers to sex hormone-related disorders in both males and females, including those specific to males and those specific to females. Such sex hormone-related disorders may or may not be directly associated with insufficient GnRH expression or function. In a non-exhaustive manner, this disclosure considers the following disorders: endometriosis, uterine fibroids, polycystic ovary syndrome, hirsutism, precocious puberty, delayed puberty, ovarian hyperstimulation syndrome, premenstrual syndrome, endometriosis, sleep apnea, irritable bowel syndrome, gonadal steroid-dependent tumors (e.g., pancreatic cancer, prostate cancer, breast cancer, uterine cancer, endometrial cancer, and ovarian cancer), pituitary gonadotropin-secreting adenomas, benign prostatic hyperplasia, uterine leiomyomas, acne vulgaris, acute intermittent porphyria, uterine fibrosis, benign prostatic hyperplasia, etc.

[0325] As used herein, “GnRH-related conditions” specifically refers to conditions that prevent the activation of GnRH receptors. Exemplary GnRH-related conditions include, but are not limited to: sex hormone-related conditions, sex hormone-dependent cancers, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, breast cancer, pituitary gonadotropin-secreting cell adenoma, endometriosis, polycystic ovary syndrome, uterine fibroids, primary hirsutism, luteinizing hormone surge, and precocious puberty.

[0326] As used herein, “pharmaceutically acceptable carrier” is intended to include any and all carriers compatible with pharmaceutical administration (particularly, parenteral administration) (e.g., any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc.). The use of such media and reagents for pharmaceutically active substances is known. Such media can be used in the compositions of this disclosure unless any conventional media or reagent is incompatible with the active compound. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., 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, 0.01–0.1 M (e.g., 0.05 M) phosphate buffer or 0.8% saline. Other common parenteral carriers include sodium phosphate solutions, Ringer's glucose solutions, glucose and sodium chloride solutions, lactated Ringer's solutions, or non-volatile oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's glucose solutions, etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. More particularly, pharmaceutical compositions suitable for injection include sterile aqueous solutions (when water-soluble) or dispersions, and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that it is readily injectable. It should be stable under the conditions of manufacture and storage and, in one embodiment, protected against contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size (in the case of dispersions), and by using surfactants. Antimicrobial activity can be achieved using various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In some embodiments, the composition contains isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0327] Unless the context clearly specifies otherwise, as used herein, the singular forms “a,” “an,” and “the” include plural references. For example, the term “pharmaceuticalally acceptable carrier” covers a variety of pharmaceutically acceptable carriers, including mixtures thereof.

[0328] Therefore, as used herein, “multiple (species)” can include “two (species)” or “two (species) or more (species)”.

[0329] As used herein, and unless otherwise indicated, the term "at least one (species)" may cover "one (species) or more (species)," or even "two (species) or more (species)" (or "more than one (species)"). For example, it may cover 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, 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, 5 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or greater than 100.

[0330] As used herein, and unless otherwise indicated, the term "less than..." may cover all values ​​from 0 to the corresponding threshold. For example, it may cover values ​​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, 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, 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, 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 (where applicable).

[0331] As used herein, the terms “comprising” or “including” may cover “consisting of”.

[0332] As used herein, the term "cell" may encompass any prokaryotic or eukaryotic cell. Cell types particularly considered are those suitable for the production and / or engineering of recombinant antibodies or their fragments or polypeptide chains. 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.

[0333] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the original transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to the parent cells in terms of nucleic acid content, but may contain mutations. Mutant progeny with the same function or biological activity as screened or selected in the original transformed cells are included herein. Host cells are any type of cell system that can be used to generate the binding proteins disclosed herein. Therefore, host cells may include cultured cells, such as cultured mammalian 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 just a few.

[0334] The term "transformation" refers to the introduction of a "foreign" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell, thereby causing the host cell to express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed." The nucleic acids of this invention can be used to produce the anti-GnRH antibody of this invention in a suitable expression system.

[0335] As used herein, the term "expression system" means a host cell and a compatible vector, under suitable conditions, for example, to express a protein encoded by foreign DNA carried by said vector and introduced into said 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, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., 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.), and primary or established mammalian cell cultures (e.g., derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in the dihydrofolate reductase gene (hereinafter referred to as the “DHFR gene”) (Urlaub G et al.; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as “YB2 / 0 cells”), and so on. The invention also relates to a method for producing recombinant host cells expressing antibodies according to the invention, the method comprising the steps of: (i) introducing the recombinant nucleic acid or vector described above into competent host cells in vitro or in vitro; (ii) culturing the obtained recombinant host cells in vitro or in vitro; and (iii) optionally, selecting cells that express and / or secrete the antibodies. Such recombinant host cells can be used to produce antibodies of the invention. The anti-GnRH antibody of the present invention can be appropriately separated from the culture medium by conventional immunoglobulin purification methods (e.g., protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0336] As used herein, the terms “vector,” “cloning vector,” and “expression vector” refer to a delivery vehicle through which a nucleic acid sequence (e.g., a DNA or RNA sequence (e.g., a foreign gene)) can be introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Typically, the nucleic acid is a DNA or RNA molecule that can be included in any suitable vector, such as a plasmid, granule, episome, artificial chromosome, bacteriophage, or viral vector.

[0337] Therefore, a further aspect of the invention relates to vectors containing the nucleic acids of the invention. Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to induce or direct the expression of said antibodies upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the early promoter and enhancer of SV40 (Mizukami T. et al., 1987), the LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al., 1987), and promoters and enhancers of the immunoglobulin H chain (Mason JO et al., 1985) and (Gillies SD et al., 1983), etc. Any expression vector for animal cells can be used, as long as it allows for the insertion and expression of a gene encoding the C region of a human antibody. 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), and pSG1 beta d2-4- (Miyaji H et al., 1990). Other examples of plasmids include replication plasmids containing the origin of replication, or integration plasmids such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenoviruses, retroviruses, herpesviruses, and AAV vectors. Such recombinant viruses can be generated using techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed experimental protocols for generating such replication-defective recombinant viruses can be found, for example, in the following documents: 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.

[0338] In particular, the term "vector" can refer to a viral particle (e.g., AAV type) that has a specific tissue tropism (e.g., muscle tropism), which may include, for example, AAV8 and AAV9.

[0339] In particular, the term "retroviral" or "retrovirus" can refer to lentivirus or lentiviral vector.

