CN117377489A - GHR106 monoclonal antibodies as GnRH antagonists - Google Patents

Abstract

A GHR-106 monoclonal antibody, or antigen-binding fragment thereof, is provided and is useful for modulating the level of a reproductive hormone in vivo when administered to a mammalian subject. GHR-106 monoclonal antibodies or antigen-binding fragments thereof can be used to control ovulation, terminate ectopic pregnancy, and/or treat reproductive conditions or disorders in a mammalian subject.

Description

GHR106 monoclonal antibodies as GnRH antagonists

Cross Reference to Related Applications

The present application claims priority and its benefits based on two U.S. provisional patent applications, one being No. 63/189852 submitted on day 18 of 5 of 2021 and the other being No. 63/242976 submitted on day 10 of 9 of 2021. The entire contents of the foregoing two applications are incorporated herein by reference.

Technical Field

Providing embodiments for the treatment of a mammalian reproductive disorder; embodiments are provided for modulation of mammalian reproductive hormones.

Background

GnRH (gonadotropin releasing hormone) is a decapeptide hormone which reacts with GnRH receptors located in the anterior lobe of the human pituitary to control the release or secretion of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH). These two reproductive hormones are essential for sexual differentiation and maturation of the reproductive system in all animals, including humans.

GHR-106 is a monoclonal antibody derived from the N1-29 oligopeptide of the extracellular domain of the anti-human gonadotropin-releasing hormone receptor. FIG. 1A shows N1-29 oligopeptides located in the extracellular domain of GnRH receptors of several species (SEQ ID NO:1 to SEQ ID NO: 6). The comparison shows that the amino acid sequence of human has high homology (between about 90-95%) with rabbit, monkey, cat and dog, and less sequence homology with mouse.

FIG. 1B shows the amino acid sequences of the heavy and light chains (SEQ ID NO:7 and SEQ ID NO:8, respectively) of an IgG4 humanized GHR-106 monoclonal antibody, and these sequences are further recognized by underlining the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain (SEQ ID NO:9 through SEQ ID NO: 11) and light chain (SEQ ID NO:12 through SEQ ID NO: 14).

Examples of humanized IgG4GHR-106 in the figure comprise the S228P mutation of the heavy chain of the antibody, as shown in SEQ ID NO:7 (note: S228 is located at position 250 in amino acid SEQ ID NO:7 according to the EU numbering system). Without being bound by theory, it is believed that the S228P mutation or other equivalent mutation prevents the antibody recombination process known as IgG4 lens arm exchange. Fab-arm exchange can lead to the formation of unwanted bispecific antibodies, which negatively affects the specificity of the antibody target receptor. See silvaet al, JBC,2015, 290 (9): 5462-5469.

Because of the high homology (> 90-95%) of amino acid sequences, human GHR-106 cross-reacts with all N1-29 peptides of monkey, rabbit, dog, cat GnRH, but not with the N1-29 peptides of mouse and rat GnRH. GHR-106 and its humanized forms have been shown to specifically react with human GnRH receptors in cancer cells or anterior pituitary.

Humans only have one functional GnRH receptor gene. The major sites of action of the GnRH receptor are located in the anterior pituitary and are responsible for the release of gonadotropins, luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) when the hypothalamus is stimulated to release GnRH. However, in reproductive-related tissues or organs such as ovaries, testes, and cancer cells, gnRH receptors can bind to GnRH or peptide analogs thereof via an autocrine/paracrine regulatory mechanism.

Drugs of GnRH analogues having antagonism on the normal function of GnRH have been used for the treatment of various sex hormone related conditions or disorders such as reproductive diseases (both male and female), infertility, assisted reproductive therapy (such as In Vitro Fertilization (IVF) or ovum donation (such as controlled ovarian stimulation), contraceptive measures including ovulation inhibition, medical transition or degenerative treatment of denatured persons (including both male and female men) whether or not in combination with degenerative surgery, endometriosis, endometrial thinning, adenomyosis, endometrial hyperplasia, uterine fibroids, premenstrual syndrome, prostatic hyperplasia, ovarian disease, polycystic ovary disease, premature sex, etc.

Since natural hormones have a relatively short half-life (2-4 minutes), the goal of synthesizing decapeptides and their derivatives is to increase their circulating half-life to the order of hours. By modifying the structure of the decapeptides and derivatives, the biological effect of stimulating or inhibiting gonadotrophin release is maintained, and the half-life is improved by orders of magnitude. Depending on their biological mechanism of action of stimulating or inhibiting gonadotrophin release, they are commonly referred to as GnRH stimulators or GnRH antagonists, respectively. Cetrorelix was marketed a few decades ago. As GnRH antagonists for fertility regulating or anticancer drugs, cetrorelix is more potent and has a longer half-life (from minutes to hours) than natural GnRH. Other examples of synthetic GnRH antagonists are antide, cetrorelix, abarelix, degarelix, ganirelix and Elagolix et al.

Previous work on GHR-106 and its humanized forms has focused on the potential clinical use for the treatment of human cancers and fertility-related diseases (see U.S. patent nos. 8163283, 9273138 and publication nos. 2020/035462, each of which is incorporated herein by reference). PCT cited herein (application publication No. WO 2019/153075) discloses the effect of the monoclonal antibody GHR-106 on the human GnRH receptor and potentially the development of long-acting GnRH antagonists. In other aspects, the peptide is also biological similar to cetrorelix or other established peptide analogues. This is because the half-life of antibody drugs is typically much longer than the half-life of peptide antagonists such as the known GnRH peptide antagonists Cetrorelix or anti, for 5-21 days. Despite the different molecular sizes (80 kDa and 1.5 kDa), both peptide analogues and GHR-106 antibodies showed similar binding affinities (Kd 1-4 nM) and specificity for human GnRH receptors. The GHR-106 monoclonal antibody has the advantage of long half-life (5-21 days), whereas peptide GnRH antagonists such as cetrorelix or Antides have half-lives of most 1-10 hours.

There are many GnRH peptide analogues or derivatives available as medicaments for the clinical treatment of cancer such as prostate and breast cancer. Clinical applications also include a number of indications related to women's health, fertility and disease conditions. For example, gnRH peptide analogues are widely used In Vitro Fertilization (IVF) to control programmed ovulation and hormone dependent diseases such as endometriosis, uterine fibroids and premenstrual or polycystic ovary syndrome.

