Patent Application
20240199754
Some embodiments relate to compositions for treating reproductive disorders in mammalian subjects. Some embodiments relate to compositions for modulating a level of reproductive hormones in mammalian subjects.
GnRH (gonadotropin releasing hormone) is a decapeptide hormone which reacts with the GnRH receptor located in the human anterior 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 reproductive systems in all animal species including humans.
GHR-106 is a monoclonal antibody generated from mouse against the N1-29 oligopeptide located in the extracellular domains of human GnRH (Gonadotropin releasing hormone) receptor. As can be seen from
The illustrated embodiment of the humanized IgG4 GHR-106 contains a S228P mutation engineered into the heavy chain of the antibody, as seen in SEQ ID NO:7 (note that S228 according to the EU numbering system is at position 250 in the amino acid SEQ ID NO:7). Without being bound by theory, it is believed that the S228P mutation or other equivalent mutation prevents the antibody from undergoing a recombinant process known as IgG4 Fab-arm exchange. Fab-arm exchange results in the formation of unwanted bispecific antibodies, which is known to have an undesirable effect on the specificity of the antibody to the target receptor. See, for example, Silva et al., JBC, 2015, 290(9):5462-5469, which is incorporated by reference herein for all purposes.
Due to high degrees of amino acid sequence homology (>90-95%), human GHR-106 cross-reacts with the N1-29 peptides of monkey, rabbit, dog or cat GnRH, but not with those of mouse and rat. GHR-106 and its humanized forms have been shown to react specifically with human GnRH receptor either in cancer cells or in the anterior pituitary.
In humans, there is only one type of functional GnRH receptor gene. The main site of action of the GnRH receptor located in the anterior pituitary is responsible for the release of gonadotropin hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), upon pulsatile stimulation of GnRH released from hypothalamus. However, in the reproductive related tissues or organs such as ovary or testis, as well as cancer cells, the presence of the GnRH receptor can serve to react with GnRH or its peptide analogs upon binding interactions through the mechanism of autocrine/paracrine regulation.
The administration of GnRH analogs that are antagonistic to the normal function of GnRH has been used for the treatment of a variety of sex hormone-related conditions or disorders such as reproductive diseases (in both males and females), infertility, assisted reproductive therapy such as in vitro fertilization (IVF) or egg donation (e.g. to control ovarian stimulation), contraception including inhibition of ovulation, medical transition for transgender people or sex reassignment therapy, whether male-to-female or female-to-male, and whether in conjunction with sex reassignment surgery or not, endometriosis, endometrial thinning, adenomyosis, endometrial hyperplasia, uterine leiomyoma (uterine fibroids), premenstrual syndrome, benign prostatic hypertropy, ovarian disorders, polycystic ovary disease, precocious puberty, and the like.
Due to the relatively short half-life of the native form of the hormone (2-4 min), numerous decapeptides and derivatives were made with the objective of increasing their circulation half-life to hours. Due to various structural modifications, some retain similar biological actions to stimulate or inhibit the release of gonadotropins and they are generally termed as GnRH agonists or GnRH antagonists, respectively, with respect to their mechanism of biological action to stimulate or inhibit the release of gonadotropin hormones. Decades ago, cetrorelix was released in the market and served as a GnRH antagonist for application as drugs for fertility regulation or as anti-cancer drugs with higher potency and longer half-life (hours vs. minutes) than the native GnRH. Examples of synthetic GnRH antagonists include, among others, antide, cetrorelix, abarelix, degarelix, ganirelix and elagolix.
