GnRH-binding polypeptide and its use

Abstract

This invention relates to the field of GnRH antagonists and anti-GnRH ligands. The invention also relates to the field of pharmaceuticals and diagnostics. In particular, this disclosure relates to the field of contraception and GnRH-related disorders.

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 fields of contraception and GnRH-related diseases.

Background Art

[0003] Various contraceptive methods are practiced in the art, particularly for livestock including pigs.

[0004] A long-established contraceptive method for pigs consisted of the live castration of piglets without any anesthesia. However, this long-established method has been considered unethical because it causes unacceptable pain, severe distress and suffering to piglets.

[0005] Animal contraceptive methods known in the art also include surgical contraception under anesthesia (e.g., tubal ligation or vasectomy), hormonal implants inserted with intrauterine contraceptive devices (e.g., GnRH analog-containing implants), use of hormonal compositions including direct injection of chemicals into the testes to destroy reproductive tissue, and methods for immunological contraception.

[0006] GnRH-binding ligands, particularly monoclonal antibodies, have been reported in the art. Known methods of immunological contraception include administration of vaccine compositions aimed at immunizing the subject against endogenous GnRH.

[0007] In particular, GnRH vaccines stimulate antibody production, which then inactivates endogenous GnRH, causing a decrease in the release of gonadotropin hormones leading to atrophy of the gonads in adult animals or lack of development in sexually immature animals. <​​

[0009] Because they function as hormones, GnRH, GnRH analogs, and GnRH-binding ligands have also been used as therapeutic agents in numerous other medical conditions and clinical applications, including the treatment or prevention of diseases beyond infertility, particularly sex hormone-dependent disorders such as delayed puberty, ovarian hyperstimulation syndrome, precocious puberty, premenstrual syndrome, endometriosis, uterine fibroids, polycystic ovary syndrome, hirsutism, acne vulgaris, acute intermittent porphyria, breast cancer, uterine cancer, uterine fibroids, endometrial cancer, prostate cancer, benign prostatic hyperplasia, and pancreatic cancer, as either GnRH agonists or GnRH antagonists.

[0010] Silversides et al. ("Monoclonal antibodies against LHRH: development and immunoactivity in vivo and in vitro"; Journal of Reproductive Immunology, 1985) reported monoclonal antibodies derived from canine immunotherapy 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 that has the ability to suppress the progression of estrus in dogs.

[0012] Li et al. ("Vectored antibody gene delivery mediates long-term contraception," Current Biology, 2015) and US10,570,200B2 teach antibody-mediated immunocontraception, including vectorized AAV-dependent anti-GnRH antibody expression.

[0013] A purified monoclonal mouse IgG1, kappa antibody, called clone number SMI41 (Absolute Antibody reference number Ab00922-1.1), which is specific to the C-terminal peptide of GnRH, has also been reported for immunocytochemistry and has been expressed in mice via injection of recombinant adeno-associated virus with no replication ability to induce prolonged infertility.

[0014] CEVA VALORA® is reported as a GnRH / LHRH-based synthetic peptide vaccine for pigs, containing three synthetic peptides as immunogens.

[0015] IMPROVAC® has been reported as a GnRH analog protein conjugate that can stimulate the porcine immune system to produce antibodies against GnRH, thereby controlling sexual development.

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

[0017] Gonadotropin-releasing hormone (GnRH), also known as LHRH, is a well-conserved mammalian hormone secreted by the pituitary gland in the central hypothalamus. It acts as a major 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 immunizing contraceptive 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 closely related species; it is generally reported as a decapeptide characterized by a conserved N-terminal region, a central region, and a conserved C-terminal region, with the following amino acid structure provided for reference in mammals: (Pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO: 152).

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

[0020] In its folded structure, both the N-terminal and C-terminal regions of GnRH are involved in receptor binding, but it appears that only the N-terminal region is involved in receptor activation.

[0021] The development of anti-GnRH ligands is challenging, particularly in the context of immunizing contraceptive vaccines, for several reasons: (i) GnRH is a small decapeptide with very low antigenicity; (ii) Despite its small size, the folded three-dimensional structure of mammalian GnRH has led to the identification of at least two crucial regions for receptor binding, with only the N-terminal region being responsible for receptor activation; (iii) This is naturally present in the body.

[0022] Therefore, after administration to animals, it is recognized by the immune system as an "autologous protein" accompanied by a weak and transient antibody response, and contains numerous structural epitopes.

[0023] Therefore, in this field, there is still a need to identify novel ligands for GnRH, or alternatively, ligands for novel epitopes of GnRH.

[0024] In this technical field, there is still a need for ligands and formulations for GnRH that remain stable and suitable for large-scale production.

[0025] In particular, in this field of technology, there is still a need for ligands for GnRH that can be used as pharmaceuticals and / or for detection or diagnostic purposes.

[0026] In particular, in this field of technology, there remains a need for GnRH antagonists for use in the treatment of disorders, especially sex hormone-related disorders and / or GnRH-related disorders.

[0027] In this field of technology, there remains a need for contraceptive methods, particularly for livestock.

[0028] In particular, in this field of technology, there is still a need for contraceptive methods that are reversible, rapid, efficient, and easy to administer.

[0029] This disclosure is intended to satisfy these needs. [Overview of the project]

[0030] A first main embodiment of the present invention relates to an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or its antigen-binding fragment, which specifically binds to both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH.

[0031] A second main embodiment of the present invention is an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, - CDR3 polypeptide sequence of sequence number 57 or 187; - CDR3 polypeptide sequence of sequence number 33 or 188; - CDR3 polypeptide sequence of sequence number 36; - CDR3 polypeptide sequence of sequence number 96; - CDR3 polypeptide sequence of sequence number 48; - CDR3 polypeptide sequences of sequence numbers 42, 75, 81, 90, and 93; or - Variants of the CDR3 polypeptide sequence having one or two substitutions This relates to the antibody or its antigen-binding fragment, which contains a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of the following.

[0032] According to another main embodiment, the present invention relates to nucleic acids encoding an antibody or an antigen-binding fragment thereof.

[0033] According to another main embodiment, the present invention relates to a vector comprising the nucleic acid of the present invention.

[0034] According to another main embodiment, the present invention relates to a host cell comprising the nucleic acid or vector of the present invention; in particular to a eukaryotic cell comprising the nucleic acid or vector of the present invention. Such a host cell may be isolated by any method known in the art.

[0035] According to another main embodiment, the present invention is - Isolated antibodies or antigen-binding fragments thereof, or nucleic acids, or vectors, or host cells according to the present invention; and - Pharmaceutically acceptable excipients This relates to a pharmaceutical composition containing [a specific ingredient / component].

[0036] According to another main embodiment, the present invention relates to an in vitro method for detecting gonadotropin-releasing hormone (GnRH), a) A step of providing a sample, particularly a biological sample or a fraction thereof; b) The step of contacting the sample with the isolated antibody or its antigen-binding fragment according to the present invention. Regarding methods including

[0037] According to another main embodiment, the present invention relates to a method for isolating an anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, a) Providing a library of single-domain antibodies (SdAbs) or their antigen-binding fragments; b) A step of subjecting the library to antigen affinity selection, characterized in that the selected SdAb or fragment thereof has specificity for both the N-terminal region of natural GnRH (pGlu-His-Trp) and the C-terminal region of natural GnRH (Pro-Gly-NH2), the above step Regarding methods including [Brief explanation of the drawing]

[0038] [Figure 1] This graph shows the results of ELISA and SPR for biotinylated biopeptides 1(A) and 2(B). The 10 highlighted amino acids represent the mammalian GnRH natural peptide (SEQ ID NO: 152). [Figure 2]This graph displays the ELISA quality control results for directly coated or captured biopeptides. The graph shows the optical concentration (OD) at 450 nm versus the log concentrations of biopeptides 1 and 2, using a nonlinear regression curve fitting of log(agonist) versus response with four parameters. A) x-axis in μg / ml, as a function of the directly coated protein. B) x-axis in nM, as a function of the captured protein. [Figure 3] This figure shows the ELISA results for biopeptide 1 with 30 purified SdAb(VHH) reads and a 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 in all panels. [Figure 4] This figure shows the SPR results for biopeptide 1 and biopeptide 2 with 30 purified SdAb(VHH) reads and a reference SMI41 antibody. For each graph, relative units (RU) are provided on the y-axis over time (s). A. Left panel: This shows the SPR signal of the SMI41(IgG1) antibody in the presence of coated biopeptide 1. Right panel: This shows the SPR signal of the SMI41(IgG1) antibody in the presence of coated biopeptide 2. B. Left panel: This shows the SPR signal of each VHH read antibody in the presence of coated biopeptide 1. Right panel: This shows the SPR signal of each VHH read antibody in the presence of coated biopeptide 2. [Figure 5A] This figure shows the innate GnRH binding of selected VHH libraries by ELISA with coated GnRH and antibody titration. The graph shows the optical concentration (OD) at 450 nm versus the log concentration (μM) of VHH antibody clones 1-12. [Figure 5B]This figure shows the innate GnRH binding of selected VHH libraries by ELISA with coated GnRH and antibody titration. The graph shows the optical concentration (OD) at 450 nm versus the log concentration (μM) of VHH antibody clones 13-24. [Figure 5C] This figure shows the innate GnRH binding of selected VHH libraries by ELISA with coated GnRH and antibody titration. The graph shows the optical concentration (OD) at 450 nm versus the log concentration (μM) of VHH antibody clones 25–36. [Figure 5D] This figure shows the innate GnRH binding of selected VHH libraries by ELISA with coated GnRH and antibody titration. Polyclonal and monoclonal VHH vs. negative control experiments with SMI41. [Figure 6] This figure shows the evaluation of the ability of five purified VHH leads to antagonize the human GnRH receptor when expressed in rat basophilic leukemia cells. The percentage of inhibition is evaluated on the y-axis as a function of the concentration of each tested VHH and the concentration of the reference SMI41 antibody. A) Concentration of each antibody expressed in μg / mL. B) Concentration of each antibody expressed in μM. [Figure 7] This figure shows the evaluation of antibody titers using reverse-phase high-performance liquid chromatography (RP-HPLC). Samples of antibodies produced by 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 area of ​​the photograph corresponding to the reference clone-derived antibody, measured by RP-HPLC. B. Main photograph of the photograph corresponding to the reference clone-derived antibody, measured by RP-HPLC. [Figure 8A] This figure shows the sequences of the Tm variants. The sequences of 01Tm1, 01Tm2, 01Tm3, and 01Tm4 (sequence variants), which are Tm variants of clone pGnRH-001, are shown, and the arrows indicate the substitutions made in the sequence variants. [Figure 8B]This figure shows the sequences of the Tm variants. The sequences of 09Tm1 and 09Tm2 (sequence variants), which are Tm variants of clone pGnRH-009, are shown, and the arrows indicate substitutions made in the sequence variants. [Figure 9] This figure shows ELISA assays for biopeptides 1 and 2. The binding properties of antibodies derived from the Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 (sequence variants) of clone pGnRH-001 were evaluated using ELISA assays for biopeptides 1 and 2. A. ELISA assay for biopeptide 1 with Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4. The corresponding optical density (OD) at 450 nm is provided on the y axis. The corresponding nM concentration of VHH is provided on the x axis. B. ELISA assay for biopeptide 2 with Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4. The corresponding optical density (OD) at 450 nm is provided on the y axis. The corresponding nM concentration of VHH is provided on the x axis. [Figure 10] This figure shows ELISA assays for biopeptides 1 and 2. The binding properties of antibodies derived from 09Tm1 and 09Tm2 (sequence variants), which are Tm variants of clone pGnRH-009, were evaluated using ELISA assays for biopeptides 1 and 2. A. ELISA assay for biopeptide 1 with Tm variants 09Tm1 and 09Tm2. The corresponding optical density (OD) at 450 nm is provided on the y axis. The corresponding nM concentration of VHH is provided on the x axis. B. ELISA assay for biopeptide 2 with Tm variants 09Tm1 and 09Tm2. The corresponding optical density (OD) at 450 nm is provided on the y axis. The corresponding nM concentration of VHH is provided on the x axis. [Figure 11]This figure illustrates the characterization of Tm variant stability. Samples of antibodies produced by clone pGnRH-001, Tm variant 01Tm2 of clone pGnRH-001, clone pGnRH-009, and Tm variant 09Tm1 of clone pGnRH-009 were subjected to five freeze / thaw cycles (5×FT) or stored at room temperature (25°C) or 40°C for two 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. For all samples and conditions shown on the Ax axis, the antibody concentration of the sample is provided on the y axis in mg / mL. For all samples and conditions shown on the Bx axis, the turbidity measured at OD500nm is provided on the y axis. [Figure 12] This figure illustrates the characterization of Tm variant stability. Samples of antibodies produced by clone pGnRH-001, clone pGnRH-001's Tm variant 01Tm2 (GnRH-001-Tm002), clone pGnRH-009, and clone pGnRH-009's Tm variant 09Tm1 (GnRH-009-Tm005) were subjected to five freeze / thaw cycles (5×FT) or stored at room temperature (25°C) or 40°C for two weeks. T0 represents the control condition and corresponds to the antibody titer of the original sample before storage. The samples were subjected to RP-HPLC. For all samples and conditions shown on the Ax axis, the total area of ​​the photographs corresponding to the clone-derived antibodies in the reference, measured by RP-HPLC, is shown on the y axis in milliabsorbance units * seconds (mAU* seconds). For all samples and conditions shown on the Bx axis, the percentage increase in high molecular weight species in the reference clone-derived sample, as measured by RP-HPLC, is shown on the y axis. [Figure 13]This figure shows the antagonist activity of Tm variants. Antagonist activity against GnRH of antibodies produced by clone pGnRH-001, clone pGnRH-001's Tm variant 01Tm2 (GnRH-001-Tm002), clone pGnRH-009, and clone pGnRH-009's Tm variant 09Tm1 (GnRH-009-Tm005). A. The percentage of inhibition is evaluated on the y-axis as a function of the respective tested VHH concentration and the reference SMI41 antibody concentration. The concentration of each antibody is expressed in nM. B. The percentage of inhibition is evaluated on the y-axis as a function of the respective tested VHH concentration and the reference SMI41 antibody concentration. The concentration of each antibody is expressed in μg / ml. [Figure 14] This figure shows a library of the top clones that were tested. 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, which are detailed in Example 4. [Figure 15] This figure shows a library of all Tm variants. A summary of the results obtained regarding the physical and chemical stability of the Tm variants, i.e., the sequence variants 01Tm1, 01Tm2, 01Tm3, 01Tm4, which are Tm variants of clone pGnRH-001, and 09Tm1, 09Tm2, which are Tm variants of clone pGnRH-009, as detailed in Example 7. [Modes for carrying out the invention]

[0039] Detailed description of the invention The inventors hereby provide an anti-GnRH antibody and its antigen-binding fragment. Surprisingly, the inventors disclose that such an anti-GnRH antibody has the ability to target both the N-terminal and C-terminal regions of native GnRH, and that these two regions are involved in receptor binding.