[0340] As used herein, the terms “recombinant AAV,” “rAAV virion,” and “AAV particle” are defined as an infectious, replication-defective virus containing an AAV protein capsid encapsulating a target heteronucleotide sequence flanked by AAV ITRs. rAAV virions are generated in suitable host cells in which sequences specifying the AAV plasmid, AAV helper functions, and accessory functions have been introduced. In this way, the host cell is enabled to encode an AAV polypeptide required for packaging the AAV plasmid (containing the target recombinant nucleotide sequence) into infectious recombinant virion particles for subsequent gene delivery.

[0341] "Recombinant virus" refers to a virus that has undergone genetic modification (e.g., by adding or inserting heterologous nucleic acid constructs into the particle).

[0342] “AAV virion” refers to a complete viral particle, such as a wild-type (wt) AAV virus particle containing a linear single-stranded AAV nucleic acid genome associated with an AAV capsid protein shell. In this respect, any complementary single-stranded AAV nucleic acid molecule (e.g., “sense strand” or “antisense strand”) can be packaged into any AAV virion, and both strands are equally infectious. Therefore, without other limitations, the term can cover any AAV selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, AAV10, and rhesus monkey-derived serotypes (including AAVrh10), as well as mixtures thereof.

[0343] "Isolated nucleic acid molecule or polynucleotide" is intended to refer to a nucleic acid molecule, DNA, or RNA that has been removed from its natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or polynucleotides purified in solution (partially or substantially). Isolated polynucleotides include polynucleotide molecules contained in cells that typically contain said polynucleotide molecules, but which are present outside the chromosome or at a chromosomal location different from their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, in positive and negative strand forms, and in double strand forms. Isolated polynucleotides or nucleic acids according to this disclosure further include synthetically produced such molecules. Additionally, polynucleotides or nucleic acids may be or may contain regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0344] As used herein, the terms “isolated” or “purified” refer to those molecules that have been altered by humans in their natural state, that is, if such molecules exist in nature, then they have been altered and / or removed from their original environment.

[0345] As used herein, the “percentage of identity” between two nucleic acid or protein (e.g., GnRH-binding antibody) sequences means the percentage of identical nucleotide or amino acid residues between the two sequences to be compared, obtained after optimal alignment, said percentage being purely statistical, and the differences between the two sequences being randomly distributed along their length. Sequence comparisons are conventionally performed by comparing the sequences after optimal alignment, which can be done segmentally or by using an “alignment window.” In addition to manual comparison, the best alignment of sequences for comparison can be achieved by means of Smith and Waterman (1981)’s local homology algorithm, Neddleman and Wunsch (1970)’s local homology algorithm, Pearson and Lipman (1988)’s similarity search method, or by means of computer software that uses these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by means of comparison software BLAST NR or BLAST P).

[0346] The percentage of identity between two sequences is determined by comparing the two optimally aligned sequences, wherein the sequences to be compared may have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. The percentage of identity is calculated by determining the number of identical nucleotide or amino acid residues located 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 of identity between the two sequences.

[0347] As used herein, the term "amino acid" refers to a natural or non-natural amino acid, in its D and L stereoisomers, for chiral amino acids. It is understood to refer to both the amino acid and the corresponding amino acid residue, for example, present in a peptide structure. Natural and non-natural amino acids are well known in the art. Common natural amino acids include, but are not limited to: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Uncommon and non-natural amino acids include, but are not limited to: allylglycine, leucine, valine, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), high arginine (hArg), high lysine (hLys), 2-naphthylalanine (2-Nal), ornithine (Orn), and pentafluorophenylalanine.

[0348] As used herein, the term "modified form" for a given amino acid encompasses any equivalent, translated or post-translational modified amino acid or non-naturally occurring amino acid (including non-standard amino acids) arising from reactions at the amino group, carboxyl group, side chain functional group, or from the substitution of any hydrogen atom by a heteroatom. Therefore, modified amino acids can encompass amino acid derivatives arising from any of the following modifications: N-glycosylation, O-glycosylation, phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin conjugation, cysteine ​​oxidation, nitrosation, and ubiquitination.

[0349] As used herein and above, "equivalent amino acid" means an amino acid that can be used to replace (i.e., by conservative substitution) another amino acid in the peptide compound according to the invention without perceptible loss of function. Those skilled in the art will recognize equivalent amino acids. Substitution of similar amino acids is based on the relative similarity of side-chain substituents, such as with respect to size, charge, hydrophilicity, and hydrophobicity as described herein. When used after a list of individual amino acids, the phrase "or its equivalent amino acid" means one or more equivalents of the individual amino acids included in the list.

[0350] For example, conservative substitutions can be defined by substitutions within the amino acid classes reflected below:

[0351] Aliphatic residues I, L, V, and M;

[0352] Cycloalkenyl-related residues F, H, W, and Y;

[0353] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y;

[0354] Negatively charged residues D and E;

[0355] Polar residues C, D, E, H, K, N, Q, R, S, and T;

[0356] Positively charged residues H, K, and R;

[0357] Small residues A, C, D, G, N, P, S, T, and V;

[0358] Very small residues A, G, and S;

[0359] The residues involved in corner formation are A, C, D, E, G, H, K, N, Q, R, S, P, and T;

[0360] Flexible residues Q, T, K, S, G, P, D, E, and R.

[0361] As used herein, “phage display” and “bacterial phage display” are considered synonyms and refer to the technique of displaying variant peptides as fusion proteins with coat proteins on the surface of bacterial phage particles (Scott, JK, and Smith, GP (1990) Science 249:386). Sorting phage libraries of random mutants requires strategies for constructing and propagating large numbers of variants, procedures for affinity purification using target receptors, and means for evaluating the results of binding enrichment. Unless otherwise indicated, the term is not limited to a specific type of bacterial phage particle, a specific type of fusion protein and / or enrichment strategy, and / or a particular phage library. Example

[0362] A. Materials and Methods

[0363] Immunization

[0364] Single-domain antibodies were obtained from llamas immunized with six injections at two-week intervals. Eight days after the final booster, serum samples were collected to determine antibody titers against GnRH peptides by ELISA. In this ELISA, 96-well plates (Maxisorp; Nunc) were coated with two biotinylated GnRH peptides. After blocking and adding diluted serum samples, the presence of anti-GnRH antibodies was demonstrated using mouse anti-llama IgG (FJB, Cat. nr. FJ1203MAB01B09) and subsequently donkey anti-mouse IgG-HRP antibody (JIR, Cat. nr. 715-035-150).