Previous work (see U.S. Pat. Nos. 8,163,283, 9,273,138, and publication No. 2020/035462, each of which is incorporated by reference herein) related to potential clinical applications of GHR-106 and its humanized forms in treatment of human cancer and possibly fertility-related diseases. PCT application publication No. WO 2019/153075, which is incorporated by reference herein, discloses that the monoclonal antibody GHR-106 acts against human GnRH receptor and can be potentially developed into a long-acting GnRH antagonist but is otherwise biosimilar to Cetrorelix or other established peptide analogues. This is due to the fact that antibody drugs generally have a much longer half-life of 5-21 days as compared to hours for peptide antagonists such as the known GnRH peptide antagonists Cetrorelix or Antide. Despite the difference in molecular size (80 kDa vs. ˜1.5 kDa), both the peptides and the GHR-106 antibody were demonstrated to exhibit similar binding affinity (Kd 1-4 nM) and specificity to human GnRH receptor. GHR-106 monoclonal antibody has a half-life of 5-21 days, whereas the half-life of peptide GnRH antagonists such as Cetrorelix or Antide is 1-10 hrs in most cases.
Numerous GnRH peptide analogs or derivatives are currently available for clinical applications as drugs in cancer treatments such as prostate and breast cancer. Clinical applications also include many indications related to women's health, fertility and disease conditions. For example, GnRH peptide analogs are widely used for in vitro fertilization (IVF) to control programmed ovulation and hormone-dependent diseases such as endometriosis, uterine fibroids and premenstrual syndrome, or polycystic ovarian syndrome.
There is a general desire for improved and/or longer acting compositions that can be used to treat reproductive disorders and/or modulate the level of reproductive hormones in a mammalian subject.
The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
GHR-106 monoclonal antibody or an antigen-binding fragment thereof can be used to regulate a level of a sex related hormone in a mammalian subject. The GHR-106 monoclonal antibody or antigen-binding fragment thereof can cause reversible suppression of at least one sex related hormone in the mammalian subject. The reversible suppression of the at least one sex related hormone can be a decrease in serum levels of the at least one sex related hormone in the subject for a period of between 3 days and 21 days after administration of the GHR-106 monoclonal antibody or antigen-binding fragment thereof to the subject. The at least one sex related hormone can be testosterone, estradiol, luteinizing hormone, progesterone, follicle stimulating hormone, or a combination thereof. The GHR-106 monoclonal antibody or antigen-binding fragment thereof can be adapted for administration at a dosage of between about 1 mg/kg to about 3 mg/kg relative to the weight of the subject. For humans, this can translate to a dose of about 50 mg to about 300 mg. the administration can be repeated at regular spaced apart intervals, for example between about every 1 week to about every 3 weeks.
In some aspects, the GHR-106 monoclonal antibody or antigen-binding fragment thereof can have a heavy chain having CDRs having each of SEQ ID NO:9, SEQ ID NO: 10 and SEQ ID NO: 11, and a light chain having CDRs having each of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
In some aspects, the GHR-106 antibody or antigen-binding fragment thereof can be used to terminate an ectopic pregnancy, can be used to control ovulation, can be used for fertility control in a male or female subject, and/or can be used in the treatment of a sex hormone-related condition or disorder. The subject can be a mammalian subject, including a human, monkey, dog, cat, rabbit, or the like. Methods embodying any of the foregoing uses are also provided.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.
Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
The inventor has now carried out investigations including proof-of-concept experiments in rabbits and quantitative gene regulation studies as disclosed herein to support the wide-spread clinical application of GHR-106 for treatment of fertility problems and other reproductive disorders in several animal species, including humans. The proof-of-concept experiments described herein conducted in rabbits revealed reversible suppressions of serum reproductive hormones (LH, testosterone or estradiol) over a period of approximately one or two weeks. This new data demonstrates the potential suitability of GHR-106 for therapeutic application in humans as well as several other animal species.
It has not previously been demonstrated that GHR-106 will also act on the GnRH receptor in the anterior pituitary in vivo in a manner similar to decapeptide GnRH antagonists, which are known to suppress the release of gonadotropins. Therefore, in this study, the rabbit was selected as a proof-of-concept animal model to demonstrate that GHR-106 acts on the pituitary GnRH receptor to suppress the release of gonadotropin hormones in vivo. Therefore, through comparisons of bio-similarity in terms of the effect on gene expression and the regulation of the levels of reproductive hormones in vivo, it is reasonable to assume that GHR-106 could serve as an alternative to the known peptide GnRH antagonists for the therapeutic treatments of many gynecological diseases or reproductive disorders, besides human cancer, but with potential benefits associated with its longer half-life.