[0040] This binding mechanism is atypical in light of existing anti-GnRH antibodies reported in the relevant field that do not share the same properties.

[0041] The inventors further provide in vitro and in vivo evidence that selected single-domain antibodies sharing the same binding mechanism are sufficient to antagonize human GnRH receptor activity in rat basophilic leukemia cells.

[0042] Unwilling to be constrained by theory, the inventors believe that the binding mechanism, which depends on the two terminal regions of natural GnRH, is the cause of its biological effect.

[0043] The present inventors further disclose a method for isolating monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) ligands that are particularly selective to other ligands having the same binding mechanism.

[0044] Unwilling to be constrained by theory, the inventors believe that a library of single-domain antibodies (SdAbs) is particularly convenient as a novel class of ligands for selecting antibodies that have atypical binding mechanisms to natural GnRH.

[0045] The experimental data shows that single-domain antibodies (SdAbs), particularly heavy chains (V), which are also known as nanobodies in this field, are used. H H) While the focus is on isolating a single variable domain on the antibody, the provided hypervariable domains are also non-V, as reported by Pekar et al. ("Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform"; Mabs, 12(1), 2020). H H can also serve as a basis for the identification and / or engineering of conventional bispecific and tripspecific antibodies.

[0046] Isolated (e.g., V HH) The following anti-GnRH antibodies are particularly contemplated as any of an antibody, its antigen-binding fragment, or its hypervariable region.

[0047] Clone 1 corresponds to the VH antibody of SEQ ID NO: 1, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 31, 32, and 33, respectively. H corresponding to the H antibody.

[0048] Clone 2 corresponds to the VH antibody of SEQ ID NO: 2, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 34, 35, and 36, respectively. H corresponding to the H antibody.

[0049] Clone 3 corresponds to the VH antibody of SEQ ID NO: 3, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 37, 38, and 39, respectively. H corresponding to the H antibody.

[0050] Clone 4 corresponds to the VH antibody of SEQ ID NO: 4, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 40, 41, and 42, respectively. H corresponding to the H antibody.

[0051] Clone 5 corresponds to the VH antibody of SEQ ID NO: 5, which contains the CDR1, CDR@, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 43, 44, and 45, respectively. H corresponding to the H antibody.

[0052] Clone & corresponds to the VH antibody of SEQ ID NO: 6, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 46, 47, and 48, respectively. H corresponding to the H antibody.

[0053] Clone 7 corresponds to the VH antibody of SEQ ID NO: 7, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 49, 50, and 51, respectively. H corresponding to the H antibody. <^

[0054] Clone 8 corresponds to the VH antibody of SEQ ID NO: 8, which contains the CDR1, CDR2, and CDR3 hypervariable regions of the sequences of SEQ ID NOs: 52, 53, and 54, respectively. It should be noted that there may be some inaccuracies in the original text, such as "CDR@" in line 28. This translation is based on the best understanding of the available content.H This corresponds to the H antibody.

[0055] Clone 9 contains the V of the sequence of sequence number 9, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 55, 56, and 57, respectively. H This corresponds to the H antibody.

[0056] Clone 10 contains the V of the sequence of sequence number 10, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 58, 59, and 60, respectively. H This corresponds to the H antibody.

[0057] Clone 11 contains the V of the sequence of sequence number 11, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 61, 62, and 63, respectively. H This corresponds to the H antibody.

[0058] Clone 12 contains the V of the sequence of sequence number 12, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 64, 65, and 66, respectively. H This corresponds to the H antibody.

[0059] Clone 13 contains the CDR1, CDR2, and CDR3 hypervariable regions of sequence number 67, 68, and 69, respectively, and is a V of sequence number 13. H This corresponds to the H antibody.

[0060] Clone 14 contains the V of the sequence of sequence number 14, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 70, 71, and 72, respectively. H This corresponds to the H antibody.

[0061] Clone 15 contains the V of the sequence of sequence number 15, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 73, 74, and 75, respectively. H This corresponds to the H antibody.

[0062] Clone 16 contains the V sequence of sequence number 16, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 76, 77, and 78, respectively. H This corresponds to the H antibody.

[0063] Clone 17 contains the V sequence of sequence number 17, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 79, 80, and 81, respectively. H This corresponds to the H antibody.

[0064] Clone 18 contains the V sequence of sequence number 18, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 82, 83, and 84, respectively. H This corresponds to the H antibody.

[0065] Clone 19 contains the V of the sequence of sequence number 19, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 85, 86, and 87, respectively. H This corresponds to the H antibody.

[0066] Clone 20 contains the V of the sequence of sequence number 20, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 88, 89, and 90, respectively. H This corresponds to the H antibody.

[0067] Clone 21 contains the V of the sequence of sequence number 21, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 91, 92, and 93, respectively. H This corresponds to the H antibody.

[0068] Clone 22 contains the V of the sequence of sequence number 22, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 94, 95, and 96, respectively. H This corresponds to the H antibody.

[0069] Clone 23 contains the V sequence of sequence number 23, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 97, 98, and 99, respectively. H This corresponds to the H antibody.

[0070] Clone 24 contains the V of the sequence of sequence number 24, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 100, 101, and 102, respectively. H This corresponds to the H antibody.

[0071] Clone 25 contains the V of the sequence of sequence number 25, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 103, 104, and 105, respectively. H This corresponds to the H antibody.

[0072] Clone 26 contains the V of the sequence of sequence number 26, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 106, 107, and 108, respectively. H This corresponds to the H antibody.

[0073] Clone 27 contains the V of the sequence of sequence number 27, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 109, 110, and 111, respectively. H This corresponds to the H antibody.

[0074] Clone 28 contains the V of the sequence of sequence number 28, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 112, 113, and 114, respectively. H This corresponds to the H antibody.

[0075] Clone 29 contains the V of the sequence of sequence number 29, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 115, 116, and 117, respectively. H This corresponds to the H antibody.

[0076] Clone 30 contains the V of the sequence of sequence number 30, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 118, 119, and 120, respectively. H This corresponds to the H antibody.

[0077] 01Tm1 is the V of sequence number 189, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 195, 196, and 197, respectively. H This corresponds to the H antibody.

[0078] 01Tm2 is the V of sequence number 190, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 31, 32, and 33, respectively. H This corresponds to the H antibody.

[0079] 01Tm3 is the V of sequence number 191, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 31, 32, and 33, respectively. H This corresponds to the H antibody.

[0080] 01Tm4 is the V of sequence number 192, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 31, 32, and 33, respectively. H This corresponds to the H antibody.

[0081] 09Tm1 is the V of sequence number 193, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 55, 56, and 57, respectively. H This corresponds to the H antibody.

[0082] 09Tm2 is the V of sequence number 194, which includes the CDR1, CDR2, and CDR3 hypervariable regions of sequence numbers 198, 199, and 200, respectively. H This corresponds to the H antibody.

[0083] In the context of the present invention, redundant sequences listed in SEQ ID NOs. 34-120 as part of separate antibody or antigen-binding fragments may be interchangeable between one equivalent CDR and another. This may include, for example, the CDR1 sequences of SEQ ID NOs. 31, 34, and 37 or the CDR2 sequences of SEQ ID NOs. 32 and 35, which are shared among several of the aforementioned clones.

[0084] Therefore, the present invention is not limited to all of the above V H This includes H clones, and / or combinations thereof of the respective CDR1, CDR2, and CDR3 hypervariable regions; and their use.

[0085] As a result, the first main embodiment of the present invention relates to an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or its antigen-binding fragment, which specifically binds to both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of native GnRH.

[0086] According to certain embodiments, an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or its antigen-binding fragment may be characterized by its specific binding to a first polypeptide sequence consisting of SEQ ID NO: 154 and a second polypeptide sequence consisting of SEQ ID NO: 153.

[0087] According to a second main embodiment, the present invention relates to an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, - CDR3 polypeptide sequence of sequence number 57 or 187; - CDR3 polypeptide sequence of sequence number 33 or 188; - CDR3 polypeptide sequence of sequence number 36; - CDR3 polypeptide sequence of sequence number 96; - CDR3 polypeptide sequence of sequence number 48; - CDR3 polypeptide sequences of sequence numbers 42, 75, 81, 90, and 93; or - Variants of the CDR3 polypeptide sequence having one or two substitutions This relates to the antibody or its antigen-binding fragment, which contains a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of the following.

[0088] According to certain embodiments, the variant of the CDR3 polypeptide sequence, where applicable, has one or two conversative substitutions.

[0089] According to certain exemplary embodiments, the isolated antibody or its antigen-binding fragment is a single-domain antibody (SdAb).

[0090] Accordingly, 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 its antigen-binding fragment comprises a variable domain having a complementarity-determining region 3 (CDR3) selected from the group defined above.

[0091] According to a particular embodiment, an isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment binds to the first polypeptide of sequence SEQ ID NO: 153 and the second polypeptide of sequence SEQ ID NO: 154.

[0092] According to a particular embodiment, the isolated antibody or antigen-binding fragment of the present invention is characterized by comprising a variable domain having a complementarity-determining region 3 (CDR3) containing a sequence selected from SEQ ID NOs. 57, 187, 33, 188, 36, and 96.

[0093] According to a more specific embodiment, the isolated antibody or antigen-binding fragment of the present invention is characterized by comprising a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence of SEQ ID NO: 57.

[0094] According to a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is the complementarity-determining region 3 (CDR3) polypeptide sequence of SEQ ID NO: 187: DX1X2X3X4GX5YYX6PDX7 (In the formula, X1 to X7 independently represent any amino acid, X1 is, for example, R. X2 is, for example, G. X3 is, for example, P. X4 is, for example, S. X6 is, for example, Y. X7 is, for example, Y. It is characterized by including a variable domain having at least one of the following:

[0095] According to a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is X1 is R, X2 is G, X3 is P, X4 is S, X6 is Y, X7 is Y. It is characterized by containing a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence of sequence number 187 as defined above.

[0096] According to a more specific embodiment, the isolated antibody or antigen-binding fragment of the present invention is characterized by comprising a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence of SEQ ID NO: 33.

[0097] According to a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is the complementarity-determining region 3 (CDR3) polypeptide sequence of SEQ ID NO: 188: DX1X2YYX3X4X5X6X7PX8 (In the formula, X1 to X8 independently represent any amino acid, X1 is, for example, D. X2 is, for example, A. X3 is, for example, P. X4 is, for example, G. X8 is preferably Q or H. It is characterized by including a variable domain having at least one of the following:

[0098] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is X1 is D, X2 is A, X3 is P, X4 is G, X8 is Q or H. It is characterized by containing a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence of sequence number 188 as defined above.

[0099] According to other specific embodiments, the isolated antibody or antigen-binding fragment of the present invention is characterized by comprising a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence selected from SEQ ID NOs. 182 to 186.

[0100] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing sequences selected from sequence numbers 55 and 82; - CDR2 containing sequences selected from sequence numbers 56, 98, 116, and 199; - CDR3 containing sequence number 187 It is characterized by including a variable domain having

[0101] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing sequences selected from sequence numbers 55 and 82; - CDR2 containing sequences selected from sequence numbers 56, 98, 116, and 199; - CDR3 containing sequence number 57 It is characterized by including a variable domain having

[0102] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing sequences selected from sequence numbers 31 and 195; - CDR2 containing the sequence selected from sequence number 32; - CDR3 containing sequence number 188 It is characterized by including a variable domain having

[0103] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing sequences selected from sequence numbers 31 and 195; - CDR2 containing the sequence selected from sequence number 32; CDR3 containing sequence number 33 It is characterized by including a variable domain having

[0104] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing the sequence selected from sequence number 34; - CDR2 containing the sequence selected from sequence number 35; - CDR3 containing sequence number 36 It is characterized by including a variable domain having

[0105] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing the sequence selected from sequence number 94; - CDR2 containing an array selected from sequence number 95; - CDR3 containing sequence number 96 It is characterized by including a variable domain having

[0106] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing the sequence selected from sequence number 46; - CDR2 containing the sequence selected from sequence number 47; - CDR3 containing sequence number 48 It is characterized by including a variable domain having

[0107] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing an array selected from sequence number 195; - CDR2 containing the sequence selected from sequence number 196; - CDR3 containing sequence number 197 It is characterized by including a variable domain having

[0108] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is - CDR1 containing the sequence selected from sequence number 198; - CDR2 containing the sequence selected from sequence number 199; - CDR3 containing the sequence of sequence number 200 It is characterized by including a variable domain having

[0109] According to a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by comprising a framework region (FR) selected from the group consisting of SEQ ID NOs: 121-151, 201-204, or 211-214.