[0365] Library Construction

[0366] RNA was extracted from PBMCs (400 ml each) of two immunized llamas. 40 μg of RNA was used for cDNA synthesis using random primers. The cDNA was amplified by primary PCR using unlabeled primers annealed at the leader sequence and the CH1 region of the hinge, followed by secondary PCR amplification introducing restriction endonuclease sites for cloning the VHH gene into the pDCL1 phage vector. The library was electroporated into TG1 *E. coli* cells, and the bacterial glycerol stock of the immunoglobulin was stored at -80°C.

[0367] Phage display selection

[0368] Will come from llamas V H Phage generation from the H library collection was used in three consecutive rounds of phage display selection using biotinylated GnRH peptide 1 (also referred to herein as "Biopeptide 1"). The first round of selection (Round I) was performed on biotinylated GnRH peptide 1 (at 5 nM), pre-captured on plates coated with neutral avidin (5 μg / mL, Thermo Fisher; Cat. nr. 31000). Non-specific phages were washed away (PBS buffer, pH 7.4), followed by phage-specific elution with trypsin (total elution). The second round of selection (Round II) was performed with a lower number of phages (1:5 of the phage volume used in Round I).

[0369] In general, the selection of Round II follows two different strategies.

[0370] According to one strategy, biotinylated GnRH peptide 1 is pre-captured on neutral avidin plates at 5 and 0.5 nM.

[0371] According to the second strategy, the same peptide was directly coated at 1 μg / mL.

[0372] Wash nonspecific phages (PBS buffer, pH 7.4), followed by elution with trypsin-specific phages (total elution).

[0373] Then, phages from the two Round II strategies were selected in a third round (Round III), where the number of phages was further reduced (1:10 of the phage volume used in the first round), and biotinylated GnRH peptide 1 was pre-captured on neutral avidin plates at 0.5 and 0.05 nM. Nonspecific phages were washed (PBS buffer, pH 7.4) followed by elution with trypsin-specific phages (total elution).

[0374] Serial dilutions of the eluted phage were performed and used to infect TG1 cells that were growing exponentially. The infected TG1 cells were plated on LBCarb 100Glu 2% plates and enrichment values ​​were calculated relative to the background (without antigen for selection).

[0375] In V H ELISA screening in the context of H-library collection selection

[0376] Individual clones from the outputs of the second and third rounds of selection conditions were picked into 96-well master plates and tested for binding to biotinylated GnRH peptides 1 and 2 (also referred to herein as "biotinylidene 2") via a combination ELISA as periplasmic extracts (PE).

[0377] For PE-binding ELISA, MaxiSorp was prepared at 4°C using 5 μg / ml neutral avidin (ThermoFisher; Cat. nr. 31000) diluted in PBS. TMHigh protein-binding capacity 96-well ELISA plates were coated overnight. The next day, the plates were washed three times with 0.05% PBS Tween (pH 7.4) and blocked for 1 hour at room temperature with 250 μL / well of 1% casein / PBS. After blocking, the plates were washed three times with 0.05% PBS Tween (pH 7.4) and incubated for 1 hour at room temperature with 100 μL / well of biotinylated GnRH peptide 1 or 2 (diluted at 5 nM in 0.1% casein / PBS (pH 7.4)). After capture, the plates were washed three times with 0.05% PBS Tween (pH 7.4) and incubated for 1 hour at RT with shaking in each well with 20 μL PE + 80 μL 0.1% casein / PBS (pH 7.4). The plate was washed three times with 0.05% PBS Tween (pH 7.4) and incubated for 1 hour at RT with shaking, containing 100 μl of anti-c-Myc antibody (Roche; Cat. nr. 11667203001) and subsequently the secondary antibody, donkey anti-mouse HRP (JIR; Cat. nr. 715-035-150) (in 0.1% casein / PBS (pH 7.4)). The plate was washed three times with 0.05% PBS Tween (pH 7.4) and the substrate solution (TMB solution) was added. The reaction was stopped with H2SO4, and the plate was read at 450 nm using a plate reader.

[0378] Screening by dissociation rate of surface plasmon resonance (SPR)

[0379] Individual clones from the second and third rounds of selection were picked into 96-well master plates, and the dissociation rate (kd) of these clones (tested as periplasmic extract (PE)) for biotinylated GnRH peptide 1 captured on a StrepAvidin (SA) sensor chip (Cytiva®, Cat. nr. BR100531) was determined by surface plasmon resonance (SPR) (Biacore 8K, GE Healthcare). Approximately 1140 to 1270 RU of biotinylated GnRH peptide 1 was immobilized onto the SA chip using a standard strepAvidin conjugation method.

[0380] QC for immobilization was performed using a commercial anti-GnRH antibody (SMI41, Absolute Antibody, Cat. nr. Ab00922-1.1) (diluted at 5 nM in HBS-EP pH 7.4 buffer). 1× HBS-EP pH 7.4 was used as the run buffer during binding kinetic measurements. 20 μl PE + 80 μl 1× HBS-EP pH 7.4 was injected at a rate of 30 μl / min for 2 minutes, followed by washing at a dissociation rate of 1 minute between injections. In each cycle, the dissociation rate was washed for 300 s after the last concentration injection. RU levels were restored to baseline after regeneration with two injections of 15 μL of 10-glycine at pH 1.5 between samples.

[0381] Data were analyzed using predefined evaluation methods for multi-cycle dynamics from Biacore Insight Evaluation Software. Sensor plots and a blank / unfixed reference flow cell were used as references. The dissociation constant was calculated using a 1:1 dissociation Langmuir combination model.

[0382] sequencing

[0383] The positive binding reagents were sent for sequencing. Based on their different HCDR3 sequences, the clones were classified into families.

[0384] Expression and purification of VHH candidate antibodies

[0385] A synthetic gene encoding the VHH variable domain with FLAG and His tags was purchased and cloned into the pET15b bacterial expression vector containing a periplasmic secretion leader sequence. *E. coli* strain BL21 DE3 was transformed and grown at 18°C ​​in ZYP-5052 self-inducing medium for 68 hours. The generated VHH antibody was captured from the clarified supernatant using Ni-NTA beads (Qiagen) on a gravity-feed column. The eluted antibody was buffer-exchanged to 1×PBS pH 7.4 and concentrated using a 10k cutoff rotary concentrator (Amicon, Cat. nr. UFC801096D). The purified VHH protein was analyzed by SDS-PAGE for the presence of the correct strand.