In some embodiments, a GHR-106 antibody or an antigen-binding fragment thereof is provided. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered to a mammal, including for example to a human, monkey, dog, cat, horse, cow, sheep, goat, rabbit or other domestic animal, to treat a reproductive condition or disorder or a sex hormone-related health condition. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is administered to a mammal in which the N1-29 terminal amino acid sequence of the GnRH receptor has an amino acid sequence with at least 90% sequence identity to the human N1-29 terminal amino acid sequence of the GnRH receptor (SEQ ID NO:1), including e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
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 target species. For example, the GHR-106 IgG4 construct disclosed herein 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 is a humanized antibody construct. In other embodiments in which the subject is a different mammalian species, chimeric antibodies engineered to contain the Fc regions of IgG4 from the subject's species could be used, for example dog IgG4-Fc for an antibody intended for administration to dogs, cat IgG4-Fc for an antibody intended for administration to cats, rabbit IgG4-Fc for an antibody intended for administration to rabbits, monkey IgG4-Fc for an antibody intended for administration to monkeys, horse IgG4-Fc for an antibody intended for administration to horses, bovine IgG4-Fc for an antibody intended for administration to cows, sheep IgG4-Fc for an antibody intended for administration to sheep, goat IgG4-Fc for an antibody intended for administration to goats, and so on.
In some embodiments, the GHR-106 antibody is provided as one or more active antigen-binding fragments of GHR-106 for use in treating sex hormone-related health conditions or disorders. In some embodiments, the fragments are single chain fragments of the variable regions of GHR-106. In some embodiments, the fragments are fragments of GHR-106 of the IgG isotype, including IgG4. In some embodiments, the fragment is an F(ab′)2 fragment. In some embodiments, the F(ab′)2 fragment has a molecular weight of 110 KDa. In some embodiments, the fragment is a Fab fragment. In some embodiments, the Fab fragment has a molecular weight of 55 KDa. In some embodiments, the fragment is an scFab fragment. In some embodiments, the scFab fragment has a molecular weight of 25 KDa. In some embodiments, the fragment is an scFv fragment. In some embodiments, the scFv fragment has a molecular weight of 25 KDa. In some embodiments, combinations of different antigen-binding fragments e.g. two or more of the fragments as described above, can be used as a drug for the treatment of a sex-hormone related condition or disorder.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof that are administered for the treatment of sex hormone-related health conditions or disorders do not possess effector functions. An antibody that does not possess effector functions cannot activate, for example, complement-dependent cytoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) pathways. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof that do not possess effector functions have an IgG4 subtype. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof inhibit complement activation. In some embodiments, the heavy chain of the antibody having the IgG4 subtype has a S228P mutation or an equivalent mutation, to prevent Fab-arm exchange. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof that do not possess effector functions are IgG antigen-binding fragments of GHR-106 antibodies. In some embodiments, the antigen-binding fragments that do not possess effector functions are F(ab′)2, Fab, scFab or scFv IgG fragments of GHR-106 antibodies. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are derived from hGHR-106.
In some embodiments, the subtype of the GHR-106 antibody is selected to modulate the effector functions of the antibody. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof is structurally modified to further modulate the effector functions of the antibody, for example by using an antigen-binding fragment of the antibody that does not possess any effector functions. In some embodiments, the Fc region of the GHR-106 antibody is of the IgG4 subtype. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof that does not possess any effector functions is used for the treatment of a sex hormone-related health condition or disorder, for reversibly suppressing a level of at least one sex related hormone in a subject, for controlling ovulation in a subject, and/or for terminating an ectopic pregnancy in a subject. In some embodiments, the GHR-106 antibody having an IgG4 subtype is used for the treatment of sex hormone-related health conditions or disorders.