[0110] In a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by containing a framework region 1 (FR1) selected from the group consisting of SEQ ID NOs: 121-137, 201-204, or 211-214.

[0111] In a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by comprising a framework region 2 (FR2) selected from the group consisting of SEQ ID NOs: 138 to 141.

[0112] In a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by comprising a framework region 3 (FR3) selected from the group consisting of SEQ ID NOs: 142 to 149.

[0113] In a more specific embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by comprising a framework region 4 (FR4) selected from the group consisting of SEQ ID NOs: 150 or 151.

[0114] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by sharing at least 80% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequence numbers 1-30, 189-194, or 205-210.

[0115] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by sharing at least 90% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequence numbers 1-30, 189-194, or 205-210.

[0116] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by sharing at least 95% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequences 1-30, 189-194, or 205-210.

[0117] According to a particular embodiment, the isolated antibody or its antigen-binding fragment of the present invention is characterized by sharing at least 99% sequence identity with anti-gonadotropin-releasing hormone (anti-GnRH) antibodies of sequence numbers 1-30, 189-194, or 205-210.

[0118] According to a particular embodiment, the isolated anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment of the present invention is characterized in that the GnRH is selected from the group consisting of human GnRH, porcine GnRH, bovine GnRH, horse GnRH, sheep GnRH, dog GnRH, and feline GnRH.

[0119] According to certain embodiments, the isolated antibody or its antigen-binding fragment of the present invention is characterized by being a recombinant protein.

[0120] According to certain embodiments, the isolated antibody or its antigen-binding fragment of the present invention specifically binds to one or more polypeptides in addition to natural GnRH.

[0121] According to that particular embodiment, the isolated antibody or its antigen-binding fragment may therefore be plurispecific, for example, bispecific or triplicate. In that respect, according to a more particular embodiment, the isolated antibody or its antigen-binding fragment is - Containing at least one antigen-binding fragment of the present invention, which specifically binds to a first polypeptide that is a natural GnRH as disclosed herein and comprises at least one of the hypervariable CDR3 regions; - A second polypeptide, such as albumin, which specifically binds to albumin. H It contains at least one antigen-binding fragment that specifically binds to a polypeptide selected from the group consisting of H antibodies or their fragments. Bispecific single-domain antibodies (SdAbs) characterized by the above, especially multispecific V H It may consist of an H antibody or its antigen-binding fragment.

[0122] According to certain embodiments, the isolated antibody or antigen-binding fragment of the present invention is characterized by being conjugated (i.e., covalently bonded to another compound or polypeptide). For example, the isolated antibody or antigen-binding fragment of the present invention may be conjugated at the N-terminus or C-terminus.

[0123] According to certain embodiments, the isolated antibody or antigen-binding fragment of the present invention may be conjugated with a detectable agent such as a radioactive substance (e.g., a radionuclide), an enzyme, a fluorescent label, or a cytotoxic drug label.

[0124] According to certain embodiments, the isolated antibody or its antigen-binding fragment of the present invention may be conjugated to an albumin polypeptide or a polypeptide that is a fragment thereof.

[0125] According to other specific embodiments, the isolated antibody or its antigen-binding fragment of the present invention is characterized by not being conjugated.

[0126] According to more specific, non-mutually exclusive embodiments, the antibody or antigen-binding fragment of the present invention is characterized by being modified to increase its biological half-life. Various approaches are possible.

[0127] The antibody of the present invention is a single-domain antibody (SdAb) or its antigen-binding fragment, more specifically V H If the antibody is an H antibody or its antigen-binding fragment, it may advantageously include, or consist of, a single-domain antibody linked to an immunoglobulin domain, such as an Fc site (e.g., human Fc or non-human Fc). The Fc site may be useful for increasing the half-life and even production of the single-domain antibody of the present invention. For example, the Fc site can bind to serum proteins, thereby increasing the half-life of the single-domain antibody.

[0128] In one embodiment, one or more of the following mutations may be introduced: T252L, T254S, and T256F, as described in U.S. Patent No. 6,277,375 by Ward. Alternatively, to increase the biological half-life, the antibody may be modified to contain a salvage receptor-binding epitope within the CH1 or CL region, isolated from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and 6,121,022 by Presta et al. Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which are factors in the transfer of maternal IgG to the fetus (Guiyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions that enhance the binding of the Fc region to FcRn. Such Fc variants include those having one or more substitutions at 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, a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0129] Another modification of the antibody or antigen-binding fragments described herein, as intended, is PEGylation. PEGylation of an antibody or antigen-binding fragment may increase, for example, the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups adhering to the antibody or antibody fragment. PEGylation may be carried out by acylation or alkylation reactions with a reactive PEG molecule (or a similar reactive water-soluble polymer). In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0130] Another modification of the antibody or antigen-binding fragment intended by the present invention is the conjugate or protein fusion of at least the antigen-binding region of the antibody of the present invention to a serum protein such as human serum albumin or a fragment thereof, in order to increase the half-life of the resulting molecule. Such an approach is described, for example, by Balance et al., EP0322094. Another possibility is the fusion of at least the antigen-binding region of the antibody of the present invention to a protein capable of binding to a serum protein such as human serum albumin, in order to increase the half-life of the resulting molecule. Such an approach is described, for example, by Nygren et al., EP0486525.

[0131] Polysialylation is another technique that uses naturally occurring polymer polyacylic acids (PSAs) to extend the activity lifetime and improve the stability of therapeutic peptides and proteins. PSAs are polymers of sialic acid (sugar). Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies.

[0132] Further aspects of the present invention relate to host cells transfected, infected, or transformed with the nucleic acids and / or vectors of the present invention.

[0133] Therefore, according to another main embodiment, the present invention relates to nucleic acids encoding the antibody or its antigen-binding fragment.

[0134] According to another main embodiment, the present invention relates to a vector comprising the nucleic acid of the present invention.

[0135] According to another main embodiment, the present invention relates to a host cell comprising the nucleic acid or vector of the present invention; in particular to a eukaryotic cell comprising the nucleic acid or vector of the present invention. Such a host cell can be isolated by any method known in the art.

[0136] According to another main embodiment, the present invention is - Isolated antibodies or antigen-binding fragments thereof, or nucleic acids, or vectors, or host cells according to the present invention; and - Pharmaceutically acceptable excipients This relates to a pharmaceutical composition containing [a specific ingredient / component].

[0137] According to certain embodiments, isolated antibodies or their antigen-binding fragments, or nucleic acids, or vectors, or host cells, or pharmaceutical compositions are for use as pharmaceuticals or for use in in vivo diagnostic methods.

[0138] In particular, isolated antibodies or their antigen-binding fragments, or nucleic acids, or vectors, or host cells, or pharmaceutical compositions are intended for use in methods for regulating reproductive capacity in humans or non-human mammals.

[0139] According to certain specific embodiments, isolated antibodies or their antigen-binding fragments, or nucleic acids, or vectors, or host cells, or pharmaceutical compositions are intended for use in methods for reducing or suppressing reproductive capacity in human or non-human mammals.

[0140] According to certain specific embodiments, isolated antibodies or their antigen-binding fragments, or nucleic acids, or vectors, or host cells, or pharmaceutical compositions are intended for pharmaceutical use in uncastrated humans or uncastrated non-human mammals.

[0141] According to certain specific embodiments, isolated antibodies or their antigen-binding fragments, or nucleic acids, or vectors, or host cells, or pharmaceutical compositions are intended for use as drugs against castrated humans or uncastrated non-human mammals.

[0142] Beyond sterilization similar to surgical procedures, the antibodies and their antigen-binding fragments of the present invention are also suitable for reducing the consequences of surgical castration (e.g., secondary effects), such as urinary incontinence and lymphoma due to LH overexpression in females whose ovaries have been removed (as described in Kutzler, 2020, Possible Relationship between Long-Term Adverse Health Effects of Gonad Removing Surgical Sterilization and Luteinizing Hormone in Dogs, Animals).

[0143] In fact, GnRH is physiologically regulated by negative feedback from estrogen / progesterone. When the ovaries of female dogs are removed (sterilization), estrogen / progesterone is not produced, and therefore GnRH production is not regulated and becomes overexpressed. This also induces overexpression of LH, which is a factor in urinary incontinence and cancer, and therefore these can be prevented or reduced.

[0144] According to certain embodiments, isolated antibodies or their antigen-binding fragments, or nucleic acids, or vectors, or host cells, or pharmaceutical compositions are for use in the treatment or prevention of sex hormone-related disorders and / or GnRH-related disorders.

[0145] The use of isolated antibodies or their antigen-binding fragments, nucleic acids, vectors, host cells, or pharmaceutical compositions of the present invention for the manufacture of pharmaceuticals is further disclosed herein.

[0146] According to another main embodiment, the present invention relates to an in vitro method for detecting gonadotropin-releasing hormone (GnRH), a) A step of providing a sample, particularly a biological sample or a fraction thereof; b) The step of contacting the sample with the isolated antibody or its antigen-binding fragment according to the present invention. Regarding methods including

[0147] According to a particular embodiment, the in vitro method of the present invention for detecting gonadotropin-releasing hormone (GnRH) further comprises the step of detecting the interaction of the isolated antibody or its antigen-binding fragment with GnRH in a sample, thereby detecting GnRH in the sample.

[0148] The sample may be any sample suspected of containing GnRH, more specifically, any natural GnRH such as human GnRH, porcine GnRH, bovine GnRH, horse GnRH, sheep GnRH, dog GnRH, and cat GnRH, for example, any biological sample.

[0149] Biological samples suspected of containing GnRH may include blood, plasma, serum, urine, amniotic fluid, tissue extracts, tissue fluid, in vitro cell culture supernatant, cell lysates, or fractions thereof.

[0150] According to a particular embodiment, the in vitro method of the present invention for detecting gonadotropin-releasing hormone (GnRH) further includes the step of comparing the amount of GnRH in a sample with a reference value.

[0151] According to another main embodiment, the present invention relates to a method for isolating an anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, a) Providing a library of single-domain antibodies (SdAbs) or their antigen-binding fragments; b) A step of subjecting the library to antigen affinity selection, characterized in that the selected SdAb or fragment thereof has specificity for both the N-terminal region of natural GnRH (pGlu-His-Trp) and the C-terminal region of natural GnRH (Pro-Gly-NH2), the above step Regarding methods including

[0152] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment is a library of the antigen-binding fragment V H It may be characterized by containing or being derived from an H polypeptide.

[0153] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment may be characterized in that the selected SdAb is selected for binding to a first polypeptide of the sequence of SEQ ID NO: 153 and to a second polypeptide of the sequence of SEQ ID NO: 154.

[0154] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment may be characterized in that the GnRH is selected from the group consisting of human GnRH, porcine GnRH, bovine GnRH, horse GnRH, sheep GnRH, dog GnRH, and feline GnRH.

[0155] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment may be characterized by comprising a series of steps comprising subjecting a library to antigen affinity selection, wherein the selected SdAb or its antigen-binding fragment is characterized by having specificity for both the N-terminal region (pGlu-His-Trp) and the C-terminal region (Pro-Gly-NH2) of natural GnRH.

[0156] Advantageously, one or more steps of subjecting the library to antigen affinity selection may further include a step of separating polypeptides that have become bound to single-domain antibodies (SdAbs) or their antigen-binding fragments from unbound ones.

[0157] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment is, step b), b1) A sub-step in which a library of single-domain antibodies (SdAb) or their antigen-binding fragments is contacted with one or more polypeptides or analogs comprising the N-terminal region of natural GnRH (pGlu-His-Trp) and the C-terminal region of natural GnRH (Arg-Pro-Gly-NH2); b2) A sub-step in which the polypeptide(s) (one or more) are subjected to at least one washing step; b3) A sub-step in which polypeptides that have become bound to a single-domain antibody (SdAb) or its antigen-binding fragment are separated from unbound polypeptides by separation through an organic phase, thereby separating candidate binding partners from other library components. It may be characterized by including

[0158] According to certain specific embodiments, a method for isolating an anti-GnRH antibody or its antigen-binding fragment may be characterized by comprising: a first step of contacting a library of single-domain antibodies (SdAbs) or their antigen-binding fragments with one or more polypeptides or analogs comprising the N-terminal region (pGlu-His-Trp) of natural GnRH; and a subsequent (e.g., second) step of contacting the library of single-domain antibodies (SdAbs) or their antigen-binding fragments with one or more polypeptides or analogs comprising the C-terminal region (Arg-Pro-Gly-NH2) of natural GnRH.

[0159] According to certain specific embodiments, a method for isolating an anti-GnRH antibody or its antigen-binding fragment may be characterized by comprising: a first step of contacting a library of single-domain antibodies (SdAbs) or their antigen-binding fragments with one or more polypeptides or analogs comprising the C-terminal region of natural GnRH (Arg-Pro-Gly-NH2); and a subsequent (e.g., second) step of contacting the library of single-domain antibodies (SdAbs) or their antigen-binding fragments with one or more polypeptides or analogs comprising the N-terminal region of natural GnRH (pGlu-His-Trp).