[0386] Dose-response ELISA using purified VHH

[0387] MaxiSorp was prepared at 4°C using 5 μg / ml neutral avidin (ThermoFisher; Cat.nr. 31000) diluted in PBS. TMHigh protein-binding capacity 96-well ELISA plates were coated overnight. The next day, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and blocked for 1 hour at room temperature with 250 μL / well of 1% casein / PBS. After blocking, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and incubated for 1 hour at room temperature with 100 μL / well of biotinylated GnRH peptide 1 (diluted at 5 nM in 0.1% casein / PBS (pH 7.4)). After capture, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and incubated with VHH in 0.1% casein / PBS (pH 7.4). The solution (pH 7.4) was diluted from 1000 to 0.01 nM in a 5-fold 7-step dilution and added to the biotinylated GnRH peptide 1 captured at RT and passed through a blocked ELISA well for 1 hour.

[0388] The plate was washed three times with 0.05% PBS Tween (pH 7.4) and incubated for 1 hour at RT with shaking, containing 100 μl of anti-c-Myc antibody (Roche; Cat. nr. 11667203001) and subsequently the secondary antibody donkey anti-mouse HRP (JIR; Cat. nr. 715-035-150) (in 0.1% casein / PBS (pH 7.4)). The plate was washed three times with 0.05% PBS Tween (pH 7.4) and substrate solution (TMB solution) was added. The reaction was stopped with H2SO4, and the plate was read at 450 nm using a plate reader. A graph of OD 450 nm value vs. log VHH concentration was plotted using a GraphPad Prism 7, with a nonlinear regression (curve fitting) of log (agonist) vs. response – variable slope (four parameters) applied, and EC50 was determined.

[0389] Affinity assay of purified VHH by Biacore

[0390] To assess the affinity of the selected purified clones for biotinylated GnRH peptides 1 and 2, the peptides were captured on a StrepAvidin (SA) sensor chip (Cytiva®, Cat.nr. BR100531) using a standard streptavidin conjugation method.

[0391] The binding kinetics of the selected single-domain antibody at pH 7.4 were determined using surface plasmon resonance (SPR) (Biacore T200, GE Healthcare). Approximately 1140 to 1270 RU of biotinylated GnRH peptides 1 and 2 were immobilized onto an SA chip. QC for immobilization was performed using a commercial anti-GnRH antibody (SMI41, Absolute Antibody, Cat. nr. Ab00922-1.1) (diluted at 5 nM in HBS-EP pH 7.4 buffer). 1× HBS-EP pH 7.4 was used as the run buffer during binding kinetic measurements. Purified VHH was injected at 100 nM in HBS-EP pH 7.4 at a rate of 10 μl / min for 5 minutes, followed by washing at a dissociation rate of 1 minute between injections. In each cycle, the dissociation rate was washed for 20 minutes after the last concentration injection. After regeneration with 15 μl of 1 M 10 Glycine at pH 1.5 between samples, RU levels were restored to baseline. Data were analyzed using the binding kinetics predefined evaluation method in Biacore InsightEvaluation Software Fitting. Sensor plots and blank / unimmobilized reference flow cell 1 were referenced. The 1:1 dissociation Langmuir binding model was used to calculate dissociation constants to determine the kinetic constants of antibody-antigen interactions, including association rate (ka), dissociation rate (kd), and affinity (KD).

[0392] Through the natural protein binding of ELISA

[0393] (Gly-OH10)-luteinizing hormone-releasing hormone (Sigma-Aldrich, Cat. nr. L8008) was coated overnight in Maxisorp plates at 4°C with 1 μg / ml in 1×PBS. The coated plates were blocked with 4% emulsion in 1×PBS. The GnRH VHH antibody group was titrated by performing a 7-step, 5-fold serial dilution (starting at 30 μM in 1% emulsion in 1×PBS).

[0394] VHH was detected using anti-c-myc HRP antibody (Roche, Cat. nr. 11 667 203 001) at a dilution of 1:200. This included titration with SMI41 monoclonal antibody (Absolute Antibody, Ab00922-1.1) and GnRH1 polyclonal antibody (Invitrogen, PA1-121). Subsequent titrations were performed using anti-mouse and anti-rabbit IgG-HRP (JIR, 715-035-150 and 111-035-144) at a dilution of 1:10,000.

[0395] The plates were developed using TMB solution (Invitrogen, 00-4201-56) followed by H2SO4 (Fisher Chemical, J / 8430 / 15).

[0396] The binding of natural porcine GnRH was evaluated by measuring the OD at 450 nm.

[0397] Cell-based GnRH function assay

[0398] To evaluate the antagonistic activity of the top 5 VHH clones, rat basophilic leukemia cells stably expressing the human GnRH receptor were used in a cell-based GnRH function assay. A fluorescence assay was used to detect the antagonistic activity of GnRH-dependent cytoplasmic calcium receptors. 2+ The VHH effect of ion migration was used to determine the inhibition function.

[0399] For this purpose, cells were suspended in HBSS buffer (Invitrogen) with 20 mM Hepes and distributed in microplates at a density of 1.186E+04 cells / well. A fluorescent probe (Fluo8 Direct, AATBioquest) was mixed with probenecid in HBSS buffer (Invitrogen, pH 7.4) supplemented with 20 mM Hepes (Invitrogen), added to each well, and equilibrated with the cells at 30°C for 60 min. Then, the five purified VHHs were titrated in a five-step, 5-fold serial dilution (starting at 100 μg / mL), added to the plates, and incubated for 5 min. Subsequently, 8 nM GnRH (1:1 to 1:800 GnRH / VHH ratio) was added to the plates, and changes in fluorescence intensity were measured using a microplate reader (FlipR Tetra, Molecular Device), relative to free cytoplasmic Ca2+. 2+Ion concentrations varied proportionally. The standard reference antagonist, cetrorexate acetate, was tested at several concentrations in each experiment, and IC50 values ​​were calculated. Commercial anti-GnRH antibodies (SMI41, Absolute Antibody, Cat. nr. Ab00922-1.1) at two different concentrations (100 and 20 μg / mL) were also included as comparators and positive controls.

