WO2019211842A1 - Antagonistes de l'hormone de croissance humaine à action prolongée et leurs procédés de production - Google Patents

Antagonistes de l'hormone de croissance humaine à action prolongée et leurs procédés de production Download PDF

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WO2019211842A1
WO2019211842A1 PCT/IL2019/050479 IL2019050479W WO2019211842A1 WO 2019211842 A1 WO2019211842 A1 WO 2019211842A1 IL 2019050479 W IL2019050479 W IL 2019050479W WO 2019211842 A1 WO2019211842 A1 WO 2019211842A1
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hgha
variant
ctp
amino acid
growth hormone
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PCT/IL2019/050479
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English (en)
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Oren HERSHKOVITZ
Ahuva BAR-ILAN
Laura Moschcovich
Lital ISRAELI YAGEV
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Opko Biologics Ltd.
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Publication of WO2019211842A1 publication Critical patent/WO2019211842A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • compositions which include human growth hormone (hGH) antagonist variants, polypeptides comprising at least one carboxy-terminal peptide (CTP) of chorionic gonadotropin attached to the carboxy terminus of a human growth hormone (hGH) antagonist and at least one CTP attached to the amino terminus of a hGH antagonist, hGHA variants linked to one or more polyethylene glycol polymers (PEG) via reversible or non-reversible linkers.
  • PEG polyethylene glycol polymers
  • Growth hormone is a protein hormone that causes, among other things, the production and release of the IGF-l hormone into the blood system.
  • IGF-l is a marker for normal growth and metabolism in children and the body mass composition in adults, related to the activity and role of growth hormone.
  • Growth hormone is produced by the pituitary gland and plays a central role in the endocrine system in the regulation and release of various hormones such as growth hormones, sex, metabolic, etc.
  • the hormone affects all body cells and causes cell division and growth in children and adolescents.
  • the clinical indication at the onset of these symptoms is defined as acromegaly, a disease characterized by disproportionate growth, which is characterized by an increase in the size of the body, of the bones and tissue swelling.
  • the acromegaly disease may be genetic but is usually caused by benign pituitary adenoma.
  • This tumor increases the secretion of growth hormone, which stimulates the secretion of insulin-like growth factor 1 (IGF-l), which is a major trigger for the pathological symptoms of the disease.
  • IGF-l insulin-like growth factor 1
  • Another therapeutic approach is drug alternatives that are applied if the surgery is unsuccessful or if it is not possible to perform it.
  • drugs analgesics for somatostatin
  • SSAs GH receptor antagonists
  • GHRAs GH receptor antagonists
  • dopamine agonists about 50% of patients treated with somatostatin do not respond to treatment effectively and therefore receive alternative therapy.
  • the Somavert®. drug is conjugated to several polyethylene glycol sequences
  • PEG polyethylene glycol
  • Somavert® is given daily. Patients treated with Somavert® often discontinue treatment due to the discomfort and constraints associated with the use of the drug, including a restrictive injection regimen based on daily injections and powdered formulation required to be dissolved before each injection.
  • Radiotherapy Another alternative to acromegaly is radiotherapy, which is often applied to patients who have not responded positively to medication. This therapeutic solution is not effective in many cases and involves the dangers that characterize exposure to radiation.
  • a drug that has a longer half-life, and that is more efficient and more convenient than the drugs currently available in the market is needed for acromegaly patients.
  • composition comprising a hGH antagonist (hGHA) variant having:
  • the at least one mutation resulting in antagonist activity is an amino acid substitution at a residue corresponding to residue 120 of human growth hormone, numbered from the N-terminus.
  • the amino acid substitution resulting in antagonist activity is at residue l20and is lysine.
  • said at least 6, 8, or 15 amino acid substitutions corresponding to residues 1-119 and 121-191 of human growth hormone, numbered from the N-terminus.
  • said at least 6 amino acid substitutions occur at residues selected from the group consisting 18, 21 , 167, 171, 174, and 179, numbered from the N-terminus.
  • amino acid sequence of said wild type hGH is set forth in SEQ ID NO: 4.
  • the hGHA variant comprises the amino acid substitutions H18D, H21N, R167N, D171S, E174S, and I179T.
  • the hGHA variant having at least 6 AA substitutions comprises the amino acid sequence of SEQ ID NO: 13.
  • the hGHA variant has at least 8 amino acid substitutions occurring at residues selected from the group consisting 18, 21, 54, 64, 167, 171 , 174, and 179, numbered from the N-terminus.
  • the hGHA variant comprises the amino acid substitutions H18D,
  • the hGHA variant having at least 8 AA substitutions comprises the amino acid sequence of SEQ ID NO: 14. [0013] In a related aspect, the at least 15 amino acid substitutions occur at residues selected from the group consisting 10, 14, 18, 21, 41 , 42, 45, 46, 54, 64, 167, 171, 174, 176, and 179, numbered from the N-terminus.
  • the hGHA variant comprises the a ino acid substitutions F10A, M14W, H18D, H21N, K41I, Y42H, L45W, Q46W, F54P, R64K, R167N, D171S, E174S, F176Y and I179T.
  • the hGHA variant having at least 15 AA substitutions comprises the amino acid sequence of SEQ ID NO: 15.
  • the hGHA variant further comprising a signal peptide.
  • the signal peptide is present at the N-terminus.
  • the amino acid sequence of said signal peptide comprises SEQ ID NO: 10.
  • a polypeptide comprising a growth hormone antagonist attached to at least two chorionic gonadotrophin carboxy terminal peptides (CTPs).
  • CTPs chorionic gonadotrophin carboxy terminal peptides
  • the CTP-modified hGHA comprises the hGHA or hGHA antagonitst described through the present application.