[0160] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment is, step b), b1) A sub-step in which a library of single-domain antibodies (SdAbs) or their antigen-binding fragments is contacted with one or more polypeptides or analogs consisting of the N-terminal region (pGlu-His-Trp) of natural GnRH; b2) A sub-step in which the polypeptide(s) (one or more) are subjected to at least one washing step; b3) A sub-step comprising separating a polypeptide that has become bound to a single-domain antibody (SdAb) or its antigen-binding fragment from an unbound polypeptide by separation through an organic phase, thereby separating the candidate binding partner from other library components; b4) A sub-step in which a candidate binding partner from the preceding step is contacted with one or more polypeptides or analogs thereof consisting of the C-terminal region of natural GnRH (Arg-Pro-Gly-NH2); b5) A sub-step in which the polypeptide(s) (one or more) are subjected to at least one washing step; b6) A sub-step comprising separating a polypeptide that has become bound to a single-domain antibody (SdAb) or its antigen-binding fragment from an unbound polypeptide by separation through an organic phase, thereby separating the candidate binding partner from other library components. It may be characterized by including

[0161] According to a particular embodiment, a method for isolating an anti-GnRH antibody or its antigen-binding fragment is, step b), b1) A sub-step in which a library of single-domain antibodies (SdAbs) or their antigen-binding fragments is contacted with one or more polypeptides or analogs consisting of the C-terminal region of natural GnRH (Arg-Pro-Gly-NH2); b2) A sub-step in which the polypeptide(s) (one or more) are subjected to at least one washing step; b3) A sub-step comprising separating a polypeptide that has become bound to a single-domain antibody (SdAb) or its antigen-binding fragment from an unbound polypeptide by separation through an organic phase, thereby separating the candidate binding partner from other library components; b4) A sub-step in which a candidate binding partner from the preceding step is contacted with one or more polypeptides or analogs thereof consisting of the N-terminal region (pGlu-His-Trp) of natural GnRH; b5) A sub-step in which the polypeptide(s) (one or more) are subjected to at least one washing step; b6) A sub-step comprising separating a polypeptide that has become bound to a single-domain antibody (SdAb) or its antigen-binding fragment from an unbound polypeptide by separation through an organic phase, thereby separating the candidate binding partner from other library components. It may be characterized by including

[0162] A library of SdAb or its antigen-binding fragments may include, or be derived from, a library of viral particles, each of which presents several different fusion proteins on its surface, and each fusion protein contains at least a portion of SdAb or its antigen-binding fragment conjugated to a fibrous phage coat protein.

[0163] A method for regulating the reproductive capacity of non-human mammals, preferably companion animals, livestock, exotic animals, wild animals and zoo animals, comprising the step of administering the anti-GnRH antibody of the present invention or its antigen-binding fragment to the animal, is further disclosed herein.

[0164] In particular, the methods described above are suitable for reducing or suppressing reproductive capacity in humans and non-human animals.

[0165] A method for adjusting, preferably reducing or suppressing, the porky odor of animal-derived meat, comprising the step of administering an anti-GnRH antibody or an antigen-binding fragment thereof to the animal, is further disclosed herein; the animal is, in particular, a pig, such as an uncastrated male pig.

[0166] A method for improving the quality of meat by reducing pork odor, comprising the step of administering an anti-GnRH antibody or an antigen-binding fragment thereof to an animal, is further disclosed herein; the animal is, in particular, a pig such as an uncastrated male pig.

[0167] general definition As used herein, the terms “GnRH” or “gonadotropin-releasing hormone” or “LHRH” or “luteinizing hormone-releasing hormone” are interchangeable. The primary antigens considered in this disclosure are mammalian GnRHs, particularly human and non-human (e.g., livestock and / or pig-related) GnRHs, but the terms are also intended to include other non-mammalian GnRHs without contrary indication. In particular, GnRHs may be selected from the group consisting of human GnRH, porcine GnRH, bovine GnRH, horse GnRH, sheep GnRH, dog GnRH, and feline GnRH. A polypeptide sequence of GnRH that may be used as a reference is shown below: (Pyr)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Sequence ID 152) It is a mammalian or porcine GnRH characterized by the following:

[0168] As used herein, the term “N-terminal region of natural GnRH” means the blocked amino-terminal region of natural GnRH, which contains a pyroglutamic acid residue (pGlu) consisting of the following reference sequence: pGlu-His-Trp.

[0169] As used herein, the term “C-terminal region of natural GnRH” means the C-terminal region of natural GnRH, including the C-terminal amidated carboxyl terminus consisting of the following reference sequence: Arg-Pro-Gly-NH2.

[0170] As used herein, the terms “antibody” and “immunoglobulin” have the same meaning and are used equally in this invention. As used herein, “antibody” refers to an immunoglobulin molecule and a molecule containing an immunoactive site of an immunoglobulin molecule, i.e., an antigen-binding site that immunologically binds to an antigen. Therefore, the term “antibody” encompasses not only the entire antibody molecule but also antibody fragments and antibody variants (including derivatives) and antibody fragments. Thus, unless otherwise specified, this term encompasses both conventional antibodies and non-conventional antibodies, including single-domain antibodies, bispecific antibodies, multispecific antibodies, and the like.

[0171] As used herein, the term “unconventional antibody” may in particular include engineered antibodies, such as bispecific antibodies and heavy chain antibodies, or single-domain antibodies, such as those selected from VHH types and those characterized as a single heavy chain variable domain of VNAR type antibodies.

[0172] As used herein, the term “single-domain antibody” has its general meaning in the art and refers to a single heavy-chain variable domain of an antibody of the type found, for example, in naturally occurring camel mammals and sharks that lack a light chain.

[0173] As used herein, a VHH-type single-domain antibody is also referred to as VHH or V H Also known as H or “nanobody (registered trademark)”. For a general description of (single) domain antibodies, see the prior art cited above, as well as EP0368684, Ward et al. (Nature, October 12, 1989; 341(6242): pp. 544-546), Holt et al., Trends Biotechnol., 2003, 21(11): pp. 484-490; and WO06 / 030220, WO06 / 003388.

[0174] Camel immunoglobulins can be genetically modified to produce small proteins with high affinity for targets, yielding low molecular weight antibody-derived proteins known as "nanobodies" or "VHHs." See U.S. Patent No. 5,759,808, issued June 2, 1998; Stijlemans, B. et al., 2004 J Biol Chem 279: pp. 1256-1261; Dumoulin, M. et al., 2003 Nature 424: pp. 783-788; Pleschberger, M. et al., 2003 Bioconjugate Chem 14: pp. 440-448; Cortez-Retamozo, V. et al., 2002 Int J Cancer 89: pp. 456-462; and Lauwereys, M. et al., 1998 EMBO J 17: pp. 3512-3520. Engineered libraries of camel antibodies and antibody fragments are commercially available, for example, from Ablynx in Ghent, Belgium. In certain embodiments herein, camel antibodies or nanobodies are naturally produced in camel animals, i.e., produced by camels after immunization with GnRH or its peptide fragments, using the techniques described herein for other antibodies, in accordance with this disclosure. Alternatively, GnRH-conjugated camel nanobodies are engineered, i.e., produced, by selection from a library of phages presenting appropriately mutagenerated camel nanobody proteins, for example, using a GnRH-targeted panning procedure.

[0175] As used herein, the term "VNAR" has its general meaning in the art and refers to a single heavy chain variable domain of an antibody type found in naturally occurring, light-chain-deficient sharks. Ig-like molecules consisting only of the IgNAR (immunoglobulin neoantigen receptor) heavy chain have been identified in all shark species studied to date. These are disulfide-bonded homodimer molecules composed of two polypeptide chains containing five constant domains and one variable region (VNAR) that bind to the antigen (Greenberg et al., Nature March 9, 1995; 374(6518): pp. 168-73). VNARs are small (12 kDa), stable, soluble, monomeric antigen-binding domains that can form a variety of therapeutic modalities. Isolation of various VNAR-based binding sites has been described (see, e.g., WO2003 / 014161 and WO2005 / 118629). They possess an extended CDR3 structure that potentially extends into cracks and cavities in the antigen.

[0176] The term “single-domain antibody” may further encompass different antibodies having at least one single-domain antibody of the present invention that specifically binds to GnRH, and at least one other binding unit (i.e., for a different epitope, antigen, target, protein, or polypeptide) that is typically also a single-domain antibody. Such polypeptides are referred to herein as “multispecific” polypeptides, in contrast to polypeptides containing the same single-domain antibody (“monospecific” polypeptides). Thus, in some embodiments, the polypeptides of the present invention may also provide at least one further binding site for any desired protein, polypeptide, antigen, antigenic determinant, or epitope. Such binding sites may be for the same protein, polypeptide, antigen, antigenic determinant, or epitope as the single-domain antibody of the present invention, or for a different protein, polypeptide, antigen, antigenic determinant, or epitope than the single-domain antibody of the present invention.

[0177] Typically, one or more additional binding sites may include one or more portions, fragments, or domains of conventional chain antibodies (and especially human antibodies) and / or heavy chain antibodies. For example, the single-domain antibody of the present invention may optionally be linked to a conventional (typically human) VH or VL via a linker sequence.

[0178] In some embodiments, the term “single-domain antibody” may further encompass at least one single-domain antibody that is optionally linked to one or more (typically mammalian or even human) CH1 and / or CH2 and / or CH3 domains via a linker sequence.

[0179] For example, a single-domain antibody linked to a suitable CH1 domain may be used, for example, with a suitable light chain to produce an antibody fragment / structural analogue of a conventional Fab fragment or F(ab')2 fragment, where one or (in the case of the F(ab')2 fragment) one or both of the conventional VH domains are replaced by the single-domain antibody of the present invention. In some embodiments, one or more single-domain antibodies of the present invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (e.g., two or three constant domains that may be used as part of an Fc site and / or form an Fc domain), an Fc site, and / or one or more antibody moieties, fragments, or domains that can confer one or more effector functions and / or the ability to bind to one or more Fc receptors to the polypeptide of the present invention. For example, for this purpose, one or more further amino acid sequences may include one or more CH2 and / or CH3 domains of an antibody, such as those derived from a heavy chain antibody, and more typically from a conventional human chain antibody; and / or may form an Fc region derived from another human Ig, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, or IgA, IgD, or IgM. For example, WO94 / 04678 describes a heavy chain antibody comprising a camel VHH domain or a humanized derivative thereof (i.e., a single-domain antibody), where the camel CH2 and / or CH3 domains are substituted with human CH2 and CH3 domains to provide an immunoglobulin consisting of two heavy chains, each containing a single-domain antibody and human CH2 and CH3 domains (but not a CHI domain), the immunoglobulin having effector functions provided by the CH2 and CH3 domains, and the immunoglobulin may function without the presence of any light chains.

[0180] Typically, the polypeptide of the present invention comprises the single-domain antibody of the present invention, which is fused to at least one further amino acid sequence at its N-terminus, C-terminus, or both N-terminus and C-terminus, to optionally provide a fusion protein.

[0181] As used herein, a polypeptide comprising a single single-domain antibody is referred to herein as a “monovalent” SdAb polypeptide. A polypeptide comprising, or essentially comprising, two or more single-domain antibodies of the present invention is referred herein as a “polyvalent” SdAb polypeptide.

[0182] As used herein, the term “antigen-binding fragment” refers to the full-length antibody or one or more fragments that possess the ability to specifically bind to GnRH. The antigen-binding function of an antibody may be performed by fragments of the full-length antibody. Examples of binding fragments that fall within the scope of the term “antigen-binding fragment” of an antibody include the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond in a hinge region; the Fd fragment, consisting of VH and CH1 domains; the Fv fragment, consisting of VL and VH domains of a single arm of the antibody; the dAb fragment, consisting of a VH domain (Ward et al., 1989 Nature 341: pp. 544-546); or any fusion protein containing such antigen-binding fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that allows the VL and VH regions to pair up to form a monovalent single-chain protein (known as single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242: pp. 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85: pp. 5879-5883). Such single-chain antibodies are also intended to be included within the scope of the term "antigen-binding fragment" of an antibody.

[0183] As used herein, the term "dsFv" refers to a VH::VL heterodimer stabilized by a disulfide bond. Bivalent and multivalent antibody fragments may be formed spontaneously by the association of monovalent scFvs, or they may be produced by coupling monovalent scFvs with a peptide linker, such as bivalent sc(Fv)2. Such single-chain antibodies may contain one or more antigen-binding sites or antibody fragments.

[0184] A unibody is another type of antibody fragment that lacks the hinge region of an IgG4 antibody. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and possesses a monovalent binding region rather than a bivalent binding region of the IgG4 antibody. Antigen-binding fragments can be incorporated into single-domain antibodies, SMIPs, maxibodies, minibodies, intrabodies, diabodies, triabodies, and tetrabodies (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, pp. 1126-1136). The terms "diabody," "triabody," or "tetrabody" refer to small antibody fragments having multivalent antigen-binding sites (2, 3, or 4), which contain a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to form a pair between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, resulting in two antigen-binding sites. The antigen-binding fragment can be incorporated into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with a complementary light chain polypeptide, forms a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10), 1995; pp. 1057-1062 and U.S. Patent No. 5,641,870).

[0185] These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as intact antibodies.

[0186] As used herein, the term "variable," as in "variable domain," refers to a specific portion of a relevant binding protein whose sequence differs extensively between or between antibodies, and is used in specific recognition of a particular target and binding to a particular antibody. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated segments called complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), also known as hypervariable regions, and is present in both the light-chain and heavy-chain variable domains in the context of conventional antibodies. The more highly conserved portion of the variable domain is called the framework (FR) region or sequence.