[0400] Example 1: V H Binding of H to GnRH and comparison with reference antibody

[0401] 1.1. Binding of SMI41 reference antibody to GnRH-derived peptide

[0402] The SMI41 reference antibody corresponds to the mouse IgG1 monoclonal anti-human LHRH (anti-huLHRH) antibody (Cat. Nr. PA1-121) sold by Absolute Antibody, which has an expected molecular weight of 155 kDa.

[0403] The second reference polyclonal antibody preparation is a rabbit polyclonal anti-amphibian / mouse GnRH1 antibody (anti-GnRH1) (Cat. Nr. PA1-121) sold by ThermoFisher, which has a target molecular weight of 150 kDa.

[0404] The objective of this experiment was to perform ELISA QC on the two biotinylated GnRH peptides (Biopeptide 1 and Biopeptide 2) by directly coating them or capturing them with neutral avidin. Monoclonal antibody SMI41 and polyclonal antibody were used for peptide detection, and the presence of biotin was confirmed by ExtrAvidin-HRP.

[0405] Biopeptide 1 was detected with both anti-GnRH antibodies (SMI41 and a polyclonal antibody), either through direct coating or capture. For the anti-GnRH monoclonal antibody SMI41, a complete titration profile was further obtained. Direct coating of biopeptide 1 allowed for the determination of the EC50 value (0.037 μg / ml). No background signal was observed, and no specific binding was detected for the second control.

[0406] Biopeptide 2 was detected solely by anti-GnRH polyclonal antibody. It exhibits a complete titration profile, allowing for EC50 determination whether the peptide is directly coated or captured.

[0407] Given that the SMI41 antibody is the current reference antibody, this experiment shows that bioactive peptide 1 is the peptide with the most relevant epitope.

[0408] ExtrAvidin-HRP confirmed the presence of biotin on both peptides. No background signal was observed, and binding was not detected in the second control only.

[0409] In summary, it was found that the monoclonal antibody labeled SMI41 could not specifically bind to biopeptide 2 in the N-terminal region of natural GnRH.

[0410] On the other hand, the polyclonal antibody combines with both an analogue of the N-terminal region (biopeptide 2) and an analogue of the C-terminal region (biopeptide 1).

[0411] 1.2. V H The binding of H to GnRH-derived peptides and 30 related V H H-clone selection

[0412] Multiple V were identified by displaying four selected outputs from phages. H H clones bind with high hit rates to capture GnRH biopeptide 1.

[0413] Using clones selected from three rounds for GnRH biopeptide 1, PE binding ELISA was performed on GnRH biopeptide 1 and biopeptide 2 captured in plates coated with neutral avidin.

[0414] All four outputs of the phage display experimental protocol achieved very high hit rates (88-100%), which means that the selected V H H clones bind to biopeptide 1 and also to biopeptide 2.

[0415] Therefore, the objective of this experiment is to determine the content of soluble V. H The PE of the H antibody and the dissociation rate constant (kd, dissociation rate) of GnRH biopeptide 1 captured on the SA sensor chip.

[0416] Of the 368 clones tested, 346 showed specific association with GnRH biopeptide 1 (RU>10), and the kd values ​​varied from 3.47E-02 to 6.61E-04 (1 / s).

[0417] Twenty-two clones did not show specific association with GnRH biopeptide 1 or had RU levels below 10, therefore kd values ​​were not determined.

[0418] No binding of irrelevant or blank controls was observed, and no RU response was detected when using buffer (1×HBS-EP+ pH 7.4) as the sample.

[0419] Of the 347 sequenced clones, a total of 339 valid V clones were obtained.H H sequence (97.7%). 103 unique V sequences were identified. H H sequences, and eight of them are considered enriched sequences with 10 or more representative clones.

[0420] Twenty-four unique HCDR3 sequences were identified, and eight of them were considered HCDR3-enriched sequences with ten or more representative clones. These enriched HCDR3 sequences were represented in all four outputs, independent of the selection round.

[0421] Therefore, based on the following criteria, a total of 30 V were selected, generated, purified, and further characterized. H H clone:

[0422] - In PE-binding ELISA, it binds to both biopeptide 1 and biopeptide 2;

[0423] - For biopeptide 1, the dissociation rate constant (kd, dissociation rate, < 1E-02 1 / s);

[0424] - Enrichment of HCDR3 sequences;

[0425] - Enrichment of VHH sequences;

[0426] - HCDR3 susceptibility.

[0427] 1.3. Selected individual V values ​​using ELISA and SPR methods H H-GnRH combination

[0428] The objective of this experiment is to determine the content of soluble V. H The PE of the H antibody and the dissociation rate constant (kd, dissociation rate) of GnRH biopeptide 1 captured on the SA sensor chip.

[0429] V H The H antibody group exhibited a wide range of EC50 values ​​with GnRH biopeptide 1, varying from 415 pM to 14 nM. Clones pGnRH-001 showed the lowest EC50 for the group, while clones pGnRH-019 showed the highest EC50.

[0430] To determine the affinity between the GnRH antibody group and the SMI41 reference antibody, GnRH biopeptide 1 and biopeptide 2 were immobilized on an SA chip.

[0431] On SPR, SMI41 bound only to GnRH biopeptide 1, confirming what was previously observed in ELISA. The following kinetic parameters were obtained:

[0432] - ka (1 / Ms): 4.76E+06

[0433] - kd (1 / s): 2.81E-03

[0434] - KD (nM): 0.59.

[0435] On the other hand, when captured on the SA chip, all clones from the antibody group are able to bind to both GnRH biopeptide 1 and GnRH biopeptide 2.

[0436] The VHH antibody group exhibited a wide range of affinity for GnRH biopeptide 1 and GnRH biopeptide 2, varying from ~7 nM to 125 nM.

[0437] Clone 1 exhibited the highest affinity in the group for both GnRH biopeptide 1 (13.5 nM) and GnRH biopeptide 2 (7.1 nM).

[0438] 1.4. The selected single V H The combination of H with natural porcine GnRH

[0439] The goal of this experiment is to determine GnRH V using ELISA. H The ability of H antibody groups to bind to native GnRH proteins.

[0440] Several previously mentioned lead VHH clones were tested, and the corresponding titration plots are shown in Figure 5.