It is generally desirable to have long-acting compositions that can be used to treat reproductive disorders and to regulate the level of reproductive hormones in mammalian subjects.

As noted above, examples and limitations of the related art are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to the art upon a reading of the details and a study of the drawings.

Disclosure of Invention

The following examples describe and illustrate the associations between systems, tools, and methods. This is by way of illustration only and is not meant to be limiting in scope. In various embodiments, some of the above problems have been solved and others will be readily apparent.

The GHR-106 monoclonal antibody or antigen-binding fragment thereof can be used to modulate the level of a sex-related hormone in a mammalian subject, and can also result in reversible inhibition of at least one sex-related hormone in the subject. This reversible inhibition occurs during 3 to 21 days after administration of the GHR-106 monoclonal antibody or antigen-binding fragment thereof to the subject, the level of at least one sex-related hormone in the serum is reduced. Such hormones may be testosterone, estradiol, luteinizing hormone, progesterone, follicle stimulating hormone or a combination thereof. The GHR-106 monoclonal antibody or antigen-binding fragment thereof is administered in an amount of 1 to 3 milligrams per kilogram of body weight. This dose is about 50 mg to 300 mg per person. The administration may be repeated periodically, for example, between about every 1 week and 3 weeks.

In certain aspects, the GHR-106 monoclonal antibody or antigen-binding fragment thereof can have a heavy chain having the amino acid sequence of SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11, the light chain has the CDRs of SEQ ID NO: 12. SEQ ID NO:13 and SEQ ID NO: 14.

In certain aspects, the GHR-106 antibody or antigen-binding fragment thereof may be used to terminate ectopic pregnancy, control ovulation, fertility control in men or women, and/or treat sex hormone related conditions or disorders. The subject may be any mammal, including a human, monkey, dog, cat, rabbit, and the like. Methods embodying the above uses are also provided herein.

In addition to the examples and embodiments described above, embodiments will be further understood by reference to the drawings and by reading the following detailed description.

Drawings

Exemplary embodiments are illustrated in the accompanying drawings. It is intended that the embodiments and figures disclosed herein are to be regarded as illustrative rather than restrictive.

FIG. 1A compares the amino acid sequences of the N1-29 oligopeptides of the extracellular domain of the human, rabbit, monkey, cat, dog and mouse GnRH receptor.

FIG. 1B shows the amino acid sequences of the heavy and light chains of the GHR-106 antibody, underlined by the Complementarity Determining Regions (CDRs).

FIG. 2 shows a bipartite plot of ΔOD and GHR-106 antibody concentration at 405nm, GHR-106 monoclonal antibodies were applied to three isolated, well-coated synthetic oligopeptides of GnRH receptor N1-29 from humans, dogs and rabbits, respectively.

FIG. 3 shows the serum LH (mIU/ml) and testosterone (ng/ml) concentrations as a function of days after adult male rabbits were subcutaneously injected at day 1 with 3mg/kg GHR-106. Hormone levels were monitored from day 1 to day 30.

FIG. 4 shows the serum LH (MIU/ml) and estradiol (E2, pg/ml) concentrations as a function of days after a single subcutaneous injection of 3mg/kg GHR-106 in adult female rabbits on day 1. Hormone levels were monitored from day 1 to day 20.

FIG. 5 quantitative RT-PCR detection of gene expression levels of OC-3-VGH ovarian cancer cells to reveal the effect of antigen (peptide antagonist) and GHR-106 on gene regulation of ovarian cancer cells.

Fig. 6 shows serum testosterone levels of 10 male rabbits, divided into three experimental groups.

FIG. 7 shows serum LH levels of 10 male rabbits, divided into three experimental groups.

Fig. 8 shows serum estradiol (E2) levels of 10 female rabbits, divided into 3 experimental groups, maintained from day 1 to day 17.

Fig. 9 shows the serum LH levels of 10 female rabbits divided into three experimental groups during the study period.

Detailed Description

In the following description, specific details are set forth in order to provide a more thorough understanding to those skilled in the art. Well known elements may not have been shown or described in detail, however, to avoid unnecessarily obscuring the disclosure. The description and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

The inventors have now conducted studies, including proof of concept experiments in rabbits and quantitative gene regulation studies as disclosed herein, to support the broad clinical use of GHR-106 in several animal species, including humans, for the treatment of fertility problems and other reproductive disorders. The proof of concept experiments described herein on rabbits showed that serum reproductive hormone (LH, testosterone or estradiol) was reversibly inhibited in about a two week period. This new data demonstrates the potential applicability of GHR-106 in therapeutic applications in humans as well as several other animals.

GHR-106 has not previously been shown to act on the anterior pituitary GnRH receptor in a manner similar to that of the decapeptides GnRH antagonists, which are known to inhibit gonadotrophin release. Thus, the present study selected rabbits as a proof of concept animal model demonstrating that GHR-106 acts on the pituitary GnRH receptor, inhibiting gonadotrophin release in vivo. Thus, by comparing the effect of GHR-106 on gene expression with the biological similarity of in vivo genital hormone level modulation, it can be reasonably assumed that GHR-106 can replace known GnRH peptide antagonists for the treatment of many gynaecological diseases or reproductive disorders other than human cancers, but with the potential benefit of a longer half-life.

In some embodiments, a GHR-106 antibody, or antigen-binding fragment thereof, is provided. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered to a mammal, including a human, monkey, dog, cat, horse, cow, sheep, goat, rabbit, or other livestock, to treat a reproductive condition or disorder or sex hormone-related health problem. In some embodiments, the GHR-106 antibody, or antigen-binding fragment thereof, is administered to a mammal wherein the N1-29 terminal amino acid sequence of the GnRH receptor has an amino acid sequence having at least 90% sequence identity, including at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to the human N1-29 terminal amino acid sequence of the GnRH receptor (SEQ ID NO: 1).