[0187] As used herein, "VH domain" or "V H The term "domain" can be used interchangeably to refer to the corresponding heavy chain immunoglobulin variable domain.

[0188] As used herein, "VL domain" or "V L The term "domain" can be used interchangeably and, where applicable, refers to the corresponding light chain immunoglobulin variable domain.

[0189] As used herein, the term “hypervariable region” means an amino acid residue of an antibody that is responsible for antigen binding. This term may be substituted for the terms “complementarity-determining region” or “CDR.”

[0190] Therefore, as used herein, “complementarity-determining region” or “CDR” refers to an amino acid sequence that defines both the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of conventional immunoglobulins each contain three CDRs, named CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, respectively. Thus, a conventional antibody-antigen-binding domain contains six CDRs, including sets of CDRs derived from the heavy and light chain variable regions, respectively.

[0191] The VHH-type antibody-antigen binding domain, which differs from conventional types, includes three CDRs, also reported herein as CDR1, CDR2, and CDR3.

[0192] Examples of non-conventional antibody-antigen binding domains of the VNAR type include two CDRs, also reported herein as CDR1 and CDR3, and CDR-like regions defined in the art as hypervariable region 2 (HV2) and hypervariable region 4 (HV4).

[0193] Furthermore, as used herein, the "framework region" (FR) refers to the amino acid sequence sandwiched between CDRs, i.e., the portion of immunoglobulin that is relatively conserved among different immunoglobulins in a single species (i.e., the light and heavy chain variable regions in conventional antibodies, or only the heavy chain variable region in single-domain antibodies).

[0194] The light and heavy chains of immunoglobulins each have four FRs, named FR-L1, FR-L2, FR-L3, FR-L4, and FR-H1, FR-H2, FR-H3, and FR-H4, respectively. Therefore, in a conventional antibody-antigen binding domain, the light chain variable domain may be named (FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), and the heavy chain variable domain may be named (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H)-(FR4-H3).

[0195] V H The amino acid sequence and structure of a single-domain antibody of type H can be considered to consist of 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 divided 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, a single-domain antibody may also be defined as an amino acid sequence having the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1-FR4 refer to framework regions 1-4, and CDR1-CDR3 refer to complementarity-determining regions 1-3, respectively. In the context of the present invention, the amino acid residues of a single-domain antibody are numbered according to the general numbering of VH domains provided by the International ImMunoGeneTics Information System amino acid numbering (http: / / imgt.cines.fr / ).

[0196] The VNAR protein scaffold consists of amino acid residues (aa)1-25 in the framework 1 (FW1) region; aa26-32 in the complementarity-determining region 1 (CDR1); aa33-43 in FW2; aa44-52 in the hypervariable 2 (HV2) region; aa53-85 in FW3; aa61-65 in HV4; and the (variable-length) CDR3 region and FW4 (11 residues starting with XGXG). Like all immunoglobulin family variable (V) domains, the VNAR contains two standard cysteine ​​residues that link FW1 and FW3 via disulfide bonds.

[0197] Residues in antibody variable domains are traditionally 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, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used herein. Kabat residue designation does not always directly correspond to the sequential numbering of amino acid residues in the sequence number sequence. The actual sequential amino acid sequence, whether the framework of the basic variable domain structure or the complementarity-determining region (CDR), may contain fewer or more amino acids than the strict Kabat numbering corresponding to the shortening or insertion of structural components. The accurate Kabat numbering of residues can be determined for a given antibody by alignment of homologous residues in the antibody sequence with the "standard" Kabat-numbered sequence. In conventional antibodies, the CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), 50-65 (H-CDR2), and 95-102 (H-CDR3) in the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), 50-56 (L-CDR2), and 89-97 (L-CDR3) in the Kabat numbering system. (http: / / www.bioinf.org.uk / abs / #cdrdef).

[0198] Using the Kabat system, V H It is also possible to depict the approximate boundaries of CDRs within antibody scaffolds belonging to antibodies that are different from conventional antibodies, such as H-type single-domain antibodies (SdAbs).

[0199] As used herein, the term “specificity” means the ability of an antibody to bind detectably to an epitope presented on an antigen, such as a native GnRH, while exhibiting relatively low detectable reactivity to a non-antigen protein or structure. Specificity can be determined relatively by binding or competitive binding assays using, for example, a Biacore instrument as described elsewhere herein. Specificity can be expressed, for example, by an affinity / binding activity ratio of approximately 10:1, 20:1, 50:1, 100:1, 10,000:1, or greater, between binding to a specific antigen and nonspecific binding to other unrelated molecules.

[0200] The term "affinity," as used herein, refers to the strength of an antibody's binding to an epitope such as natural GnRH. Antibody affinity is provided by the 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 the unbound antibody, and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Coligan et al., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92: pp. 589-601 (1983), and these references are incorporated herein by reference in their entirety. A preferred standard method well known in the art for determining the affinity of mAbs is the use of a Biacore apparatus.

[0201] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," and "mAb" refer to a single-molecule antibody composition preparation. Monoclonal antibody compositions exhibit single-binding specificity and affinity for specific epitopes.

[0202] As used herein, the term “humanized” means a conventional antibody or a non-conventional antibody, such as a VHH-type single-domain antibody of the present disclosure, where the amino acid sequence corresponding to the amino acid sequence of a naturally occurring antibody (e.g., a conventional antibody or VHH domain) is “humanized” by substituting one or more amino acid residues in the amino acid sequence (and particularly in the framework sequence) of the naturally occurring antibody sequence with one or more amino acid residues occurring at the corresponding positions (one or more) in a VH domain derived from a conventional human-derived chain antibody. Methods for humanizing single-domain antibodies are well known in the art. Typically, the humanized substitution should be selected such that the resulting humanized (e.g., conventional or single-domain) antibody still retains the desirable properties of the anti-GnRH antibody of the present invention. Those skilled in the art can determine and select an appropriate humanized substitution or a suitable combination of humanized substitutions.

[0203] As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.

[0204] As used herein, the terms “treat,” “treating,” and “treatment” are intended to include alleviating or suppressing one or more symptoms of a disorder, disease, or condition, or a disorder, disease, or condition; or alleviating or eliminating one or more causes of the disorder, disease, or condition itself.

[0205] As used herein, the terms “prevent,” “preventing,” and “prevention” mean reducing the risk of developing or delaying the development of a given phenomenon, namely, in the present invention, cancer and / or dysplasia, and more specifically, a precancerous condition, early-stage cancer, or non-metastatic cancer. The term “prevention” also includes “reducing the likelihood of development” or “reducing the likelihood of recurrence.”

[0206] As used herein, the terms “subject” or “patient” may, without distinction, refer to all animals expressing GnRH or its analogues, and in particular include humans and non-human mammals. Mammalian species that could benefit from the methods of treatment disclosed include, but are not limited to, humans, apes, chimpanzees, monkeys and orangutans and other non-human primates, domestic animals including dogs and cats, zoo animals and livestock such as horses, cattle, dairy cows, pigs, sheep and goats, or, but are not limited to, rodents such as mice and rats, guinea pigs, rabbits and hamsters and other mammalian species.

[0207] As used herein, the term “sex hormone-related disorders” refers to both male-specific and female-specific sex hormone-related disorders. Such sex hormone-related disorders may or may not be directly related to a deficiency in GnRH expression or function. In a non-exclusive manner, the following disorders are considered in this disclosure: endometriosis, uterine fibroids, polycystic ovary disease, hirsutism, precocious puberty, delayed puberty, ovarian hyperstimulation syndrome, premenstrual syndrome, endometriosis, sleep apnea, irritable bowel syndrome, gonadosteroid-dependent neoplasia such as cancer of the pancreas, prostate, breast, uterus, endometrium and ovaries, gonadotropin-producing cell pituitary adenoma, benign prostatic hyperplasia, uterine leiomyoma, acne vulgaris, acute intermittent porphyria, uterine fibrosis, benign prostatic hyperplasia, etc.

[0208] As used herein, “GnRH-related disorder” more specifically refers to a condition that prevents the activation of GnRH receptors. Exemplary GnRH-related disorders 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 gonadotrophe adenoma, endometriosis, polycystic ovary syndrome, uterine fibroids, primary hirsutism, luteinizing hormone hypertension, and precocious puberty.

[0209] As used herein, “pharmaceutically acceptable carrier” is intended to include all carriers (such as any solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders) that are suitable for the administration of pharmaceuticals, particularly parenteral administration. The use of such media and agents for pharmaceutically active substances is well known. Such media may be used in the compositions of this disclosure unless any conventional media or agent is not suitable for the active compound. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions or suspensions, including physiological saline and buffered media. In a non-exclusive 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% physiological saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate solution, or non-volatile oils. Intravenous vehicles include fluid and nutrient replenishers and electrolyte solutions, such as those based on Ringer's dextrose. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In such cases, the composition must be sterile and fluid enough to allow for easy syringeability. It must be stable under manufacturing and storage conditions, and in some embodiments, it is stored in a manner that prevents contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof.For example, proper fluidity can be maintained by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In certain embodiments, the composition contains isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0210] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise clearly indicated by the context. For example, the term "pharmaceutically acceptable carriers" includes multiple pharmaceutically acceptable carriers, including mixtures thereof.

[0211] As used herein, “multiple” may therefore include “two” or “more than two.”

[0212] As used herein, and unless otherwise indicated, the term “at least one” may include “one or more” or even “two or more” (or “plural”). For example, this term may include 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 , may include 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 more than 100.

[0213] As used herein, and unless otherwise indicated, the term “less than” may encompass all values ​​from 0 up to the corresponding threshold. For example, this term may include, where applicable, 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, less than 100 or less than 100.

[0214] As used herein, “comprise” or “comprising” may include “composed of” or “consisting of.”

[0215] As used herein, the term “cell” may encompass any prokaryotic or eukaryotic cell. Cell types of particular consideration are those suitable for the production and / or engineering of recombinant antibodies, or their fragments, or polypeptide chains. In a less comprehensive 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.

[0216] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cell. Host cells include "transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not have exactly the same nucleic acid content as the parental cell and may contain mutations. Mutated progeny having the same function or biological activity, screened or selected in the original transformed cell, may also be included. A host cell can be any type of cell line that can be used to produce the binding protein of the present disclosure. Thus, host cells can include cultured cells, such as mammalian cultured cells, for example, only to name a few, CHO cells, HEK cells, BHK cells, NS0 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.

[0217] The term "transformed" means the introduction of an "exogenous" (i.e., foreign or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell expresses 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 has been "transformed". Using the nucleic acids of the present invention, the anti-GnRH antibodies of the present invention can be generated in a suitable expression system.

[0218] As used herein, the term "expression system" means a host cell and a compatible vector that are under conditions suitable for the expression of a protein encoded by exogenous DNA carried by a vector and introduced into a host cell. General 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 (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (produced from, for example, lymphocytes, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples 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" in this specification) (Urlaub G et al.; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells" in this specification), etc. The present invention also relates to a method for generating a recombinant host cell that expresses an antibody of the present invention, the method comprising: (i) introducing the above-described recombinant nucleic acid or vector into competent host cells in vitro or ex vivo; (ii) culturing the obtained recombinant host cells in vitro or ex vivo; and (iii) optionally, selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used for the production of the antibody of the present invention. The anti-GnRH antibody of the present invention is appropriately separated from the medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0219] As used herein, “vector,” “cloning vector,” and “expression vector” mean a vehicle capable of introducing a nucleic acid sequence, such as DNA or RNA (e.g., an exogenous gene), into host cells in order 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 and may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0220] Therefore, further aspects of the present invention relate to vectors comprising nucleic acids of the present invention. Such vectors may include regulatory elements such as promoters, enhancers, and terminators for inducing or directing the expression of the antibody upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the initial promoter and enhancer of SV40 (Mizukami T. et al., 1987), the LTR promoter and enhancer of Moloney's mouse leukemia virus (Kuwana Y et al., 1987), and the promoter (Mason JO et al., 1985) and enhancer (Gillies SD et al., 1983). Any expression vector for animal cells can be used, as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al., 1990), pAGE103 (Mizukami T et al., 1987), pHSG274 (Brady G et al., 1984), pKCR (O'Hare K et al., 1981), and pSG1 beta d2-4- (Miyaji H et al., 1990). Other examples of plasmids include replication plasmids or integration plasmids containing replication origins, such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenovirus, retrovirus, herpesvirus, and AVV vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfecting packaging cells or transient introduction of helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94 / 19478.

[0221] In particular, the term "vector" may refer to viral particles that have specific tissue tropisms, such as muscle tropism (e.g., AAV types), and may include, for example, AAV8 and AAV9.

[0222] In particular, the terms "retrovirus" or "retrovirus" may refer to lentiviruses or lentiviral vectors.

[0223] As used herein, the terms “recombinant AAV,” “rAAV virion,” and “AAV particle” are defined herein as an infectious replication-deficient virus containing an AAV protein shell in which the AAV ITR capsidizes the desired heterogeneous nucleotide sequence on both sides. rAAV virions are produced by a suitable host cell that possesses a sequence identifying the AAV plasmid, AAV helper function, and introduced auxiliary function. In this manner, the host cell becomes capable of encoding the AAV polypeptide necessary to package the AAV plasmid (containing the desired recombinant nucleotide sequence) into an infectious recombinant virion particle for subsequent gene delivery.

[0224] "Recombinant viruses" refer to viruses that have been genetically modified, for example, by adding or inserting heterologous nucleic acid constructs into particles.