Without being bound by theory, it is believed that because the IgG4 antibody subtype does not activate complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC), use of the IgG4 antibody subtype for treatment of sex hormone-related health conditions or disorders or otherwise to modulate a level of a sex-related hormone in a subject, including treating fertility disorders, will minimize or eliminate the possibility of CDC and ADCC reactions upon the GHR-106 antibody binding to the anterior pituitary. See for example Vidarsson et al., Front. Immunol., 2014, 5:520, which is incorporated by reference herein for all purposes.
Further, it has been demonstrated that IgG4 antibodies can actually inhibit complement activation (see e.g. van der Zee et al., Clin. Exp. Immunol., 1986, 64(2):415-422, which is incorporated by reference herein for all purposes). Thus, in some embodiments, the GHR-106 monoclonal antibody or antigen-binding fragment thereof is selected to inhibit complement activation. In some embodiments, the 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 circulation half-life of the GHR-106 antibody is approximately 3 to 21 days, including any value therebetween e.g. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 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. By contrast, the circulation half life cetrorelix is in a range of approximately 10 to 63 hours. GHR-106 has a much longer half life compared to the decapeptide GnRH antagonist cetrorelix, and therefore may require less frequent administration, which may improve patient compliance and/or the feasibility of a proposed treatment regime.
In some embodiments, the IgG antigen-binding fragments that are derived from GHR-106, e.g. F(ab′)2, Fab, ScFab or ScFv, each has a circulation half-life of approximately 12 to 20 hours, including any value therebetween e.g. 13, 14, 15, 16, 17, 18 or 19 hours. The antigen-binding fragments of mGHR-106 or hGHR-106 have a shorter half-life compared to the mGHR-106 or hGHR-106 antibodies. In some embodiments, protein engineering is used to provide GHR-106 antibodies or antigen-binding fragments thereof that have a half-life within a desired range.
In some embodiments, the GHR-106 antibody or the antigen-binding fragment thereof has a heavy chain with the amino acid sequence according to SEQ ID NO:7 and a light chain with the amino acid sequence according to SEQ ID NO:8. In some embodiments, the GHR-106 antibody or antigen-binding fragment thereof has a heavy chain having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:7 and a light chain having an amino acid sequence with at least 90% sequence identity to SEQ ID NO:8, including e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOS:7 and 8, respectively.
In further embodiments, the GHR-106 antibody or antigen-binding fragment thereof has a heavy chain having the following complementarity determining regions (CDRs): a CDR1 region having the amino acid sequence according to SEQ ID NO:9 (RYSVH), a CDR2 region having the amino acid sequence according to SEQ ID NO: 10 (MIWGGGSTDYNPSLKSR), and a CDR3 region having the amino acid sequence according to SEQ ID NO:11 (GYYSFA). In further embodiments, the GHR-106 antibody or antigen-binding fragment thereof has a light chain having the following CDRs: a CDR1 region having the amino acid sequence according to SEQ ID NO: 12 (KSSQSLLNSRTRKNYLA), a CDR2 region having the amino acid sequence according to SEQ ID NO: 13 (WASTRES), and a CDR3 region having the amino acid sequence according to SEQ ID NO: 14 (KQSYNLYT).
The GHR-106 antibodies or antigen-binding fragments thereof described herein can be formulated in any suitable manner for administration as a medicament. Thus, they can be combined with pharmaceutically acceptable excipients or other pharmaceutically suitable compounds to provide pharmaceutical compositions useful for the modulation of levels of sex hormones and/or treatment of sex hormone-related health conditions or disorders.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are administered in a therapeutically effective amount for the treatment of sex hormone-related health conditions or disorders and/or in an amount effective to modulate a level of one or more sex related hormones in a mammal, including a human, monkey, dog, cat, rabbit, horse, cow, sheep, goat or other domestic animal. The mammal may be a male or a female. In some embodiments, the sex hormone-related health condition or disorder is a reproductive disease (in a male or female subject), medical transition for transgender people including male-to-female (MTF) or female-to-male (FTM) sex reassignment therapy, whether or not accompanied by sex reassignment surgery, in vitro fertilization (IVF) or egg donation (e.g. to control ovarian stimulation), contraception including inhibition of ovulation in female subjects or of sperm production in male subjects, endometriosis, endometrial thinning, adenomyosis, endometrial hyperplasia, uterine leiomyoma (uterine fibroids), premenstrual syndrome, benign prostatic hypertrophy, ovarian disorders, polycystic ovary disease, precocious puberty, and the like.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are administered to a male subject for fertility control (e.g. birth control). Without being bound by theory, the data contained in the examples of this application support that administration of GHR-106 antibodies or antigen-binding fragments thereof to a male subject can decrease levels of sex related hormones such as testosterone to a level that is likely to interfere with the production of sperm by the male subject, thereby providing fertility control for a male subject (i.e. birth control for a male subject).