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is a chimeric antibody that is engineered to minimize the likelihood of cross-reactivity of the antibody in the species of interest. For example, a polypeptide disclosed herein having the amino acid sequence of SEQ ID NO:7 and the heavy chain of the amino acid sequence of SEQ ID NO:8 is a humanized antibody construct. In other embodiments, where the subject is a different mammalian species, chimeric antibodies containing an IgG4Fc region from the subject species may be used, for example, dog IgG4-Fc for antibodies to dogs, cat IgG4-Fc for antibodies to cats, rabbit IgG4-Fc for antibodies to rabbits, monkey IgG4-Fc for antibodies to monkeys, horse IgG4-Fc for antibodies to horses, cow IgG4-Fc for antibodies to cows, sheep IgG4-Fc for antibodies to sheep, sheep IgG4-Fc for antibodies to goats, and the like.

In some embodiments, the GHR-106 antibody is provided as one or more active antigen-binding fragments of GHR-106 for use in treating a sex hormone related health condition or disease. In some embodiments, the fragment is a single-stranded fragment of the GHR-106 variable region. In some embodiments, the fragment is a fragment of GHR-106 of IgG isotype, including IgG4. In some embodiments, the fragment is a F (ab') 2 fragment. In some embodiments, the molecular weight of the F (ab') 2 fragment is 110kDa. In some embodiments, the fragment is a Fab fragment. In some embodiments, the Fab fragment has a molecular weight of 55kDa. In some embodiments, the fragment is a scFab fragment. In some embodiments, the scFab fragment has a molecular weight of 25kDa. In some embodiments, the fragment is an scFv fragment. In some embodiments, the scFv fragment has a molecular weight of 25kDa. In some embodiments, a combination of different antigen binding fragments, such as two or more of the fragments described above, can be used as a medicament for treating a sex hormone related condition or disorder.

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof for use in treating a sex hormone related health condition or disorder has no effector function. Antibodies that do not have effector functions are unable to activate Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Cytotoxicity (ADCC) pathways. In some embodiments, the non-effector GHR-106 antibody or antigen-binding fragment thereof has an IgG4 subtype. In some embodiments, the GHR-106 antibody, or antigen-binding fragment thereof, inhibits complement activation. In some embodiments, the heavy chain of an antibody having an IgG4 subtype has an S228P mutation or an equivalent mutation to prevent Fab-arm exchange. In some embodiments, the non-effector functional GHR-106 antibody or antigen-binding fragment thereof is an IgG antigen-binding fragment of a GHR-106 antibody. In some embodiments, the antigen binding fragment that does not have effector function is a F (ab') 2, fab, scFab, or scFvIgG fragment of a GHR-106 antibody. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is derived from hGH R-106.

In some embodiments, a subtype of GHR-106 antibody is selected to modulate the effector function of the antibody. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is structurally modified to further modulate the effector function of the antibody, for example by using an antigen-binding fragment of an antibody that does not have any effector function. In some embodiments, the Fc region of the GHR-106 antibody is an IgG4 subtype. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof that does not have any effector function is used for treating a sex hormone related health condition or disorder, for reversibly inhibiting the level of at least one sex hormone in a subject, for controlling ovulation in a subject, and/or for terminating ectopic pregnancy in a subject. In some embodiments, the GHR-106 antibody having an IgG4 subtype is used to treat a sex hormone related health condition or disorder.

Without being bound by theory, it is believed that the use of the IgG4 antibody subtype to treat sex hormone-related health conditions or disorders or to otherwise modulate sex hormone levels in a subject, including treating fertility disorders, will minimize or eliminate the likelihood of developing CDC and ADCC reactions by GHR-106 antibodies bound to the anterior pituitary, due to the IgG4 antibody subtype not activating Complement Dependent Cytotoxicity (CDC) or antibody dependent cytotoxicity (ADCC). See vidarssonet, front. 520.

Furthermore, igG4 antibodies have been shown to actually inhibit complement activation (see vanderZee et al, clin. Exp. Immunol.,1986, 64 (2): 415-422). Thus, in some embodiments, a GHR-106 monoclonal antibody or antigen-binding fragment thereof is selected to inhibit complement activation. In some embodiments, a GHR-106 monoclonal antibody or antigen-binding fragment thereof that inhibits complement activation is used to treat a sex hormone related condition or disorder.

In some embodiments, the circulating half-life of the GHR-106 antibody is about 3 to 21 days, including any value therebetween, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, or 20 days, or 72 to 500 hours, including any value therebetween, e.g., 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, or 475 hours. In contrast, cetrorelix (cetrorelix) has a circulation half-life of about 10 to 63 hours. GHR-106 has a longer half-life than the decapeptide GnRH antagonist cetrorelix and therefore may require less frequent dosing, which may improve patient compliance and/or feasibility of proposed treatment regimens.

In some embodiments, igG antigen binding fragments from GHR-106, such as F (ab') 2, fab, scFab, or ScFv, each have a circulatory half-life of about 12 to 20 hours, including any value therebetween, such as 13, 14, 15, 16, 17, 18, or 19 hours. The mGHR-106 or antigen-binding fragment of hGHR-106 has a shorter half-life than the mGHR-106 or hGHR-106 antibody. In some embodiments, protein engineering is used to provide GHR-106 antibodies or antigen-binding fragments thereof with half-lives within a desired range.

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof has an amino acid sequence according to SEQ ID NO:7 and a heavy chain according to the amino acid sequence of SEQ ID NO:8, and a light chain of the amino acid sequence of seq id no. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof has a sequence identical to SEQ ID NO:7 and a heavy chain having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:8, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7 and 8 have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In a further embodiment, the GHR-106 antibody or antigen-binding fragment thereof has a heavy chain of the complementarity determining region: has the sequence according to SEQ ID NO:9 (RYSVH), having a CDR1 region according to the amino acid sequence of SEQ ID NO:10 (MIWGGGSTDYNPSLKSR) and a CDR2 region having an amino acid sequence according to SEQ ID NO:11 A CDR3 region of the amino acid sequence of (GYSFA). In a further embodiment, the GHR-106 antibody or antigen-binding fragment thereof has a light chain with the following CDRs: has the sequence according to SEQ ID NO:12 (KSSQSLLNSRTRKNYLA) CDR1 region of an amino acid sequence according to SEQ ID NO:13 A CDR2 region of the amino acid sequence of (WASTRES) and a polypeptide having a sequence according to SEQ ID NO:14 A CDR3 region of the amino acid sequence of (KQSYNLIT).

The GHR-106 antibodies or antigen-binding fragments thereof described herein can be formulated in a suitable manner for use as a medicament. Thus, they may be combined with pharmaceutically acceptable excipients or other pharmaceutically suitable compounds to provide medicaments useful for modulating sex hormone levels and/or treating sex hormone related health conditions or disorders.