[0225] The term "AAV virion" refers to a complete viral particle, such as a wild-type (wt) AAV virus particle (including a linear single-chain AAV nucleic acid genome associated with the AAV capsid protein coat). In this respect, a single-chain AAV nucleic acid molecule of either a complementary sense, e.g., "sense" or "antisense" chain, may be packaged in any one AAV virion, and both chains are equally infectious. Thus, without further limitation, the term may encompass any AAV selected from the group consisting of serotypes derived from rhesus monkeys, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, AAV10, and AAVrh10, as well as mixtures thereof.

[0226] "Isolated nucleic acid molecules or polynucleotides" refers to nucleic acid molecules, DNA, or RNA that have been removed from their natural environment. For example, recombinant polynucleotides encoding polypeptides contained in vectors are considered to be isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides (partially or substantially) purified in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain polynucleotide molecules, but which are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-strand and negative-strand forms, and double-strand forms. Isolated polynucleotides or nucleic acids of this disclosure further include synthetically produced molecules. Polynucleotides or nucleic acids may also be regulatory elements such as promoters, ribosome-binding sites, or transcription terminators, or may contain such regulatory elements.

[0227] As used herein, the terms “isolated” or “purified” mean molecules that have been modified by humans in their natural state, i.e., molecules that have been altered and / or removed from their original environment if they exist in nature.

[0228] As used herein, the “identity percentage” between two sequences of nucleic acids or proteins (e.g., GnRH-binding antibodies) means the percentage of identical nucleotide or amino acid residues between the two sequences being compared, obtained after optimal alignment. This percentage is purely statistical, and the difference between the two sequences is randomly distributed along their lengths. Sequence comparisons are traditionally performed by comparing the sequences after optimal alignment, and such comparison may be performed by segmentation or by using an “alignment window.” The optimal sorting of sequences for comparison can be performed not only by manual comparison, but also by the local homology algorithm of Smith and Waterman (1981), the local homology algorithm of Neddleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), or by computer software using these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI's GAP, BESTFIT, FASTA and TFASTA, or the comparison software BLAST NR or BLAST P).

[0229] The identity percentage between two sequences is determined by comparing two optimally aligned sequences, where the sequence being compared may have additions or deletions compared to a reference sequence, for optimal alignment between the two sequences. The identity percentage is calculated by determining the number of positions where nucleotide or amino acid residues are identical between the two sequences, preferably between the two complete sequences; dividing the number of identical positions by the total number of positions in the alignment window; and multiplying the result by 100 to obtain the identity percentage between the two sequences.

[0230] As used herein, the term “amino acid” means natural or non-natural amino acids, including their D and L stereoisomers for chiral amino acids. It is understood to include both amino acids and their corresponding amino acid residues, for example, those present in a peptidyl 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). Unusual non-natural amino acids include, but are not limited to, allylglycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphthylalanine (2-Nal), ornithine (Orn), and pentafluorophenylalanine.

[0231] As used herein, a “modified form” of a given amino acid includes any equivalent translationally modified or post-translationally modified amino acids or non-naturally occurring amino acids, including non-standard amino acids, resulting from reactions at the amino group, carboxyl group, side-chain functional group, or substitution with any hydrogen heteroatom. Accordingly, modified amino acids may include amino acid derivatives resulting from any one of the modifications selected from N-linked glycosylation, O-linked glycosylation, phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin linkage, cysteine ​​oxidation, nitrosylation, and ubiquitination.

[0232] As used herein and above, “equivalent amino acid” means an amino acid that can be substituted (i.e., through conservative substitution) for another amino acid in the peptide compound of the present invention without any apparent loss of function. Equivalent amino acids are recognizable to those skilled in the art. Substitutions of similar amino acids are made on the basis of the relative similarity of the side-chain substituents with respect to size, charge, hydrophilicity and hydrophobicity, as described herein, for example. The term “or its equivalent amino acid” when used following a list of individual amino acids means one or more equivalents of the individual amino acids included in the list.

[0233] Exemplary examples, a conservative substitution can be defined by a substitution within a class of amino acids, as reflected below: Aliphatic residues I, L, V, and M Cycloalkenyl-related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G and S Residues A, C, D, E, G, H, K, N, Q, R, S, P involved in rotation, and residue T involved in formation. Flexible residues Q, T, K, S, G, P, D, E and R

[0234] As used herein, the terms "phage display" and "bacteriophage display" are considered synonymous and refer to the technique of presenting variant polypeptides as fusion proteins on the coat proteins on the surface of bacteriophage particles (Scott, J.K. and Smith, G.P. (1990) Science 249: 386). Selecting a phage library of random mutants requires a strategy for constructing and growing a large number of variants, a procedure for affinity purification using a target receptor, and a means for evaluating the results of binding enrichment. Unless otherwise indicated, the term is not limited to one particular type of bacteriophage particle, nor to one particular type of fusion protein and / or enrichment strategy and / or particle phage library.

Example

[0235] A. Materials and Methods Immunization Single domain antibodies were obtained from llamas immunized with six injections at two-week intervals. Eight days after the last boost, serum was collected and the antibody titer against the GnRH peptide was determined by ELISA. In this ELISA, 96-well plates (Maxisorp; Nunc) were coated with two biotinylated GnRH peptides. After blocking and addition of diluted serum samples, the presence of anti-GnRH antibodies was demonstrated by using mouse anti-llama IgG (FJB, catalog number FJ1203MAB01B + 09) and then donkey anti-mouse IgG-HRP antibody (JIR, catalog number 715-035-150).

[0236] Library Construction 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 used in initial PCR amplification using untagged primers annealing at the leader sequence and hinge CH1 region, and then in secondary PCR amplification to introduce restriction endonuclease sites for cloning the VHH gene in a pDCL1 phagemide vector. The library was electroporated into TG1 E. coli cells, and bacterial glycerol stocks of the immunotherapy library were stored at -80°C.

[0237] Phage display selection In three consecutive rounds of phage display selection using biotinylated GnRH peptide 1 (also known herein as "biopeptide 1"), Rama V H Phage generation from a 1H library pool was used. The first round of selection (Round I) was performed on biotinylated GnRH peptide 1, pre-captured on a 5 nM neutraavidin (5 μg / mL, ThermoFisher; catalog number 31000) coated plate. Nonspecific phages were washed away (PBS buffer, pH 7.4), and then specific phages were eluted with trypsin (total elution). A second round of selection (Round II) was performed with a smaller number of phages (1:5 the volume of phages used in Round I).

[0238] Overall, the second round of selection was achieved by following two different strategies.

[0239] Following one strategy, biotinylated GnRH peptide 1 was pre-captured on neutraavidin plates at concentrations of 5 and 0.5 nM.

[0240] Following the second strategy, the same peptide was directly coated at 1 μG / mL.

[0241] Nonspecific phages were washed (PBS buffer, pH 7.4), and then specific phages were eluted with trypsin (total elution).

[0242] Phages derived from both round II strategies were then selected under the third round (round III), where the number of phages was further reduced (1:10 of the volume of phages used in the first round), and biotinylated GnRH peptide 1 was pre-captured on neutraavidin plates at 0.5 and 0.05 nM. Nonspecific phages were washed (PBS buffer, pH 7.4), and then specific phages were eluted with trypsin (total elution).

[0243] Serial dilutions of eluted phages were used to infect exponentially growing TG1 phages. Infected TG1 phages were seeded onto LBCarb100Glu2% plates, and the enrichment value was calculated relative to the background (no antigen for selection).

[0244] V H ELISA screening in the context of H library pool selection Individual clones from the second and third rounds of the selection criteria output were selected into a 96-well master plate and tested for binding to biotinylated GnRH peptides 1 and 2 (also known herein as "biopeptide 2") via binding ELISA as periplasm extracts (PE).

[0245] For PE-binding ELISA, MaxiSorp® high protein-binding capacity 96-well ELISA plates were coated with 5 μg / ml neutraavidin (ThermoFisher; catalog no. 31000) and diluted in PBS overnight at 4°C. The following day, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and blocked with 1% casein / PBS at 250 μL / well for 1 hour at room temperature. After blocking, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and incubated with 100 μL / well of biotinylated GnRH peptide 1 or 2 at 5 nM diluted in 0.1% casein / PBS (pH 7.4) for 1 hour at room temperature. After capture, the plate was washed three times with PBS Tween 0.05% (pH 7.4) and incubated with 20 μl of PE and 80 μl of 0.1% casein / PBS (pH 7.4) per well at room temperature for 1 hour with shaking. The plate was washed three times with PBS Tween 0.05% (pH 7.4) and incubated with 100 μl of anti-c-Myc antibody (Roche; catalog no. 11667203001) in 0.1% casein / PBS (pH 7.4) and then with the secondary antibody donkey anti-mouse HRP (JIR; catalog no. 715-035-150) at room temperature for 1 hour with shaking. The plate was washed three times with PBS Tween 0.05% (pH 7.4) and substrate solution (TMB solution) was added to the plate. The reaction was stopped with H2SO4 and the plate was read at 450 nm on a plate reader.

[0246] Off-rate screening using surface plasmon resonance (SPR). Individual clones from the second and third rounds of the selection criteria output were selected and placed in a 96-well master plate. The offrate (kd) of the clones tested as periplasmic extract (PE) for biotinylated GnRH peptide 1 captured on a streptavidin (SA) sensor chip (Cytiva®, catalog no. BR100531) was determined via surface plasmon resonance (SPR) (Biacore 8K, GE Healthcare). Approximately 1140–1270 RU of biotinylated GnRH peptide 1 was immobilized on the SA chip via a standard streptavidin coupling method.

[0247] Fixed QC was performed using a commercially available antibody anti-GnRH (SMI41, Absolute Antibody, catalog number Ab00922-1.1) diluted in HBS-EP pH7.4 buffer at a concentration of 5 nM. 1×HBS-EP pH7.4 was used as the running buffer during the binding kinetics measurement. 20 μl of PE + 80 μl of 1×HBS-EP pH7.4 was injected at 30 μl / min for 2 minutes, followed by a 1-minute off-rate wash between injections. The off-rate wash occurred 300 seconds after the final concentration injection in each cycle. Between samples, RU levels were restored to base levels after regeneration with two injections of 15 μL of 10-glycine at pH1.5.

[0248] The data was analyzed using the multi-cycle kinetics predefined evaluation method of the Biacore Insight Evaluation software. Sensorgrams referenced blank / unfixed reference flow cells. Dissociation constants were calculated using a 1:1 dissociation Langmuir binding model.

[0249] Sequencing Those that showed a positive binding result (positive binder) were sent for sequencing. The clones were classified into families according to their different HCDR3 sequences.

[0250] Expression and purification of VHH candidate antibodies Synthetic genes classifying the VHH variable domain with FLAG and His tags were purchased and cloned into the pET15b bacterial expression vector along with the periplasmic secretion reader sequence. E. coli strain BL21 DE3 was transformed and grown in ZYP-5052 autoinducible medium at 18°C ​​for 68 hours. The produced VHH antibody was captured from the clarified supernatant using Ni-NTA beads (Qiagen) on a gravity-feed column. The eluted antibody was buffered with 1×PBS pH 7.4 and concentrated using a 10k cutoff spin concentrator (Amicon, catalog no. UFC801096D). The purified VHH protein was analyzed by SDS-PAGE for the presence of the correct strand.

[0251] Dose-response ELISA in purified VHH A MaxiSorp® high protein binding capacity 96-well ELISA plate was coated with 5 μg / ml neutraavidin (ThermoFisher; catalog number 31000) and diluted in PBS overnight at 4°C. The following day, the plate was washed three times with PBS Tween 0.05% (pH 7.4) and blocked with 1% casein / PBS at 250 μL / well for 1 hour at room temperature. After blocking, the plate was washed three times with PBS Tween 0.05% (pH 7.4) and incubated with 5 nM biotinylated GnRH peptide 1 diluted in 0.1% casein / PBS (pH 7.4) at 100 μL / well for 1 hour at room temperature. After capture, the plate was 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 fivefold in seven steps from 1000 to 0.01 nM, and added to the captured biotinylated GnRH peptide 1. The ELISA wells were then blocked at room temperature for 1 hour.

[0252] The plate was washed three times with PBS Tween 0.05% (pH 7.4), and incubated with 100 μl of anti-c-Myc antibody (Roche; catalog number 11667203001) in 0.1% casein / PBS (pH 7.4) with the secondary antibody donkey anti-mouse HRP (JIR; catalog number 715-035-150) at room temperature for 1 hour with shaking. The plate was washed three times with PBS Tween 0.05% (pH 7.4), and substrate solution (TMB solution) was added to the plate. The reaction was stopped with H2SO4, and the plate was read at 450 nm using a plate reader. The OD450 nm value versus the log concentration of VHH was plotted using GraphPad Prism 7 with nonlinear regression (curve fitting) of log (agonist) versus response-variable slope (4 parameters) applied, and EC50 was determined.

[0253] Affinity determination of purified VHH using Biacore The affinity of selected and purified clones to biotinylated GnRH peptides 1 and 2, captured on a streptavidin (SA) sensor chip (Cytiva®, catalog number BR100531) by a standard streptavidin coupling method, is evaluated.