[0441] All VHH antibody groups successfully bound to native porcine GnRH protein at the highest concentration (30 μM), when measured at 3 times the GFP VHH OD value at the highest concentration. On the other hand, the SMI41 monoclonal antibody did not readily bind to native porcine GnRH protein. The polyclonal antibody bound correctly to native GnRH (as a positive control). This demonstrates a specific binding mechanism in the selected ligand, which is not present in SMI41 monoclonal antibodies reported in the prior art.

[0442] Example 2: V for GnRH H H antagonist activity

[0443] Based on five selected V H A cell-based GnRH function assay was established using H clones (including clone 1, clone 2, clone 9, clone 22, and clone 24). Selection criteria included:

[0444] - Minimum EC50;

[0445] - Higher affinity (lower KD) and slowest dissociation rate (lower KD).

[0446] - Faster binding rate (higher ka);

[0447] - Yield.

[0448] The five purified V antibodies were validated by expressing the human GnRH receptor in rat basophilic leukemia cells. H H antagonist activity. Their activity against agonist-induced cytoplasmic calcium was measured using a fluorescence assay. 2+ The effect of ion migration.

[0449] The results are expressed as the percentage of inhibition of the control response to 8 nM GnRH.

[0450] Cetrorexate acetate was used as a standard reference antagonist, and tests were conducted at several concentrations to generate concentration-response curves from which its IC50 value was calculated and then reported. Figure 6 middle.

[0451] All top five V H Both H clones were able to inhibit GnRH in cell-based functional assays. Clones 9 and 1 exhibited the best characterization profiles, with clone 9 showing superior characterization compared to the reference antibody SMI41.

[0452] Example 3: Alanine scanning screening of the selected VHH clones

[0453] VHH clones 1 and 9 (SEQ ID NO: 1 and 9, respectively) were further screened by alanine scanning (ELISA) to identify key residues involved in GnRH binding.

[0454] Therefore, neutral avidin (Thermo Fisher Scientific, Cat. nr. 31000) was coated overnight in Maxisorp plates at 4°C with 5 μg / ml in 1×PBS. The coated plates were blocked with 1% casein in 1×PBS. GnRH bioactive peptide 1 (SB-Peptide, Lot nr. 10869) with sequence SEQ ID NO: 155 was captured at 5 nM in 0.1% casein in 1×PBS. Clones 1 and 9, as well as their corresponding variants (where the CDR3 hypervariable region was replaced by sequences SEQ ID NO: 159 to 186), were titrated using a 7-step 5-fold serial dilution method, starting at 1 μM in 0.1% casein in 1×PBS. V was detected with anti-c-myc antibody (Roche, Cat. nr. 11 667 203 001) at a 1:200 dilution in 0.1% casein in 1×PBS, and subsequently with anti-mouse IgG-HRP (JIR, Cat. nr. 715-035-150) at a concentration of 0.1% casein in 1×PBS. H H. Binding to GnRH biopeptide 1 was evaluated by measuring the optical density at 450 nm.

[0455] The results were obtained using clone 1 and the CDR3 variant tested.

[0456] Table 1 – Alanine scans for clone 1 and CDR3 variants.

[0457] The results were obtained using clone 9 and the CDR3 variant tested.

[0458] Table 2 – Alanine scans for clone 9 and CDR3 variants.

[0459] A mutation different from alanine was further introduced at a selected position within the CDR3 of clone 1. Surprisingly, replacing the final "D" residue with histidine (H) resulted in improved affinity for the antigen.

[0460] Table 3 – Mutations tested in clone 1.

[0461] Based on the above, the following common CDR3 regions are obtained for sequences SEQ ID NO: 187 and 188:

[0462] Example 4: Susceptibility of several target clones

[0463] To assess their susceptibility, more specifically, antibodies generated from clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 were tested for their physical and chemical stability after storage at different temperatures.

[0464] Each antibody composition was subjected to temperature variations to assess its stability, specifically:

[0465] - Under one test condition, the sample was subjected to 5 rounds of freeze / thaw cycles (5x FT);

[0466] - Under other testing conditions, the samples were stored at room temperature (25°C) for 2 weeks and at 40°C for 2 weeks.

[0467] Physical stability was assessed by measuring concentration, turbidity (OD500), and SE-HPLC before and after stress testing. Chemical stability was assessed by analyzing samples on RP-HPLC before and after stress testing. Additionally, unstressed samples were analyzed using HIC (hydrophobic interaction chromatography) to rank molecules based on their hydrophobicity.

[0468] Concentration and turbidity

[0469] Use the following methods for measurement:

[0470] - Instrument: UV / Vis spectrophotometer (Unchained Labs Lunatic)

[0471] - Plate: Lunatic plate (2 μL, 0.5 mm path, measurement range 0.03-40 OD; 10 mm).

[0472] SE-HPLC

[0473] The sample was centrifuged at 14,000 g for 5 minutes prior to injection. The sample was analyzed on an Agilent 1100 HPLC system. Instrument control and data analysis were performed using Chemstation for LC Systems (rev. B.04.03-SP1).

[0474] RP-HPLC

[0475] Samples were analyzed on an Agilent 1200 HPLC system. Instrument control and data analysis were performed using Chemstation for LC Systems (rev. B.04.03-SP1).

[0476] HIC

[0477] Samples were analyzed on an Agilent 1100 HPLC system. Instrument control and data analysis were performed using Chemstation for LC Systems (rev. B.04.03-SP1).

[0478] The results are presented in Figure 7 A (depicting the area of ​​the HPLC peak corresponding to the analyzed antibody) and Figure 7 B (which depicts the % of the main HPLC peak corresponding to the analyzed antibody) is included. The results of antibody stability analysis are summarized in... Figure 14 The table shown in the diagram is included.

[0479] Based on the obtained data, the following conclusions can be drawn:

[0480] - All antibodies generated from clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 were stable after undergoing 5 freeze / thaw cycles;

[0481] - All antibodies generated from clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 were stable when stored at 25°C for 2 weeks.

[0482] - When stored at 40°C for 2 weeks:

[0483] - Antibody samples from pGnRH-009 and pGnRH-024 showed some protein loss;

[0484] - Antibody samples from pGnRH-001, pGnRH-002, and pGnRH-022 showed an increase in HMW (high molecular weight) species, which is typically a sign of antibody degradation;

[0485] - Antibody samples from pGnRH-001, pGnRH-002 and pGnRH-022 showed an increase of more than 5% in a single variant on RP-HPLC.