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are administered to terminate an ectopic pregnancy. Without being bound by theory, it is believed that the drop in reproductive hormone levels caused by the administration of a GHR-106 antibody or antigen-binding fragment thereof will be deleterious to the fetus, and/or that GnRH receptor and GnRH are highly expressed in human placenta in parallel with the amount of human chorionic gonadotropin secretion, resulting in the rapid termination of the ectopic pregnancy while minimizing negative effects on the subject.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are used to regulate ovulation in a subject. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are administered to a female subject for fertility control (e.g. birth control). In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are used to regulate a level of one or more sex related hormones in a subject, including by causing a reversible decrease in a serum concentration of the one or more sex related hormones. In some embodiments, the sex related hormone is testosterone, estradiol, lutenizing hormone, progesterone, follicle stimulating hormone, or a combination thereof. In some embodiments, alteration of the level of the sex related hormone alters the fertility status of the subject.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof act as GnRH antagonists in the treatment of any condition that can be treated by known GnRH antagonists including antide or cetrorelix. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are used in the treatment of a condition in which a longer half-life than that of known GnRH antagonists, including antide or cetrorelix, is desirable.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are administered to a subject at dosage levels of 0.5-10 mg/kg, including any value therebetween, including e.g. about 1 to about 3 mg/kg in some embodiments, e.g. 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 including any value or subrange therebetween. In some embodiments in which the binding affinity and/or specificity of the GHR-106 antibody or antigen-binding fragment thereof has been modified, the dosage level of the modified antibody or antigen-binding fragment thereof is modified appropriately.
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 mg and about 300 mg, including any value therebetween, e.g. 75, 100, 125, 150, 175, 200, 225, 250 or 275 mg.
In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof are administered at repeated spaced apart intervals, for example 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 therebetween, e.g. every 2, 3, 4, 5, 6 or 7 weeks, or every 2-6 months or any value therebetween, e.g. every 3, 4 or 5 months. In some embodiments, the GHR-106 antibody or antigen-binding fragment there of is administered at repeated spaced apart intervals of between about every 1 week to about every 3 weeks. In some embodiments, the GHR-106 antibodies or antigen-binding fragments thereof that are administered to a human are humanized GHR-106 antibodies or antigen-binding fragments thereof. In some embodiments, the humanized GHR-106 antibody is hGHR-106 IgG4 having a heavy chain having the amino acid sequence of SEQ ID NO:7 and a light chain having the amino acid sequence of SEQ ID NO:8.
A typical route of administration of pharmaceutical compositions comprising antibodies is via injection, typically intravenous or intramuscular. However, any suitable mode of administration can be used in various embodiments.
Certain embodiments are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature.
Proof-of-concept experiments were performed in rabbits to demonstrate reversible suppression of serum reproductive hormones upon a single injection of humanized monoclonal antibody against GnRH receptor, GHR-106(hIgG4) having a heavy chain having the amino acid sequence of SEQ ID NO:7 and a light chain having the amino acid sequence of SEQ ID NO:8.
A single subcutaneous injection of 1 mg/kg or 3 mg/kg of the antibody to the male rabbit was shown to decrease in parallel the serum LH and testosterone concentrations by 80 to 90% of the normal level for a period of seven to ten days. The reproductive hormones returned to normal levels approximately two weeks after the initial injection. Similar observations were obtained with the female rabbits, in which the serum LH and estradiol concentrations were reversibly suppressed and recovered upon the same dose of single injection with the same antibody. These experiments support that GHR-106(hIgG4) can act on the anterior pituitary GnRH receptor as an antibody-based GnRH antagonist similar to Elagolix or Antide, except that GHR-106(hIgG4) has a much longer half-life (days vs. hours).