[0254] The binding kinetics of selected single-domain antibodies were determined at pH 7.4 using surface plasmon resonance (SPR) (Biacore T200, GE Healthcare). Biotinylated GnRH peptides 1 and 2, approximately 1140–1270 RU, were immobilized on SA tips. Immobilization QC was performed using a commercially available anti-GnRH antibody (SMI41, Absolute Antibody, catalog no. Ab00922-1.1) at 5 nM diluted in HBS-EP pH 7.4 buffer. 1× HBS-EP pH 7.4 was used as the running buffer during binding kinetics measurements. Purified VHH was injected at 10 μl / min for 5 minutes at 100 nM in HBS-EP pH 7.4, followed by a 1 minute off-rate wash between injections. The off-rate wash was performed 20 minutes after the last concentration injection in each cycle. Between samples, RU levels were restored to base levels after regeneration with 15 μL of 1 M 10-glycine at pH 1.5. Data were analyzed using the Biacore Insight Evaluation software fitting's predefined binding kinetics evaluation method. The sensorgram referenced blank / unfixed reference flow cell 1. Dissociation constants were calculated using a 1:1 dissociation Langmuir binding model to determine the reaction rate constants of antibody-antigen interactions, including association rate (ka), dissociation rate (kd), and affinity (KD).

[0255] Natural protein binding by ELISA (Gly-OH10)-Luteinizing hormone-releasing hormone (Sigma-Aldrich, catalog number L8008) was used to coat Maxisorp plates at 1 μg / ml in 1×PBS overnight at 4°C. The coated plates were blocked with 4% milk in 1×PBS. The GnRH VHH antibody panel was titrated by 5-fold serial dilutions in 7 steps, starting with 30 μM in 1% milk in 1×PBS.

[0256] VHH was detected at a 1:200 titration with anti-c-myc HRP antibody (Roche, catalog no. 11 667 203 001). This included titrations with SMI41 monoclonal (Absolute Antibody, Ab00922-1.1) and GnRH1 polyclonal antibody (Invitrogen, PA1-121). This was followed by 1:10,000 titrations with anti-mouse and anti-rabbit IgG-HRP (JIR, 715-035-150 and 111-035-144).

[0257] The plate was developed using TMB solution (Invitrogen, 00-4201-56) followed by H2SO4 (Fisher Chemical, J / 8430 / 15).

[0258] Binding to natural porcine GnRH was evaluated by measuring the OD at 450 nm.

[0259] GnRH cell-based functional assay Using rat basophilic leukemia cells that stably express the human GnRH receptor, the antagonist activity of the top five VHH clones will be evaluated using a GnRH cell-based functional assay. Fluorescence quantitative detection will be used to assess GnRH-dependent cytoplasmic calcium (C) levels. 2+ The inhibitory function was determined by measuring the VHH effect on ion mobilization.

[0260] To this end, cells were suspended in HBSS buffer (Invitrogen) supplemented with 20 mM Hepes and distributed in microplates at a density of 1.186E+04 cells / well. A fluorescent probe (Fluo8 Direct, AAT Bioquest) was mixed with probenicid in HBSS buffer (Invitrogen) supplemented with 20 mM Hepes (Invitrogen, pH 7.4) and added to each well, and the cells were equilibrated at 30°C for 60 minutes. Next, five purified VHH solutions were titrated in five steps of 5-fold serial dilution starting at 100 μg / mL, added to the plate, and incubated for 5 minutes. Subsequently, 8 nM GnRH (1:1 to 1:800 ratio GnRH / VHH) was added to the plate, and free cytoplasmic Ca was added. 2+ The change in fluorescence intensity, which fluctuates in proportion to the ion concentration, was measured using a microplate reader (FlipR Tetra, Molecular Device). The standard reference antagonist, cetrorelics acetate, was tested at several concentrations in each experiment, and the IC50 value was calculated. A commercially available antibody, anti-GnRH (SMI41, Absolute Antibody, catalog number Ab00922-1.1), was also included at two different concentrations of 100 and 20 μg / mL as a comparer and positive control.

[0261] (Example 1) V to GnRH H H binding and comparison with reference antibody 1.1. Binding of SMI41 reference antibody to GnRH-derived peptide The SMI41 reference antibody corresponds to the mouse IgG1 monoclonal anti-human LHRH (anti-huLHRH) antibody sold by Absolute Antibody (number PA1-121) with a predicted molecular weight of 155 kDa.

[0262] The second reference polyclonal antibody preparation is a rabbit polyclonal anti-amphibian / mouse GnRH1 antibody (anti-GnRH1) sold by ThermoFisher (catalog number PA1-121), with a predicted molecular weight of 150 kDa.

[0263] The aim of this experiment is to perform ELISA QC on two biotinylated GnRH peptides (biopeptide 1 and biopeptide 2) by either direct coating or capture with neutraavidin. Monoclonal antibody SMI41 and polyclonal antibody are used for peptide detection, and the presence of biotin is confirmed by ExtrAvidin-HRP.

[0264] Biopeptide 1 was detected by both anti-GnRH antibodies (SMI41 and polyclonal antibodies), either directly coated or captured. A complete titration curve was obtained for the anti-GnRH SMI41 monoclonal antibody. Biopeptide 1 was directly coated, allowing for the determination of the EC50 value (0.037 μg / ml). No background signal was observed, and no specific binding was detected for the secondary control.

[0265] Biopeptide 2 is detected only by anti-GnRH polyclonal antibody. This provided complete titration curves and enabled EC50 determination, both when the peptide was directly coated or captured.

[0266] If the SMI41 antibody is the current reference antibody, this experiment shows that biopeptide 1 is the peptide with the most relevant epitope.

[0267] Biotin was confirmed in both peptides from ExtrAvidin-HRP. No background signal was observed, and binding was not detected for the secondary control only.

[0268] In conclusion, the monoclonal antibody labeled as SMI41 is unable to specifically bind to biopeptide 2, which mimics the N-terminal region of natural GnRH.

[0269] On the other hand, polyclonal antibodies bind to both the analogue of the N-terminal region (biopeptide 2) and the analogue of the C-terminal region (biopeptide 1).

[0270] (Example 1.2) V to GnRH-derived peptides H H bond and 30 related V H H clone selection Through four outputs of phage display selection, multiple V H The H clone was identified.

[0271] Using clones derived from three rounds of selection for GnRH biopeptide 1, PE-conjugated ELISA was performed on captured GnRH biopeptide 1 and biopeptide 2 in neutraavidin-coated plates. A very high hit rate (88-100%) was obtained for all four outputs of the phage display protocol, and selected V was found bound to biopeptide 1. H This means that the H clone is also bound to biopeptide 2.

[0272] Next, the aim of this experiment is also to determine the soluble V in relation to GnRH biopeptide 1 captured on the SA sensor chip. H The objective was to determine the dissociation rate constant (kd, off-rate) of PE containing H antibody. Of the 368 clones tested, 346 showed specific association to GnRH biopeptide 1 (RU>10), and the kd values ​​ranged from 3.47E-02 to 6.61E-04 (1 / sec).

[0273] The kd values ​​could not be determined for 22 clones because they either did not show specific association to GnRH biopeptide 1 or had RU levels less than 10.

[0274] No binding was observed for unrelated controls or blank controls, and no RU response was detected when buffer (1×HBS-EP+pH7.4) was used as the sample.

[0275] From 347 sequenced clones, a total of 339 valid V H H sequence (97.7%) was obtained. 103 unique V H The H sequences were identified, and eight of them were considered to be enriched sequences containing more than 10 representative clones.

[0276] Twenty-four unique HCDR3 sequences were identified, eight of which were considered enriched HCDR3 sequences possessing more than ten representative clones. These enriched HCDR3 sequences were shown in all four outputs, regardless of the selection round.

[0277] The following criteria: - Binding to both biopeptide 1 and biopeptide 2 in PE-conjugated ELISA; - Dissociation rate constant (kd, off-rate, <1E-02 1 / sec) for biopeptide 1; - Enriched HCDR3 sequence; - Enriched VHH sequences; - HCDR3 tendencies (liabilities) Based on this, a total of 30 V H H clones were selected, generated, purified, and further characterized in this manner.

[0278] (Example 1.3) Selected individual Vs to GnRH via ELISA and SPR methods H H bond The aim of this experiment is also to determine the soluble V in relation to GnRH biopeptide 1 captured on the SA sensor chip.H The objective was to determine the dissociation rate constant (kd, offrate) of PE containing H antibody.

[0279] V H The H antibody panel showed a wide range of EC50 values ​​for GnRH biopeptide 1, from 415 pM to 14 nM. Clone pGnRH-001 showed the lowest EC50 value in the panel, while clone pGnRH-019 showed the highest.

[0280] To determine the affinity of the GnRH antibody panel and the SMI41 reference antibody, GnRH biopeptide 1 and biopeptide 2 were immobilized on the SA chip.

[0281] SMI41 bound only GnRH biopeptide 1 to SPR, as previously observed in ELISA. The following kinetic parameters were obtained: - ka(1 / Msec):4.76E+06 - kd(1 / sec):2.81E-03 - KD(nM):0.59

[0282] On the other hand, all clones derived from the antibody panel were able to bind to both GnRH biopeptide 1 and GnRH biopeptide 2 when captured on the SA chip.

[0283] The VHH antibody panel showed a wide range of affinities to GnRH biopeptide 1 and GnRH biopeptide 2, from approximately 7 nM to 125 nM.

[0284] Clone 1 showed the highest affinity in the panel for both GnRH biopeptide 1 (13.5 nM) and GnRH biopeptide 2 (7.1 nM).

[0285] (Example 1.4) Selected individual V to natural porcine GnRH H H bond The aim of this experiment is to use ELISA to determine GnRH VH The objective is to determine the ability of the H antibody panel to bind to the native GnRH protein.

[0286] The aforementioned VHH clones were tested, and the corresponding titration graphs are disclosed in Figure 5.

[0287] All VHH panels, when determined as three times the GFP VHH OD value at the highest concentration, successfully bound to native porcine GnRH protein at the highest concentration (30 μM). In contrast, the SMI41 monoclonal antibody did not readily bind to native porcine GnRH protein. The polyclonal antibody correctly bound to native GnRH (as a positive control). This demonstrates a specific binding mechanism for the selected ligand that is not present in SMI41 monoclonal antibodies reported in the art.

[0288] (Example 2) V for GnRH H H antagonist activity Five selected V clones, including clone 1, clone 2, clone 9, clone 22, and clone 24. H Based on H clones, a cell-based GnRH function assay was established. The selection criteria were: - Minimum EC50; - Higher affinity (lower KD) and slowest offrate (lower KD) - Fast on-rate (higher ka) - Production yield It included.

[0289] By expressing the human GnRH receptor in rat basophilic leukemia cells, these five purified V H The antagonist activity of H was confirmed. Using a fluorescence detection method, agonist-induced cytoplasmic Ca was detected. 2+ We measured their effects on ion mobilization.

[0290] The results were expressed as the inhibition percentage of the control response to 8nM GnRH.

[0291] Cetrorelics acetate was used as a standard reference antagonist, and concentration-response curves were generated by testing at several concentrations and calculating their IC50 values, which are then reported in Figure 6.

[0292] Top 5 V H All H clones were able to inhibit GnRH in cell-based functional assays. Clones 9 and 1 showed the best profiles, with clone 9 showing a better profile than the reference antibody SMI41.

[0293] (Example 3) Alanine scanning screening of selected VHH clones To determine the key residues involved in GnRH binding, VHH clones 1 and 9 (SEQ ID NOs: 1 and 9, respectively) were further screened by alanine scanning (ELISA).

[0294] Therefore, neutraavidin (Thermo Fisher Scientific, catalog number 31000) was used to coat Maxisorp plates at 5 μg / ml in 1×PBS overnight at 4°C. The coated plates were blocked with 1% casein in 1×PBS. GnRH biopeptide 1 (SB-Peptide, lot number 10869) with the sequence of SEQ ID NO: 155 was captured at 5 nM in 0.1% casein in 1×PBS. Clones 1 and 9, and their corresponding variants, in which the CDR3 hypervariable region was substituted with the sequences of SEQ ID NOs: 159-186, were titrated by seven 5-fold serial dilutions starting at 1 μM in 0.1% casein in 1×PBS. In 0.1% casein in 1×PBS, a 1:200 dilution of anti-c-myc antibody (Roche, catalog number 11 667 203 001) was used, followed by 0.16 μg / mL of anti-mouse IgG-HRP (JIR, catalog number 715-035-150) in 0.1% casein in 1×PBS. HH was detected. Binding to GnRH biopeptide 1 was evaluated by measuring the optical density at 450 nm.

[0295] Corresponding results were obtained for clone 1 and its CDR3 variant that was tested.

[0296] [Table 1]

[0297] Corresponding results were obtained with clone 9 and its CDR3 variant that was tested.

[0298] [Table 2]

[0299] In clone 1, at a selected location within CDR3, we induced further mutations, separate from alanine. Surprisingly, the substitution of the final "D" with histidine (H) resulted in improved affinity for the antigen.

[0300] [Table 3]

[0301] Regarding the above, we obtained the following two CDR3 consensus regions for sequence sequence numbers 187 and 188.

[0302] [Table 4]

[0303] (Example 4) Some clone tendencies To evaluate these liabilities, antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 were more specifically tested for their physical and chemical stability after storage at various temperatures.

[0304] Each antibody composition was subjected to temperature changes to evaluate its stability, and in particular: - Under one test condition, the sample was subjected to 5 rounds of freeze / thaw cycles (5 × FT); - Under other test conditions, samples were stored at room temperature (25°C) for two weeks and at 40°C for two weeks.

[0305] Physical stability was evaluated by measuring concentration, turbidity (OD500), and SE-HPLC before and after stress testing. Chemical stability was evaluated by analyzing samples with RP-HPLC before and after stress testing. In addition, HIC (hydrophobic interaction chromatography) analysis was performed on non-stressed samples to rank molecules based on their hydrophobicity.