[0486] Example 5: Sequence optimization of some target clones

[0487] Sequence optimization was performed on antibodies generated from clones pGnRH-001 and pGnRH-009 (which were considered the target clones).

[0488] Regarding the cloning of pGnRH-001, four different sequence variants were generated: 01Tm1 (corresponding to SEQ ID NO: 205), 01Tm2 (corresponding to SEQ ID NO: 206), 01Tm3 (corresponding to SEQ ID NO: 207), and 01Tm4 (corresponding to SEQ ID NO: 208). Sequence diagrams of these variants are illustrated in... Figure 8A In the image, the arrow points to the amino acid residues that have been mutated compared to the original clone pGnRH-001.

[0489] Regarding the cloning of pGnRH-009, two distinct sequence variants were generated: 09Tm1 (corresponding to SEQ ID NO: 209) and 09Tm2 (corresponding to SEQ ID NO: 210). Sequence diagrams of these variants are illustrated in... Figure 8B In the image, the arrow points to the amino acid residues that have been mutated compared to the original clone pGnRH-009.

[0490] Example 6: Binding of sequence variants from clones pGnRH-001 and pGnRH-009 to GnRH (biopeptides 1 and 2).

[0491] The ELISA assays for bioactive peptides 1 and 2, as described in the “Materials and Methods” section, were used to evaluate the binding properties of antibodies from Tm variants 01Tm1, 01Tm2, 01Tm3, 01Tm4, 09Tm1, and 09Tm2 (sequence variants) from clones pGnRH-001 and pGnRH-009, respectively, and to compare them with the binding properties of antibodies from the original clones. The goal was to identify whether sequence mutations (substitutions) included to remove physical and chemical susceptibility had any effect on the binding ability of the clones to the GnRH peptides.

[0492] The ELISA assay setup is as follows:

[0493] - Neutral avidin (Thermo Fisher Scientific, Cat. nr. 31000) was coated overnight in Maxisorp plates at 4°C with 5 μg / ml of 1×PBS.

[0494] - Block the coated plates with 1% casein in 1×PBS;

[0495] - GnRH biopeptide 1 (SB-Peptide, Lot nr. 10869) and biopeptide 2 (SB-Peptide, Lot nr. 10870) were captured at 5 nM in 0.1% casein in 1×PBS;

[0496] - The pGnRH_001 and pGnRH_009 VHH Tm variants were titrated by performing a 7-step, 5-fold serial dilution (starting at 1 μM in 0.1% casein in 1×PBS);

[0497] - VHH was detected with anti-VHH antibody (JIR, Cat. nr. 128-005-230) at a 1:1000 dilution in 0.1% casein in 1×PBS, and subsequently with anti-goat IgG-HRP (JIR, Cat. nr. 705-035-147) at a 1:10000 dilution in 0.1% casein in 1×PBS;

[0498] - A coating control was performed using 1 μg / mL of anti-GnRH polyclonal antibody in 0.1% casein in 1×PBS, followed by anti-rabbit IgG-HRP (JIR, Cat. nr. 111-035-144) at a 1:10000 dilution in 0.1% casein in 1×PBS;

[0499] - The binding to GnRH biopeptide 1 was evaluated by measuring the OD at 450 nm.

[0500] The results are presented in Figure 9 (Sequence variants of cloned pGnRH-001: 01Tm1, 01Tm2, 01Tm3, 01Tm4), Table 4. Figure 10 (Sequence variants 09Tm1 and 09Tm2 of cloned pGnRH-009) and in Table 5.

[0501] Table 4 – Binding characteristics of 01Tm1, 01Tm2, 01Tm3, and 01Tm4 with biopeptides 1 and 2.

[0502] Table 5 – Binding characteristics of 09Tm1 and 09Tm2 with biopeptides 1 and 2.

[0503] As in Figure 9 As shown in Table 4, sequence optimization of the sequence variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 that led to the clone pGnRH-001 allowed for improved binding of all these variants to biopeptide 1.

[0504] As in Figure 10 As shown in Table 5, sequence optimization of the sequence variants 09Tm1 and 09Tm2 that led to the clone pGnRH-009 allowed for improved affinity of all these variants for biopeptide 1.

[0505] Example 7: Susceptibility and stability of cloned sequence variants of pGnRH-001 and pGnRH-009.

[0506] The Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 of clone pGnRH-001 and the Tm variants 09Tm1 and 09Tm2 of clone pGnRH-009 were expressed in Escherichia coli, purified by Ni-NTA affinity chromatography, and formulated in PBS.

[0507] The physical and chemical stability was tested using the same experimental protocol described in Example 4. Specifically, each antibody composition was subjected to temperature variations to assess its stability, more precisely:

[0508] - Under one test condition, the sample is subjected to 5 rounds of freezing / thawing steps (5x FT);

[0509] - Under other testing conditions, the samples were stored at room temperature (25°C in this case) for 2 weeks and at 40°C for 2 weeks.

[0510] For all these conditions, antibody concentration was measured (results illustrated in the diagram). Figure 11 In section A), turbidity was measured at OD 500 nm in parallel for all samples and conditions (results illustrated in the figure). Figure 11 (B in the middle).

[0511] The sample was subjected to RP-HPLC, and the total area of ​​each peak was measured to determine the increase in high molecular weight species. The results are illustrated graphically. Figure 12 middle.

[0512] For the purposes of these experiments, the antibodies were renamed as shown in Table 6 below.

[0513] Table 6 – A table showing the correspondence between the notes and names used in the Examples section and the variants disclosed herein.

[0514] The results of antibody stability analysis are summarized in Figure 15 The table shown in the diagram is included.

[0515] Based on the obtained data, the following conclusions can be drawn:

[0516] - All antibodies generated from clones pGnRHTm-001, pGnRHTm-002, pGnRHTm-003, pGnRHTm-004, pGnRHTm-005, and pGnRHTm-006 were stable after undergoing 5 freeze / thaw cycles;

[0517] - All antibodies generated from clones pGnRHTm-001, pGnRHTm-002, pGnRHTm-003, pGnRHTm-004, pGnRHTm-005 and pGnRHTm-006 were stable when stored at 25°C for 2 weeks.