In some embodiments, an effective amount of a GHR-106 antibody or antigen-binding fragment thereof to treat a hormone-related health condition or disorder and/or to modulate the level of one or more sex-related hormones in a mammal (including humans, monkeys, dogs, cats, rabbits, horses, cattle, sheep, goats, or other livestock) is provided. The mammal may be male or female. In some embodiments, the sex hormone related health condition or disorder is a reproductive disease (in a male or female subject), and the medical shift in a denatured person includes male-to-female (MTF) or female-to-male (FTM) degenerative treatment, whether accompanied by degenerative surgery, in Vitro Fertilization (IVF) or egg donation (e.g., controlled ovarian stimulation), contraceptive measures, including inhibiting ovulation in a female subject or sperm production in a male subject, endometriosis, endometrial thinning, adenomyosis, endometrial hyperplasia, uterine fibroids, premenstrual syndrome, benign prostatic hypertrophy, ovarian disease, polycystic ovarian disease, sexual precocity, and the like.

In some embodiments, the male subject is fertility controlled with a GHR-106 antibody or antigen-binding fragment thereof. Without being bound by theory, the data contained in the examples of the present application support that administration of GHR-106 antibodies or antigen-binding fragments thereof to a male subject can reduce the level of sex-related hormones, such as testosterone, to a level that may interfere with male sperm production, thereby providing fertility control to the male subject.

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered to terminate ectopic pregnancy. Without being bound by theory, it is believed that the decrease in reproductive hormone levels caused by administration of the GHR-106 antibody or antigen-binding fragment thereof will be detrimental to the fetus and/or that the GnRH receptor and GnRH are highly expressed in the human placenta with concomitant amounts of human chorionic gonadotrophin secretion, resulting in rapid termination of ectopic pregnancy while minimizing negative effects on the subject.

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is used to modulate ovulation in a subject. The GHR-106 antibody or antigen-binding fragment thereof is administered to a female for fertility control (e.g., birth control). In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is used to modulate the level of one or more sex-related hormones in a subject, including by reversibly reducing the serum concentration of the one or more sex-related hormones. In some embodiments, the sex-related hormone is testosterone, estradiol, luteinizing hormone, progesterone, follicle stimulating hormone, or a combination thereof. In some embodiments, the change in sex-related hormone level alters the fertility status of the subject.

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof acts as a GnRH antagonist in the treatment of any disease treatable by a known GnRH antagonist, including anti or Cetrorelix. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is used to treat a condition in which a longer half-life is desired than known GnRH antagonists (including anti-ides or Cetrorelix).

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered at a dosage level of 0.5-10 mg/kg, including any value therebetween, such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 mg/kg. In some embodiments, for example, about 1 to about 3 mg/kg, including any values or sub-values therebetween. In some embodiments where the binding affinity and/or specificity of the GHR-106 antibody or antigen-binding fragment thereof has been modified, the dosage level of the antibody or antigen-binding fragment thereof is appropriately modified.

In some embodiments in which the subject is a human, the GHR-106 antibody or antigen-binding fragment thereof is administered at a dose of between about 50 milligrams to about 300 milligrams, including any value in between, such as 75, 100, 125, 150, 175, 200, 225, 250, or 275 milligrams.

In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered at a recurring interval, e.g., every 5-30 days or any value therebetween, e.g., every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 days; every 1-8 weeks or any value in between, e.g. every 2, 3, 4, 5, 6 or 7 weeks, or every 2-6 months or any value in between, e.g. every 3, 4 or 5 months. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered at a repeating interval of between about every 1 week and about every 3 weeks. In some embodiments, the GHR-106 antibody, or antigen-binding fragment thereof, administered to a human is a humanized GHR-106 antibody, or antigen-binding fragment thereof. In some embodiments, the humanized GHR-106 antibody is hGHR-106IgG4 having the amino acid sequence of SEQ ID NO:7 and a heavy chain having the amino acid sequence of SEQ ID NO:8, and a light chain of the amino acid sequence of seq id no.

The typical route of administration of the pharmaceutical compositions containing the antibodies is by injection, usually intravenous or intramuscular. However, any suitable mode of administration may be used in various embodiments.

Examples

Certain embodiments are further described with reference to the following examples, which are intended to be illustrative rather than limiting.

A proof of concept experiment was performed in rabbits to demonstrate the reversible inhibition of serum genital hormone by a single injection of humanized monoclonal antibody GHR-106 (hig 4) against GnRH receptor, GHR-106 (hig 4) having a heavy chain with the amino acid sequence of seq id No. 7 and a light chain with the amino acid sequence of seq id No. 8.

A single subcutaneous injection of 1 mg/kg or 3 mg/kg of antibody to male rabbits reduced serum LH and testosterone levels in parallel by 80-90% of normal levels over 7-10 days. The reproductive hormones return to normal levels approximately two weeks after the initial injection. Serum LH and estradiol concentrations in female rabbits were reversibly inhibited and recovered at the same dose of the same antibody as observed for female rabbits, and serum LH and estradiol concentrations in female rabbits were reversibly recovered at the same dose of the same antibody. These experiments supported that GHR-106 (hIgG 4) could act as an antibody-based GnRH antagonist similar to Elagolix or anti on the anterior pituitary GnRH receptor, except that GHR-106 (hIgG 4) had a much longer half-life (days versus hours).

In addition to the rabbit proof of concept experiments, quantitative RT-PCR experiments were performed and as a tool, in vitro studies demonstrated nearly identical intracellular gene regulation between GHR-106 and the decapeptide GnRH antagonist. Thus, it is reasonable to consider that antibody-based and peptide-based GnRH antagonists are highly similar in terms of the biological mechanisms of antagonizing cancer cells and reversibly inhibiting anterior pituitary gonadotrophin release, except that GHR-106 has a significantly longer half-life.

Production of GHR-106 related antibody drugs

Various isomers of GHR-106, including murine-dog or murine-cat chimeric forms, can be mass produced based on knowledge and methods known in the art, including the U.S. patents cited herein. For example, human (variable region) dog (constant Fc region) chimeric antibodies and murine (variable region) dog (constant Fc region) chimeric antibodies can be mass produced based on the established knowledge of the dosage of GHR-106 antibodies to dogs.