Besides proof-of-concept experiments in rabbits, quantitative RT-PCR experiments were performed and used as a tool to demonstrate almost a complete identity of the intracellular gene regulation between GHR-106 and decapeptide GnRH antagonists through in vitro studies. Therefore, it is reasonable to conclude that antibody-based and peptide-based GnRH antagonists are highly similar in terms of biological mechanisms of action, both against cancer cells and in the reversible suppressions of gonadotropin release by anterior pituitary, except that GHR-106 has a significantly longer half-life.
GHR-106 of various isoforms including mouse-dog or mouse-cat chimeric forms can be mass-produced based on the knowledge and methods as are known in the art, including the US patents cited herein. For example, human (variable region) dog (constant Fc region) chimeric antibody, and mouse (variable region) dog (constant Fc Region) chimeric antibody can be mass produced based on the established knowledge for a GHR-106 antibody intended for administration to dogs.
Murine GHR-106 can be produced and purified through ascites fluid in mice or by in vitro culture methods of hybridoma cell lines. Humanized GHR-106 can be produced by permanent cell lines established previously. These include mGHR-106 (murine origin) GHR-106(hIgG1), and GHR-106(hIgG4) as well as different antibody fragments such as Fab, (Fab′)2 or single chain fragments of the variable region.
GHR-106 is a monoclonal antibody derived from the immunization of mouse against the N1-29 oligopeptide of human GnRH receptor. The amino acid sequences of the heavy chain and light chain of GHR-106 are shown in
Based on the comparisons of N1-29 peptide sequences from different animal species, it can be concluded that GHR-106 may have a high degree of binding cross-reactivity between humans and several other animal species including rabbit, cat, dog and monkey. Therefore, GHR-106 can be potentially used as GnRH antagonist, not only for human applications, but also for those of several other animal species (mammals) including rabbit, dog and cat. The person skilled in the art can carry out established techniques (for example those used in the design of humanized antibodies) to design an antibody that is suitable for use in a different mammalian species to minimize the likelihood of undesirable cross-reactivity of the antibody.
Binding studies between GHR-106 and N1-29 peptides derived from the animal species mentioned are essential to demonstrate comparable binding affinity of GHR-106 to N1-29 oligopeptides derived from human, dog, and rabbit, respectively. Therefore, comparative binding ELISA studies were performed to estimate the relative binding affinity between GHR-106 and microwell-coated N1-29 oligopeptides derived from human, dog and rabbit, respectively. The results of such binding studies are presented and compared in
In previous studies, the inventor had showed that the three isoforms of GHR-106 including murine GHR-106, humanized GHR-106 and humanized GHR-106(hIgG4) are essentially identical in their respective binding affinity and specificity to human GnRH receptor as well as its N1-29 oligopeptide with dissociation constants on the order of 1-5 nM.
Based on ELISA binding studies presented in
Previous in vitro studies with human cancer cells have indicated that the apoptosis of cultured cancer cells can be induced after 24 to 72 hours following co-incubation with 1-10 μg/ml of GHR-106 monoclonal antibody in different isoforms. The degrees of induced apoptosis were comparable to those of the decapeptide GnRH antagonist, Antide, although the antibody is fifty times higher in molecular size.
To demonstrate that GHR-106 interacts with pituitary GnRH receptor similar to that of 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 regularly following a single subcutaneous injection with 3 mg/kg of hGHR-106.