[0518] - When stored at 40°C for 2 weeks:

[0519] - Antibody samples from pGnRHTm-002, pGnRHTm-003, pGnRHTm-004 and pGnRHTm-005 showed an increase in high molecular weight species, but this increase was less significant than that observed for the original clones 1 and 9 under similar conditions;

[0520] - Antibody samples from pGnRHTm-001 and pGnRHTm-003 showed an increase of more than 4% and 5% in single variants, respectively, on RP-HPLC.

[0521] Example 8: Antagonistic activity of variants GnRH_001_Tm002 and GnRH_009_Tm005

[0522] Their effect on agonist-induced cytoplasmic calcium was measured using a fluorescence assay. 2+ The effect of ion migration was used to determine the antagonistic activity of antibodies produced by clones GnRH_001_Tm002 and GnRH_009_Tm005 against human GnRH receptor expressed in rat basophilic leukemia cells.

[0523] The setup for this experiment is as follows:

[0524] - Rat basophilic leukemia cells resuspended in HBSS buffer (Invitrogen) were distributed in microplates at a density of 1.186E+04 cells / well;

[0525] - Then, a fluorescent probe (Fluo8 Direct, AAT Bioquest) mixed with probenecid in HBSS buffer (Invitrogen) supplemented with 20 mM Hepes (Invitrogen, pH 7.4) was added to each well and equilibrated with the cells at 30°C for 60 min.

[0526] - Add the most promising mutant versions of GnRH_001 and GnRH_009 monovalent VHH, GnRH-001-Tm002 and GnRH-009-Tm005, to the cells and incubate for 5 minutes;

[0527] - SMI41 positive control was tested at concentrations of 100, 20, 4, 0.8, and 0.16 μg / mL;

[0528] - Also included were E. coli production of GnRH_001 and GnRH_009 monovalent VHH as controls;

[0529] Then, 8 nM GnRH was added to the cells, and changes in fluorescence intensity were measured, comparing it to free cytoplasmic Ca2+. 2+ The ion concentration changes proportionally;

[0530] - Express the results as the percentage of inhibition of the control response to 8 nM GnRH;

[0531] - Cetrorexate acetate was used as a standard reference antagonist and tested at several concentrations to generate concentration-response curves from which its IC50 value was calculated.

[0532] The results are illustrated in the diagram. Figure 13 Examples of neutralization are illustrated in Table 7 below.

[0533] Table 7 – IC50 of Tm variants and original clones.

[0534] Based on these results, the following conclusions can be drawn:

[0535] - The GnRH_001_Tm002 candidate showed a similar characteristic curve to GnRH_001, which suggests that the introduced mutation can improve the chemical susceptibility and stability of the parent clone without affecting its functional activity.

[0536] - The GnRH_009 candidate produced for this experiment exhibited different characteristic curves and IC50 than the antibody obtained during the lead product identification phase;

[0537] - The GnRH_009_Tm005 candidate showed a similar characteristic curve to GnRH_009, suggesting that the introduced mutation can improve the chemical susceptibility and stability of the parent clone without affecting its functional activity.

[0538] In summary, the data confirm the benefits of mutations implemented in the Tm variants, particularly in the sequences of GnRH_001_Tm002 and GnRH_009_Tm005, in terms of physical and chemical stability, while maintaining their ability to bind to GnRH and antagonize antibodies.

Claims

1. An isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or its antigen-binding fragment, wherein the antibody or antigen-binding fragment specifically binds to both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH.

2. An isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of: - CDR3 polypeptide sequence SEQ ID NO: 33 or 188; - CDR3 polypeptide sequence SEQ ID NO: 57 or 187; - CDR3 polypeptide sequence SEQ ID NO: 36; - CDR3 polypeptide sequence SEQ ID NO: 96; - CDR3 polypeptide sequence SEQ ID NO: 48; - CDR3 polypeptide sequence SEQ ID NO: 42, 75, 81, 90, 93; or - A variant of the CDR3 polypeptide sequence with one or two substitutions.

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

4. The isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that, It contains 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 its antigen-binding fragment according to any one of claims 1 or 2, characterized in that, It contains a variable structure domain, which has: - CDR1, which contains sequences selected from SEQ ID NO: 55 and 82; - CDR2, which contains sequences selected from SEQ ID NO: 56, 98, 116 and 199; - CDR3, which contains the sequence SEQ ID NO:

57.

6. The isolated antibody or its antigen-binding fragment according to any one of claims 1 or 2, characterized in that, It contains a variable structure domain, which has: - CDR1, which contains sequences selected from SEQ ID NO: 31 and 195; - CDR2, which contains a sequence selected from SEQ ID NO: 32; - CDR3, which contains the sequence SEQ ID NO:

33.

7. The isolated antibody or its antigen-binding fragment according to any one of claims 1 or 2, characterized in that, It contains a variable structure domain, which has: - CDR1 containing a sequence selected from SEQ ID NO: 34, CDR2 containing a sequence selected from SEQ ID NO: 35, and CDR3 containing a sequence selected from SEQ ID NO: 36; or - CDR1 containing a sequence selected from SEQ ID NO: 94, CDR2 containing a sequence selected from SEQ ID NO: 95, and CDR3 containing a sequence selected from SEQ ID NO: 96; or - CDR1 containing a sequence selected from SEQ ID NO: 46, CDR2 containing a sequence selected from SEQ ID NO: 47, and CDR3 containing a sequence selected from SEQ ID NO:

48.

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

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

10. A vector comprising the 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 its antigen-binding fragment according to any one 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 - Pharmaceutically acceptable excipients.

13. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a nucleic acid according to claim 9, or a vector according to claim 10, or a host cell according to claim 11, or a pharmaceutical composition according to claim 12, for use as a drug or for use in in vivo diagnostic methods.

14. An in vitro method for detecting gonadotropin-releasing hormone (GnRH), comprising the following steps: a) Provide samples, especially biological samples or fractions thereof; b) Contact the sample with the isolated antibody or its antigen-binding fragment according to any one of claims 1 to 8.

15. A method for isolating an anti-gonadotropin-releasing hormone (anti-GnRH) antibody or an antigen-binding fragment thereof, comprising the following steps: a) Provide a library of single-domain antibodies (SdAbs) or fragments thereof; b) Subject the library to antigen affinity selection, wherein the selected SdAb or fragment thereof is characterized by specificity for both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH. c) 。

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