The mouse GHR-106 may be produced and purified by in vitro culture of mouse ascites or hybridoma cell lines. Humanized GHR-106 can be produced by established permanent cell lines. These include mGR-106 (mouse origin), GHR-106 (hIgG 1) and GHR-106 (hIgG 4) as well as different antibody fragments, such as single chain fragments of Fab, (Fab') 2 or variable regions.

Example 1: proof of concept experiments in rabbits and the meaning of wide clinical applications in humans and/or domestic animals.

GHR-106 is a monoclonal antibody against the human GnRH receptor N1-29 oligopeptide obtained from immunized mice. The amino acid sequences of the heavy and light chains of GHR-106 are shown in FIG. 1B with the CDRs underlined. Animal models were chosen to demonstrate that GHR-106 interaction with the pituitary GnRH receptor can lead to reversible inhibition of reproductive hormones in vivo. Thus, N1-29 oligopeptides from different animal species including human, monkey, dog, cat, rabbit and mouse (SEQ ID NO:1 to SEQ ID NO: 6) were compared and sequence homology is shown in FIG. 1A. Based on the assumption that highly similar sequence identity will lead to highly similar in vivo binding activity and comparable biological activity of their respective GnRH receptors, rabbits were selected as a suitable animal model for in vivo proof of concept experiments. In particular, given that the amino acid sequence similarity of the N1-N29 peptides of GnRH receptors is greater than 95% in humans and rabbits, the inventors predicted that their apparent KD for binding to GHR-106 (hig 4) was similar, and rabbits were selected as suitable animals demonstrating reversible inhibition of reproductive hormones such as LH, E2 and testosterone.

By comparison of the N1-29 peptide sequences of different animals, it is speculated that GHR-106 may have a high degree of binding cross-reactivity between humans and animals such as rabbits, cats, dogs, monkeys, etc. Thus, GHR-106 can be applied not only to humans as a GnRH antagonist, but also to several other animals (mammals), including rabbits, dogs, and cats. Those skilled in the art can implement established techniques (e.g., techniques used in humanized antibody design) to design antibodies suitable for use in different mammalian species to minimize the likelihood of undesired cross-reactivity of the antibodies.

EXAMPLE 2 ELISA comparative study

The binding studies between GHR-106 and the N1-29 peptide from the above animal species were critical to demonstrate the affinity comparison of GHR-106 with N1-29 oligopeptides from humans, dogs and rabbits, respectively. Thus, the present study uses a binding ELISA method to compare the affinity of GHR-106 binding to the N1-29 oligopeptide of humans, dogs and rabbits to determine the affinity of GHR-106 binding to the N1-29 oligopeptide. The results of these binding studies are given in figure 2 and compared.

FIG. 2 shows a bipartite plot of ΔOD at 405nm versus GHR-106 antibody concentration, wherein GHR-106 monoclonal antibodies were applied to three isolated, well-coated synthetic oligopeptides of GnRH receptor N1-29 in humans, dogs and rabbits, respectively. RP215 monoclonal antibody was used as a negative control. Goat anti-human IgG labeled with alkaline phosphatase was used as a secondary antibody for signal detection. P-nitrophenyl phosphate was used as substrate and monitored at 405nm and a bipartite graph was drawn. The results show that the binding affinity of the GHR-106 and N1-29 peptides derived from human, dog and rabbit respectively is comparable to the binding affinity of the N1-29 peptide compared to the binding affinity of the N1-29 peptide of human, dog and rabbit compared to the negative reference of RP215 without affinity.

In previous studies, the inventors have shown that three isoforms of GHR-106, including mouse GHR-106, humanized GHR-106 and humanized GHR-106 (hIgG 4), are substantially identical in their binding affinity and specificity to the human GnRH receptor and its N1-29 oligopeptide, respectively, with dissociation constants on the order of 1-5 nm.

Based on the ELISA binding studies shown in FIG. 2, GHR-106 has been demonstrated to have similar binding to human or rabbit GnRH receptor or N1-29 oligopeptide thereof. Thus, rabbits were selected for proof of concept experiments to demonstrate the reversible inhibition of reproductive hormones in vivo by a single treatment with GHR-106.

Example 3: serum LH and testosterone concentrations in male rabbits injected with GHR-106

Previous in vitro studies on human cancer cells show that 1-10 mug/ml GHR-106 monoclonal antibodies of different subtypes are incubated for 24-72 hours, and can induce apoptosis of the cancer cells. Apoptosis was induced to a degree comparable to that of the decapeptide GnRH antagonist antibody, although the molecular size of the antibody was 50 times that of the former.

To demonstrate that GHR-106 interacts with the pituitary GnRH receptor similarly to the decapeptide GnRH antagonist, proof of concept experiments were performed in rabbits. In the case of male rabbits, serum concentrations of reproductive hormones including Luteinizing Hormone (LH) and testosterone were monitored periodically after subcutaneous injection of 3 mg/kg hGH-106.

Serum LH and testosterone concentrations were measured separately using EIA kit and plotted as a function of time over days 1-30. The results of hormone distribution are shown in figure 3, which shows a plot of serum LH (mIU/ml) and testosterone (ng/ml) concentrations versus days after subcutaneous injection of 3mg/kg GHR-106 on day 1 in an adult male rabbit.

As shown in FIG. 3, the male rabbits were immediately inhibited (from 3.5mIU/mI down to <0.5 mIU/mL) by serum LH and testosterone after injection for 24-48 hours. The low LH level lasts at least one to two weeks. The LH level fluctuation then rises until a steady normal range of values (2.9-5.0 mIU/ml) is reached on weekends 3. Without being bound by theory, it is noted that fluctuations in the level of the mammalian reproductive hormone are typically observed on a daily basis in mammals, for example, due to endocrine, environmental or physiological causes, so some fluctuations in the level of the reproductive hormone are expected. However, in these examples, it is also apparent that the trend of reversible inhibition of the reproductive hormone is consistent after administration of GHR-106.

Likewise, the serum testosterone concentration of male rabbits was reduced from 0.95 nanograms per milliliter to less than or equal to 0.1 nanograms per milliliter over the first two weeks at a single injection of 3 milligrams per kilogram dose, by more than 80%. Serum testosterone levels are time dependent with LH levels. In the third week after injection, the fluctuating changes in LH and testosterone levels were parallel to each other and the hormone levels did not return to normal range until day 30 (fig. 3).