The serum LH and testosterone concentrations were determined, respectively, by EIA kits and plotted as a function of time during the day 1 to 30 time period. The results of the hormonal profiles are presented in
As shown in
Similarly, upon a single injection of 3 mg/kg dose to a male rabbit, the serum testosterone concentrations decreased by more than 80% from 0.95 ng/ml to ≤0.1 ng/ml during the first two weeks. The time-dependent serum levels of testosterone are synchronised with those of LH. During the third week after the injections, the fluctuating changes of LH and testosterone levels are parallel to each other until day 30 when the hormone levels are within the normal range (
The hormonal profiles of serum luteinizing hormone (LH) and Estradiol (E2) in female rabbits were monitored upon a single injection with a 3 mg/kg dose of hGHR-106. The serum concentrations of LH and E2 were also determined regularly from Day 1 to Day 20 and presented in
The suppression of LH levels was observed immediately during the first few days after the antibody injection (from 3 mlU/ml to ≤1 mlU/ml). Similarly, the serum E2 concentrations were decreased in parallel during the same time period (from 50 pg/ml to ≤20 pg/ml) until day 10.
From day 10 to day 20, both LH and E2 concentrations increased with time and reached 6 mIU/ml and 120 pg/ml, respectively after day 18 and 20.
At a lower dose injection of 1 mg/kg to separated females, the overall profiles of LH and E2 concentrations were similar to those of the high dose during the same observation period (data not presented).
Quantitative Gene Regulation Studies were performed to justify a complete identity of molecular mechanisms between GHR-106 and the peptide GnRH antagonist antide. Quantitative changes of gene expression upon binding to human GnRH receptor were presented by using either GHR-106 or the peptide GnRH antagonist antide after incubation with human cancer cells. To make further comparisons between GHR-106 and decapeptide GnRH antagonists, ten regulatory genes were selected for quantitation by RTPCR methods and results are presented and compared in
Testing reveals the high specificity of GHR-106 to human GnRH receptor as compared to many other known and available anti GnRH receptor monoclonal antibodies. In particular, the ability of GHR-106 to detect overexpression of GnRHR in a reference cell line is tested and compared with four different commercially available antibodies. It is found that only the GHR-106 antibody is able to detect overexpression of GnRHR in the reference cell line.
Given this tissue-specificity in humans that is not observed in other antibodies, GHR-106 may be one of the best antibodies generated to react with human GnRH receptor, respectively, as well as the N1-29 oligopeptide of this receptor.
In addition, the other examples described herein also reveal a high degree of species cross-reactivity among several different animal species. Therefore, GHR-106 should be recognized as a third class of therapeutic, i.e. an antibody-based GnRH antagonist which is comparable to organic chemical or decapeptide-based GnRH antagonists.
Humanized forms of GHR-106 can only be utilized for human clinical application either in cancer therapy or in fertility control, due to the intrinsic and restricted immunogenicity upon applications in humans. This concern may be considered so suitable modifications to the antibody, and in particular the Fc regions of the antibody, can be made for clinical applications in other animal species, including other mammalian species. For example, in order to avoid allergic reactions to allogeneic injections, pure-bred mouse-derived antibodies can be replaced with chimeric antibodies from another species, e.g. dogs or cats (a receptor-constant region). To minimize heterologous immune response, mouse (variable, VR)-dog (constant, Fc) chimeric IgG can be produced and used for applications in dogs. Similarly, mouse-cat chimeric antibody can be generated according to known methods for application in cats. Similar modifications can be made for application of the GHR-106 antibody as a therapeutic agent in other species.
To demonstrate that GHR-106(hIgG4) acts as a GnRH antagonist in vivo, large scale proof of concept experiments were performed in rabbits and data presented in these additional examples.
Judging from the data from the above examples, reversible suppression of reproductive (i.e. sex related) hormones including luteinizing hormone, testosterone and estradiol was observed upon single injections of male or female rabbits. The serum levels of reproductive hormones returned to normal ranges following one to two weeks after injection.