[0306] Concentration and turbidity - Equipment: UV / Vis spectrophotometer (Unchained Labs Lunatic) - Plate: Lunatic plate (2 μL, 0.5 mm optical path length, measurement range 0.03~40 OD; 10 mm) The concentration and turbidity were measured using [a specific method / tool].

[0307] SE-HPLC Prior to injection, the sample was centrifuged at 14000g for 5 minutes. The sample was analyzed using an Agilent 1100 HPLC system. Instrument control and data analysis were performed using ChemStation for LC system (rev.B.04.03-SP1).

[0308] RP-HPLC Samples were analyzed using an Agilent 1200 HPLC system. Instrument control and data analysis were performed using ChemStation for LC system (rev.B.04.03-SP1).

[0309] HIC Samples were analyzed using an Agilent 1100 HPLC system. Instrument control and data analysis were performed using ChemStation for LC system (rev.B.04.03-SP1).

[0310] The results are shown in Figure 7A (representing the area of ​​the HPLC image corresponding to the analyzed antibody) and Figure 7B (representing the percentage of the main HPLC image corresponding to the analyzed antibody). The results of the antibody stability analysis are summarized in the table shown in Figure 14.

[0311] Based on the data obtained, - All antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 are stable after being subjected to 5 freeze / thaw cycles; - All antibodies produced by clones pGnRH-001, pGnRH-002, pGnRH-009, pGnRH-022, and pGnRH-024 are stable when stored at 25°C for two weeks; - If stored at 40°C for 2 weeks, - Antibody samples derived from pGnRH-009 and pGnRH-024 showed some protein loss; - Antibody samples derived from pGnRH-001, pGnRH-002, and pGnRH-022 typically showed an increase in HMV (high molecular weight species), which is characteristic of antibody degradation; - Antibody samples derived from pGnRH-001, pGnRH-002, and pGnRH-022 showed a single variant increase of more than 5% by RP-HPLC. This was the conclusion reached.

[0312] (Example 5) Sequence optimization of clones for specific purposes Sequence optimization was continued for antibodies produced by clones pGnRH-001 and pGnRH-009, which were considered to be the desired targets.

[0313] Regarding clone 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). The sequences of these variants are shown in Figure 8A, and the mutated amino acid residues are indicated by arrows compared to the sequence of the original clone pGnRH-001.

[0314] Regarding clone pGnRH-009, two different sequence variants were generated: 09Tm1 (corresponding to sequence number 209) and 09Tm2 (corresponding to sequence number 210). The sequences of these variants are shown in Figure 8B, and the mutated amino acid residues are indicated by arrows compared to the sequence of the original clone pGnRH-009.

[0315] (Example 6) Binding of sequence variants derived from clones pGnRH-001 and pGnRH-009 to GnRH (biopeptides 1 and 2). To identify whether the sequence mutations (substitutions) included to remove physical and chemical tendencies have any effect on the clonal binding ability to the GnRH peptide, the binding properties of antibodies derived from the Tm variants, 01Tm1, 01Tm2, 01Tm3, 01Tm4, 09Tm1, and 09Tm2 (sequence variants) of clones pGnRH-001 and pGnRH-009 were evaluated using ELISA assays for biopeptides 1 and 2, as described in the Materials and Methods section, and compared with those of antibodies derived from the original clone.

[0316] The ELISA assay configuration was as follows: - MaxiSorp plates were coated with nutraavidin (Thermo Fisher Scientific, catalog number 31000) at a concentration of 5 μg / ml in 1×PBS overnight at 4°C. - Coated plates were blocked with 1% casein in 1×PBS. - GnRH biopeptide 1 (SB-Peptide, lot number 10869) and biopeptide 2 (SB-Peptide, lot number 10870) were captured at 5 nM in 0.1% casein in 1× PBS. - The pGnRH_001 and pGnRH_009 VHH Tm variants were titrated by a 7-step 5-fold serial dilution starting with 1 μM in 0.1% casein in 1×PBS. VHH was detected using anti-VHH antibody (JIR, catalog number 128-005-230) at a 1:1000 dilution in 0.1% casein in 1×PBS, and then using anti-goat IgG-HRP (JIR, catalog number 705-035-147) at a 1:10000 dilution in 0.1% casein in 1×PBS. - Coating control was performed using 1 μg / mL of anti-GnRH polyclonal antibody in 0.1% casein in 1×PBS, followed by a 1:10000 dilution of anti-rabbit IgG-HRP (JIR, catalog number 111-035-144) in 0.1% casein in 1×PBS. - Binding to GnRH biopeptide 1 was evaluated by measuring the OD at 450 nm.

[0317] The results are shown in Figure 9 (sequence variants 01Tm1, 01Tm2, 01Tm3, 01Tm4 of clone pGnRH-001), Table 4, Figure 10 (sequence variants 09Tm1, 09Tm2 of clone pGnRH-009), and Table 5.

[0318] [Table 5]

[0319] [Table 6]

[0320] As shown in Figure 9 and Table 4, sequence optimizations leading to sequence variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 of clone pGnRH-001 enable improved binding of biopeptide 1 to all of these variants.

[0321] As shown in Figure 10 and Table 5, sequence optimizations leading to sequence variants 09Tm1 and 09Tm2 of clone pGnRH-009 enable improved affinity of biopeptide 1 to all of these variants.

[0322] (Example 7) Trends and stability of sequence variants derived from clones pGnRH-001 and pGnRH-009. Tm variants 01Tm1, 01Tm2, 01Tm3, and 01Tm4 of clone pGnRH-001 and Tm variants 09Tm1 and 09Tm2 of clone pGnRH-009 were expressed in E. coli, purified via Ni-NTA affinity chromatography, and formulated in PBS.

[0323] Physical and chemical stability was tested according to the same protocol detailed in Example 4. In particular, each antibody composition was subjected to temperature changes to evaluate its stability, and more precisely, - Under one test condition, the sample was subjected to 5 rounds of freeze / thaw cycles (5 × FT); - Under other test conditions, samples were stored at room temperature (25°C in this case) for two weeks and at 40°C for two weeks.

[0324] Antibody concentrations were measured for all of these conditions (results shown in Figure 11A) to assess potential protein loss. In parallel, turbidity was measured at OD500nm for all samples and conditions (results shown in Figure 11B).

[0325] The samples were subjected to RP-HPLC, and the total area of ​​each photograph was measured to determine the increase in high molecular weight species. The results are explained in Figure 12.

[0326] For the purposes of these experiments, the antibody names were changed as follows in Table 6.

[0327] [Table 7]

[0328] The results of the antibody stability analysis are summarized in the table shown in Figure 15.

[0329] Based on the data obtained, - All antibodies produced by clones GnRHTm-001, pGnRHTm-002, pGnRHTm-003, pGnRHTm-00, pGnRHTm-005, and pGnRHTm-006 are stable after being subjected to 5 freeze / thaw cycles; - All antibodies produced by clones pGnRHTm-001, pGnRHTm-002, pGnRHTm-003, pGnRHTm-00, pGnRHTm-005, and pGnRHTm-006 are stable when stored at 25°C for two weeks; - If stored at 40°C for 2 weeks, - Antibody samples derived from pGnRHTm-002, pGnRHTm-003, pGnRHTm-004, and pGnRHTm-005 showed some increase in high molecular weight species, but this increase was not as significant as that observed under similar conditions for the original clones 1 and 9; - Antibody samples derived from pGnRHTm-001 and pGnRHTm-003 showed single variant growth of 4% or 5%, respectively, by RP-HPLC. This was the conclusion reached.

[0330] (Example 8) Antagonist activity of variants GnRH_001_Tm002 and GnRH_009_Tm005 The antagonist activity of antibodies produced by clones GnRH_001_Tm002 and GnRH_009_Tm005 at the human GnRH receptor expressed in rat basophilic leukemia cells was determined using a fluorescence detection method, specifically focusing on agonist-induced cytoplasmic Ca2+ activity. 2+ This was determined by measuring their effects on ion mobilization.

[0331] The structure of this experiment was as follows: - Rat basophilic leukemia cells, resuspended in HBSS buffer (Invitrogen), were distributed into microplates at a density of 1.186E+04 cells / well; Next, 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 the cells were equilibrated at 30°C for 60 minutes; - The most promising monovalent VHH mutants of GnRH_001 and GnRH_009, GnRH-001-Tm002 and GnRH-009-Tm005, were added to the cells and incubated for 5 minutes. - SMI41-positive controls were tested at 100, 20, 4, 0.8, and 0.16 μg / mL; - E. coli production of monovalent VHH of GnRH_001 and GnRH_009 was also included as a control; Next, 8 nM GnRH was added to the cells, and free cytoplasmic Ca was added. 2+ The change in fluorescence intensity, which fluctuates in proportion to the ion concentration, was measured. - Results were expressed as the inhibition percentage of the control response to 8nM GnRH. - Cetrorelics acetate was used as a standard reference antagonist, and concentration-response curves were generated by testing at several concentrations and calculating its IC50 value.

[0332] The results are shown in Figure 13 and Table 7 below.

[0333] [Table 8]

[0334] Based on these results, - The GnRH_001_Tm002 candidate was shown to have a similar profile to GnRH_001, suggesting that the introduced mutation was able to mitigate the chemical tendencies and stability of the parental clone without affecting its functional activity; - The GnRH_009 candidate produced specifically for this experiment showed a different profile and IC50 from the antibodies obtained during the read identification stage; - The GnRH_009_Tm005 candidate was shown to have a similar profile to GnRH_009, suggesting that the introduced mutation was able to mitigate the chemical tendencies and stability of the parental clone without affecting its functional activity. It can be concluded that...

[0335] Overall, the data confirm the benefits of mutations made to the Tm variants, particularly GnRH_001_Tm002 and GnRH_009_Tm005 sequences, in terms of physical and chemical stability while maintaining their GnRH binding ability and antibody antagonist activity.

[0336] [Table 9] TIFF2026514390000010.tif241169 TIFF2026514390000011.tif241169 TIFF2026514390000012.tif241169 TIFF2026514390000013.tif241169 TIFF2026514390000014.tif237169 TIFF2026514390000015.tif243169 TIFF2026514390000016.tif232169 TIFF2026514390000017.tif235169 TIFF2026514390000018.tif18169

Claims

1. Isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) single-domain antibody (SdAb) or its antigen-binding fragment, comprising the N-terminal region of native GnRH (pGlu-His-Trp) and the C-terminal region of native GnRH (Pro-Gly-NH ２ An antibody or its antigen-binding fragment that specifically binds to both of the following:

2. An isolated monoclonal anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, - CDR3 polypeptide sequence of sequence number 33 or 188; - CDR3 polypeptide sequence of sequence number 57 or 187; - CDR3 polypeptide sequence of sequence number 36; - CDR3 polypeptide sequence of sequence number 96; - CDR3 polypeptide sequence of sequence number 48; - CDR3 polypeptide sequences of sequence numbers 42, 75, 81, 90, and 93; or - Variants of the CDR3 polypeptide sequence having one or two substitutions An isolated antibody or its antigen-binding fragment comprising a variable domain having a complementarity-determining region 3 (CDR3) selected from the group consisting of the following.

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

4. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized by comprising a variable domain having at least one complementarity-determining region 3 (CDR3) polypeptide sequence of SEQ ID NO: 57 or 187.

5. - CDR1 containing sequences selected from sequence numbers 55 and 82; - CDR2 containing sequences selected from sequence numbers 56, 98, 116, and 199; - CDR3 containing the sequence of sequence number 57 An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized by comprising a variable domain having a variable domain.

6. - CDR1 containing sequences selected from sequence numbers 31 and 195; - CDR2 containing the sequence selected from sequence number 32; - CDR3 containing the sequence of sequence number 33 An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized by comprising a variable domain having a variable domain.

7. - CDR1 containing the sequence selected from sequence number 34, CDR2 containing the sequence selected from sequence number 35, CDR3 containing the sequence of sequence number 36; or - CDR1 containing the sequence selected from SEQ ID NO: 94, CDR2 containing the sequence selected from SEQ ID NO: 95, CDR3 containing the sequence of SEQ ID NO: 96; or - CDR1 containing the sequence selected from sequence number 46, CDR2 containing the sequence selected from sequence number 47, and CDR3 containing the sequence from sequence number 48. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized by comprising a variable domain having a variable domain.

8. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the GnRH is selected from the group consisting of human GnRH, porcine GnRH, bovine GnRH, horse GnRH, sheep GnRH, dog GnRH, and feline GnRH.

9. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid described in claim 9.

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

12. - 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; and - Pharmaceutically acceptable excipients A pharmaceutical composition containing the following:

13. For use as a pharmaceutical or for use in a method of in vivo diagnosis, 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.

14. An in vitro method for detecting gonadotropin-releasing hormone (GnRH), a) A step of providing a sample, particularly a biological sample or a fraction thereof; b) The step of contacting the sample with an isolated antibody or its antigen-binding fragment according to any one of claims 1 to 8. A method that includes this.

15. A method for isolating an anti-gonadotropin-releasing hormone (anti-GnRH) antibody or its antigen-binding fragment, a) Providing a library of single-domain antibodies (SdAbs) or fragments thereof; b) A step of subjecting the library to antigen affinity selection, wherein the selected SdAb or fragment thereof is the N-terminal region of the natural GnRH (pGlu-His-Trp) and the C-terminal region of the natural GnRH (Pro-Gly-NH ２ The process is characterized by having specificity for both of the following: A method that includes this.

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