Example 4: serum LH and estradiol concentrations at one injection of hGHR-106 in female rabbits.

The hormone levels of female rabbits serum Luteinizing Hormone (LH) and estradiol (E2) were monitored by a single injection of hGH-106 at a dose of 3 mg/kg. Serum LH and E2 concentrations were measured periodically from day 1 to day 20 as shown in fig. 4.

Inhibition of LH levels (from 3mIU/mI to 1 mIU/mL) was immediately observed during the first few days after antibody injection. Likewise, serum E2 concentrations decreased in parallel over the same period (from 50pg/ml to. Ltoreq.20 pg/ml) until day 10.

On days 10 to 20, the LH and E2 concentrations increased with time, reaching 6mIU/ml and 120pg/ml on days 18 and 20, respectively.

In another experiment, females were injected with a low dose of 1 mg/kg, and the overall distribution of LH and E2 concentrations was similar to that of the high dose during the same observation period (data not shown).

EXAMPLE 5 quantitative Gene regulatory studies

Quantitative gene regulation studies were performed to demonstrate that the molecular mechanism between GHR-106 and the peptide GnRH antagonist antibody is identical. After incubation with GHR-106 or GnRH peptide antagonist anti with human cancer cells, the amount of gene expression that binds to the human GnRH receptor was altered. To further compare GHR-106 with the decapeptide GnRH antagonist, we selected 10 regulatory genes, quantified using the RTPCR method, and the results are shown and compared in FIG. 5. The results of these comparative studies show that GHR-106 has strong similarity in mechanism of action with anti-tumor drugs.

Example 6 high specificity of GHR-106 monoclonal antibody

GHR-106 has a high degree of specificity for the human GnRH receptor as compared to many known and available anti-GnRH receptor monoclonal antibodies. In particular, GHR-106 was tested for its ability to detect GnRHR overexpression in a reference cell line and compared to four different commercially available antibodies. It was found that only GHR-106 antibodies were able to detect overexpression of GnRHR in the reference cell line.

In view of this tissue specificity in humans, not observed in other antibodies, GHR-106 may be one of the best antibodies to react with the human GnRH receptor and the N1-29 oligopeptide of that receptor, respectively.

In addition, other examples described herein also disclose high species cross-reactivity between several different animal species. Thus, GHR-106 should be considered a third class of therapeutic agents. I.e., an antibody-based GnRH antagonist, comparable to organic chemistry or decapeptides-based GnRH antagonists.

Humanized GHR-106 can only be used in human clinical, whether cancer therapy or fertility control, due to inherent and limited immunogenicity in human applications. Suitable modifications of antibodies, particularly the Fc region of antibodies, are contemplated for clinical use in other animal species, including other mammalian species. For example, to avoid allergic reactions to allogeneic injections, chimeric antibodies from other species (e.g., dogs or cats) may be substituted for the pure mouse-derived antibodies (receptor constant regions). To reduce the heterologous immune response, mouse (variable, VR) -dog (constant, fc) chimeric IgG may be prepared and used in dogs. Similarly, murine-feline chimeric antibodies can be produced according to known methods for use in cats. Similar modifications may be made for the use of GHR-106 antibodies as therapeutic agents in other species.

EXAMPLE 7 proof of concept Large Scale experiments in rabbits

To demonstrate that GHR-106 (hign 4) acts as a GnRH antagonist in vivo, extensive proof of concept experiments were performed on rabbits and data are provided in these additional examples.

From the data of the above examples, reversible inhibition of reproductive (i.e., sex-related) hormones including luteinizing hormone, testosterone and estradiol was observed at the time of a single injection in either male or female rabbits. The serum reproduction hormone level returns to normal 1-2 weeks after injection.

Based on preliminary observations made on individual rabbits, large-scale rabbit experiments (n.gtoreq.30) including negative controls were performed using the same protocol. In particular, to further demonstrate the role of GHR-106 as a GnRH antagonist, extensive experiments were performed in male and female rabbits. Statistical analysis was performed on the data generated for each experimental group. The mean and standard deviation of the rabbit hormone levels for each experimental group are shown in figures 6-9 and the corresponding statistical analysis and individual comparison with negative controls are shown in tables 1-4.

In the case of male rabbits, the concentration of reproductive hormones such as LH and testosterone in the serum of 10 male rabbits was monitored after subcutaneous injection of 1mg/kg GHR-106 (low dose, n=3) or 3mg/kg GHR-106 (high dose, n=3) or no injection of GHR-106 (negative control, n=4) on day 1, respectively. Serum testosterone levels were determined and the mean (standard deviation) from day 1 to day 17 was determined, and serum LH levels (standard deviation) from day 1 to day 13 were determined. The results of testosterone profile are shown in figure 6. The results of LH distribution are shown in FIG. 7. Selected statistical analyses and separate comparisons with the negative controls of fig. 6 and 7 are shown in tables 1 and 2, respectively.

As shown in fig. 6 and 7, a single injection of either low or high dose GHR-106 reversibly inhibited serum testosterone and LH levels in male rabbits on day 1. The low-dose or high-dose treatment can immediately lower serum testosterone and LH levels compared with a negative reference, and has statistical significance. Notably, similar inhibition magnitudes were observed in the low and high dose groups. Serum testosterone and LH in the low and high dose groups recovered to similar levels to the negative control group after several days of inhibition.

TABLE 1 statistical analysis of selection of male Rabbit testosterone profiles in FIG. 6

TABLE 2 statistical analysis of selection of male rabbit LH profile in FIG. 7

Taking female rabbits as an example, after subcutaneous injection of 1mg/kg GHR-106 (low dose, n=3) or 3mg/kg GHR-106 (high dose, n=3) or no injection of GHR-106 (negative control, n=4) on day 1, the concentration of serum reproductive hormones including LH and estradiol (E2) in 10 female rabbits was monitored. Serum E2 levels were measured from day 1 to day 17 and serum LH levels were measured from day 1 to day 13. The results of the estradiol (E2) curve are shown in fig. 8. The results of LH distribution are shown in FIG. 9. The statistical analysis of the selection and the individual comparison with the negative control of fig. 8 and 9 are shown in tables 3 and 4, respectively.