With preliminary observations in single rabbit data, large scale rabbit experiments (n≥30) including negative controls were conducted with identical protocols. Specifically, to further confirm that GHR-106 acts as a GnRH antagonist, large scale experiments were performed in male and female rabbits. The data generated in each experimental group were analyzed statistically. The means and standard deviations of hormone levels from rabbits in each experimental group are presented in
In the case of male rabbits, the serum concentrations of reproductive hormones including LH and testosterone of ten male rabbits were monitored and measured after a single subcutaneous injection with 1 mg/kg of GHR-106 (low dosage, n=3) or 3 mg/kg of GHR-106 (high dosage, n=3) on day 1, or after no injection (negative control, n=4). Serum testosterone levels were determined and the averages (with standard deviations) were determined from day 1 to day 17, and serum LH levels were determined starting on day 1 and ending on day 13 (with standard deviations) The results of the testosterone profile are found in
As seen in
In the case of female rabbits, the serum concentrations of reproductive hormones including LH and estradiol (E2) of ten female rabbits were monitored and measured after a single subcutaneous injection with 1 mg/kg of GHR-106 (low dosage, n=3) or 3 mg/kg of GHR-106 (high dosage, n=3) on day 1, or after no injection (negative control, n=4). Serum E2 levels were determined starting on day 1 and ending on day 17, and serum LH levels were determined starting on day 1 and ending on day 13. The results of the estradiol (E2) profile are found in
As seen in
In general, both single or multi-rabbit experiments revealed reversible suppression of reproductive hormones upon a single injection of GHR-106(hIgG4) with either 1 mg/kg or 3 mg/kg dose. The hormone levels return to normal ranges as in the negative control group after 1-3 weeks, indicating the suppression of the sex related hormones is reversible. Thus, consistent with the individual rabbit data described above, effects of reversible hormone suppression were observed among the larger experimental group.
In conclusion, multi-rabbit and individual rabbit experiments led to the same conclusion in terms of time-dependent reversible sex related hormone suppression upon GHR-106 antibody treatments. Therefore, the inventor has demonstrated that GHR-106(hIgG4) is an antibody-based long-acting GnRH antagonist, which exhibits biological effects comparable with the decapeptide GnRH antagonist such as cetrorelix, currently in use clinically, but with potential benefits of a longer period of activity given its longer half life.
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 specification as a whole.
Without limiting the foregoing, various embodiments include a number of aspects including the following. These aspects are based on the examples disclosed in this application, which demonstrate factors including (1) a high degree of amino acid sequence homology and broad degrees of species-cross reactivity of GHR-106 to several animal species; (2) proof-of-concept experiments providing direct evidence of strong interactions between GHR-106 and GnRH receptor in humans or other mammalian species; and (3) a high degree of identity and consistency in quantitative gene expression level changes between the long acting antibody-based and short-lived peptide-based GnRH antagonists.
In a first aspect, GHR-106 of various isoforms or species cross-reacts with GnRH receptors of several different animal species (dog, cat and rabbit and monkey) and can be used as a GnRH antagonist, as long as they share with humans a high degree of sequence homology in N1-29 oligopeptide of their respective receptors (≥90-95%).
In a second aspect, as a GnRH antagonist, GHR-106 can be used to reversibly suppress the endogeneous reproductive hormones (eg: LH, FSH, testosterone, estradiol and progesterone etc.) of humans or any other animal species which meet the criteria under the first aspect.
In a third aspect, as a GnRH antagonist, GHR-106 in humanized IgG4 isotype [GHR-106(hIgG4)] can act directly on human anterior pituitary for reversible suppression of reproductive hormones upon GHR-106 treatment to manipulate GnRH receptor-controlled fertility regulations or disorders, similar to the drug actions of decapeptide analogs such as cetrorelix.
In a fourth aspect, as a GnRH antagonist, GHR-106 in various isoforms or species can be used in cancer therapy of almost all receptor-positive cancer not only in humans, but also several other animal species, including dog, cat and rabbit, etc.
This application claims priority to, and the benefit of, U.S. provisional patent application No. 63/189,852 filed 18 May 2021 and No. 63/242,976 filed 10 Sep. 2021, both entitled Applications of GHR-106 Monoclonal Antibody as GnRH Antagonist. Both of the foregoing applications are hereby incorporated by reference in their entireties for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050777 | 5/17/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63189852 | May 2021 | US | |
| 63242976 | Sep 2021 | US |