As shown in fig. 8 and 9, serum estradiol (E2) and LH levels of female rabbits were reversibly inhibited by a single injection of low-dose or high-dose GHR-106 on day 1. Either low or high dose treatment resulted in a significant decrease in serum estradiol (E2) and LH immediately compared to the negative control group. Notably, similar inhibition magnitudes were observed in the low and high dose groups. Serum estradiol (E2) and LH in the low and high dose groups recovered to similar levels to the negative control group after several days of inhibition.

TABLE 3 statistical analysis of selection of female Rabbit estradiol (E2) spectra of FIG. 8

TABLE 4 statistical analysis of selection of mother rabbit LH maps in FIG. 9

Overall, experiments with single or multiple rabbits showed that inhibition of reproductive hormone by single injection of GHR-106 (hig 4) at doses of 1 mg/kg or 3 mg/kg was reversible. The hormone level was restored to the normal range after 1-3 weeks, as in the negative control group, indicating that inhibition of sex-related hormone was reversible. Thus, consistent with the individual rabbit data described above, the effects of reversible hormone inhibition were observed in a larger experimental group.

In conclusion, the multiple rabbit and individual rabbit experiments draw the same conclusion in terms of time-dependent reversible associated hormone inhibition upon GHR-106 antibody treatment. Thus, the present inventors have demonstrated that GHR-106 (hig 4) is an antibody-based long-acting GnRH antagonist that exhibits a similar biological effect as the currently clinically used decapeptide GnRH antagonists, such as cetrorelix, but, due to its longer half-life, its potential benefit is a longer active period.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the entire specification.

Without limiting the foregoing, various embodiments include aspects, including the following. These aspects are based on the examples disclosed in this application, which demonstrate factors including (1) high amino acid sequence homology and broad species cross-reactivity of GHR-106 to several animal species; (2) Proof of concept experiments provide direct evidence of strong interactions of human or other mammalian GHR-106 with GnRH receptors; (3) The antibody-based long-acting GnRH antagonists and the peptide-based short-acting GnRH antagonists have a high degree of identity and consistency in quantifying the level of gene expression changes.

In a first aspect, the GHR-106 of each isomer or species cross-reacts with GnRH receptors of several different animal species (dog, cat, rabbit and monkey) and can be used as GnRH antagonists as long as they have a high degree of sequence homology (. Gtoreq.90-95%) with humans on the N1-29 oligopeptides of the respective receptors.

In a second aspect, GHR-106 may be used as a GnRH antagonist for reversibly inhibiting endogenous reproductive hormones (e.g., LH, FSH, testosterone, estradiol, progesterone, etc.) of a human or any other animal species meeting the criteria of the first aspect.

In a third aspect, GHR-106 in the humanized IgG4 isotype [ GHR-106 (hIgG 4) ] acts directly on the anterior pituitary of humans and reversibly inhibits reproductive hormones during GHR-106 therapy to manipulate GRH receptor-controlled fertility regulation or dysfunction similar to the pharmacological effects of decapeptide analogs such as Cetrorelix.

In a fourth aspect, GHR-106 of different subtypes or species is useful as a GnRH antagonist not only in humans, but also in the treatment of nearly all receptor positive cancers in other animal species, including dogs, cats, rabbits, and the like.

Sequence listing

<110> Vancouver biotechnology Co., ltd (VANCOUVER BIOTECH LTD.)

Use of <120> GHR106 monoclonal antibodies as GnRH antagonists

<130> V101 0043/JAM

<150> US63/189852

<151> 2021-05-18

<150> US63/242976

<151> 2021-09-10

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 30

<212> PRT

<213> person (Homo sapiens)

<400> 1

Met Ala Asn Ser Ala Ser Pro Glu Gln Asn Gln Asn His Cys Ser Ala

1 5 10 15

Ile Asn Asn Ser Ile Pro Leu Met Gln Gly Asn Leu Pro Thr

20 25 30

<210> 2

<211> 30

<212> PRT

<213> Rabbit (Oryctolagus cuniculus)

<400> 2

Met Glu Asn Ser Ala Ser Pro Glu Gln Asn Gln Asn His Cys Ser Ala

1 5 10 15

Ile Asn Asn Ser Ile Pro Leu Thr Gln Gly Asn Leu Asn Thr

20 25 30

<210> 3

<211> 30

<212> PRT

<213> chimpanzee (Pan troglodines)

<400> 3

Met Ala Asn Ser Ala Leu Pro Glu Gln Asn Gln Asn His Cys Ser Val

1 5 10 15

Ile Asn Asn Ser Ile Pro Leu Met Gln Gly Asn Leu Pro Thr

20 25 30

<210> 4

<211> 29

<212> PRT

<213> cat (Felis catus)

<400> 4

Met Ala Ser Ala Pro Pro Glu Gln Asn Gln Asn His Cys Ser Ala Ile

1 5 10 15

Asn Asn Ser Ile Pro Leu Met Gln Gly Asn Leu Pro Thr

20 25

<210> 5

<211> 29

<212> PRT

<213> domestic dogs (Canis family)

<400> 5

Met Ala Ser Ala Ser Pro Glu Gln Asn Gln Asn His Cys Ser Ala Val

1 5 10 15

Asn Asn Ser Asn Met Leu Met Gln Gly Asn Leu Pro Thr

20 25

<210> 6

<211> 30

<212> PRT

<213> mice (Mus musculus)

<400> 6

Met Ala Asn Asn Ala Ser Leu Glu Gln Asp Pro Asn His Cys Ser Ala

1 5 10 15

Ile Asn Asn Ser Ile Pro Leu Ile Gln Gly Lys Leu Pro Thr

20 25 30

<210> 7

<211> 468

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 7

Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser

1 5 10 15

Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Glu Ser Gly

20 25 30

Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val

35 40 45

Ser Gly Phe Ser Leu Ser Arg Tyr Ser Val His Trp Ile Arg Gln Pro

50 55 60

Pro Gly Lys Gly Leu Glu Trp Ile Gly Met Ile Trp Gly Gly Gly Ser

65 70 75 80

Thr Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp

85 90 95

Asn Ser Lys Ser Gln Val Phe Leu Lys Met Ser Ser Val Thr Ala Ala

100 105 110

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Asn Asp Gly Tyr Tyr Ser

115 120 125

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

145 150 155 160

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr

210 215 220

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

225 230 235 240

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

340 345 350

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Leu Gly

465

<210> 8

<211> 239

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 8

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr

100 105 110

Tyr Cys Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 9

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 9

Arg Tyr Ser Val His

1 5

<210> 10

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 10

Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

Arg

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 11

Gly Tyr Tyr Ser Phe Ala

1 5

<210> 12

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 12

Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 13

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Artificial structure (Artificial Construct)

<400> 14

Lys Gln Ser Tyr Asn Leu Tyr Thr

1 5

Claims (28)

1. Use of a GHR-106 monoclonal antibody or antigen-binding fragment thereof to modulate sex-related hormone levels in a mammalian subject.

2. The use of claim 1, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof results in reversible inhibition of at least one sex-related hormone in a subject.

3. The use of claim 2, wherein the reversible inhibition of the at least one sex-related hormone comprises a decrease in serum level of the at least one sex-related hormone in the subject within 3 to 21 days after administration of the GHR-106 monoclonal antibody or antigen-binding fragment thereof.

4. The use of any one of claims 1-3, wherein the at least one sex-related hormone is testosterone, estradiol, luteinizing hormone, progesterone, follicle stimulating hormone, or a combination thereof.

5. Use according to any one of claims 1-4 for terminating ectopic pregnancy.

6. The use of any one of claims 1-4, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is used to control ovulation in a female subject.

7. The use of any one of claims 1 to 4 or 6, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is for fertility control in a female subject.

8. The use of any one of claims 1-4, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is for fertility control in a male subject.

9. The GHR-106 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4 for use in treating a sex hormone related condition or disorder in a subject.

10. The use of claim 9, wherein the sex hormone related condition or disorder is a reproductive disease, medical transition in a denatured human, infertility, assisted reproductive therapy, contraception, endometriosis, endometrial thinning, adenomyosis, endometrial hyperplasia, uterine fibroids, premenstrual syndrome, benign prostatic hypertrophy, ovarian disease, polycystic ovary disease, or precocious puberty.

11. Use according to any one of claims 9 or 10, wherein the sex hormone related condition or disorder is a disease treatable by administration of a known GnRH antagonist, including known GnRH antagonists such as known antide or Cetrorelix.

12. The use of any one of claims 9-11, wherein the sex hormone related condition or disorder is a disease in which the half-life of an active therapeutic agent in the circulation is longer than known decapeptide GnRH antagonists.

13. The use of any one of claims 1 to 12, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is adapted for administration at a dose of about 1mg/kg to about 3mg/kg relative to the weight of the subject.

14. The use of any one of claims 1 to 13, wherein the subject is a human, and wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is suitable for administration at a dose of about 50 mg to about 300 mg.

15. The use of any one of claims 1 to 14, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is adapted for administration at a repeating interval of between about every 1 week and about every 3 weeks.

16. The use of any one of claims 1-15, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof has a heavy chain having a sequence identical to SEQ ID NO:7 has an amino acid sequence having at least 90% sequence identity; and/or wherein the GHR-106 antibody has a sequence identical to SEQ ID NO:8, and a light chain having an amino acid sequence with at least 90% sequence identity to the amino acid sequence of seq id no.

17. The use of any one of claims 1 to 16, wherein:

a) The heavy chain CDR1 region or antigen binding fragment of GHR-106 monoclonal antibody has the amino acid sequence RYSVH (SEQ ID NO: 9) The method comprises the steps of carrying out a first treatment on the surface of the A kind of electronic device with high-pressure air-conditioning system

b) The heavy chain CDR2 region of GHR-106 monoclonal antibody or antigen binding fragment thereof has the amino acid sequence MIWGGGSTDYNPSLKSR (SEQ ID NO:

10 A) is provided; a kind of electronic device with high-pressure air-conditioning system

c) The heavy chain CDR3 region of GHR-106 monoclonal antibody or antigen binding fragment thereof has the amino acid sequence GYYSFA (SEQ ID NO:11 A) is provided; a kind of electronic device with high-pressure air-conditioning system

d) The GHR-106 monoclonal antibody light chain CDR1 region or antigen binding fragment has the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO:12 A) is provided; a kind of electronic device with high-pressure air-conditioning system

e) The light chain CDR2 region of GHR-106 monoclonal antibody or antigen binding fragment thereof has the amino acid sequence waste (SEQ ID NO:13 A) is provided; a kind of electronic device with high-pressure air-conditioning system

f) The CDR3 region of the GHR-106 monoclonal antibody light chain or antigen binding fragment has the amino acid sequence KQSYNLYT (SEQ ID NO: 14).

18. The use of any one of claims 1 to 17, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is formulated in any suitable manner for administration as a medicament, including in combination with a pharmaceutically acceptable excipient or other pharmaceutically suitable compound to provide a pharmaceutical composition.

19. The use of any one of claims 1-18, wherein the GHR-106 antibody or antigen-binding fragment thereof functions similarly to a known decapeptide GnRH antagonist.

20. The use of claim 19, wherein the known decapeptide GnRH antagonist comprises an anti or cetrorelix.

21. The use of any one of claims 1 to 4 or 8 to 20, wherein the subject is a male.

22. The use of any one of claims 1 to 7 or 9 to 20, wherein the subject is a female.

23. The use of any one of claims 1-22, wherein the antigen-binding fragment of the GHR-106 monoclonal antibody comprises an IgG antibody fragment, wherein the IgG antibody fragment optionally comprises F (ab') 2, fab, scFab, or scFv.

24. The use of any one of claims 1 to 23, wherein the subject is a human, and wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof comprises a humanized GHR-106 monoclonal antibody or antigen-binding fragment thereof.

25. The use as defined in any one of claims 1 to 23 wherein the subject is a monkey, rabbit, cat or dog.

26. The use of any one of claims 1-23, wherein the subject is a mammal, wherein the N1-29 amino acid sequence of the GnRH receptor hybridizes to SEQ ID NO:1 has at least 90% sequence identity.

27. The use of any one of claims 25 or 26, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody engineered to comprise the Fc region of IgG4 in a subject species.

28. The use of any one of claims 1 to 27, wherein the GHR-106 monoclonal antibody or antigen-binding fragment thereof has a half-life in the human circulation of 3 days to 21 days.