  • the first CTP of said at least two CTPs is attached to the amino terminus of said growth hormone antagonist, and the second CTP of said at least two CTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • the amino acid sequence of the CTP-modified hGHA comprises SEQ ID NO: 5.
  • CTP-modified hGHA further comprises at least three CTPs attached to said growth hormone antagonist.
  • one CTP is attached to the amino terminus of said growth hormone antagonist, and two CTPs are attached to the carboxy terminus of said growth hormone antagonist.
  • -23 wherein the amino acid sequence of CTP-modified hGHA comprises any one of SEQ ID NO: 7, 8, or 9.
  • the CTP-modified hGHA further comprises at least four CTPs attached to said growth hormone antagonist.
  • one CTP is attached to the amino terminus of said growth hormone antagonist, and three CTPs are attached to the carboxy terminus of said growth hormone antagonist.
  • the amino acid sequence of CTP-modified hGHA comprises SEQ ID NO: 6.
  • CTP-modified hGHA further comprises a signal peptide.
  • the signal peptide is present at the N-terminus.
  • the amino acid sequence of said signal peptide comprises SEQ ID NO: 10.
  • the amino acid sequence of the CTP- modified hGHA containing a signal peptide comprises SEQ ID NO: 11.
  • At least one CTP of the CTP-modified hGHA is glycosylated.
  • a pharmaceutical composition comprising the CTP-modified hGHA described throughout the present application.
  • the nucleotide sequence encoding the CTP-modified hGHA comprises SEQ ID NO: 12.
  • PEG is a polyethylene glycol polymer
  • R2 is H, O-CH 3 , or SO 3 H
  • hGHA is a human growth hormone antagonist or hGHA variant.
  • the PEGylated variant comprises any of the hGHA or hGHA variants described throughout the present application.
  • the PEGylated hGHA variant is linked via an amino group of said hGHA variant.
  • the linker containing a maleimide group is further reacted with a thiol- containing molecule.
  • the thiol-containing molecule is cysteine or cysteamine.
  • the reacting results in the reduction of the MAL-linker-hGHA such as maleimide hydrogenation, and/or the coupling of the thiol-containing molecule to the linker-hGHA.
  • the PEG is a PEG polymer linked to a thiol group on said PEG polymer having the formula CH3-(0-CH2-CH2)n-S- wherein n is 5, 30, 40, or 60.
  • the PEG is PEG2, PEG5, PEG10, PEG20, PEG30, PEG40, or PEG60.
  • the PEG has a molecular weight in the range of 2,000 to 50,000 Da.
  • FIG. 1 is a schematic presentation of the interactions between the growth hormone (GH)/ growth hormone antagonist (GHA) to the growth hormone receptor (GHR).
  • the GH having a mutation in amino acid at position 120 (GHA) at site 2, binds to the receptor and prevents biological activity, while the additional mutations (not indicated in the schematic) at site 1, increase the antagonist's affinity to the receptor and contribute to neutralizing the signal.
  • Figure 2 shows the dose-dependent binding curves of different MOD-1201 variants and Somavert® to the GHR (cell proliferation inhibition).
  • Figure 3 shows a comparison of the PK profiles of the five-MOD-l20l variants and Somavert® after subcutaneous (SC) injection at 3 mg/kg.
  • Figure 4 shows a dose dependent graph of MOD-12014 serum levels following single injection at three different dose levels in rats.
  • Figure 5 shows a dose depended graph of IGF-l profiles, as measured by the averaged percent decrease compare to pre-dose, following single SC injection of MOD-12014 at three different dose levels compared to Somavert in rats
  • Figure 6 shows a dose dependent IGF-l profiles, as measured by the percent decrease compared to pre-dose, following SC injections of variant 2 and 4 at two dose levels compare to Somavert , in rabbits.
  • Figure 7 shows a dose dependent IGF-l profiles, as measured by the percent decrease compare to pre-dose, following three SC injections at 3.2 and 7.2mg/kg or single dose at 25mg/kg, in rabbits.
  • Figure 8 shows a schematic presentation of a vector containing hGHA sequence and one or more CTP copies used to produce MOD-1201 variants.
  • Figure 9 shows a schematic presentation of MOD-1201 variant 4 (MOD-12014) with 3 CTPs copies, 8 amino acid substitutions for increasing binding affinity, and the AA G120K substitution for antagonistic characteristic.
  • Figure 10 shows a flow Chart of MOD- 12014 DSP Process.
  • the present invention provides a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • cgCTPs chorionic gonadotrophin carboxy terminal peptides
  • the present invention provides a polypeptide comprising a growth hormone antagonist.
  • the growth hormone comprises a human growth hormone.
  • the hGH antagonist is mutated to improve its binding characteristics (See Lowman, Henry B., and James A. Wells. "Affinity maturation of human growth hormone by monovalent phage display.” Journal of molecular biology 234.3 (1993): 564-578).
  • the number of mutations of the hGH antagonist are selected to improve its binding characteristics.
  • the hGH antagonist contains 6 mutations to improve its binding characteristics.
  • the hGH antagonist contains 8 mutations to improve its binding characteristics.
  • the hGH antagonist contains 15 mutations to improve its binding characteristics.
  • the presence of 6 mutations in the hGHA is the most conservative approach.
  • the presence of 8 mutations in the hGHA increases the binding to the receptor without increasing the immune risk in humans.
  • the presence of 15 mutations in the hGHA results in highest binding affinity.
  • the presence of 15 mutations in the hGHA increases the risk of immunogenic response in humans.
  • the growth hormone antagonist comprises a growth hormone sequence wherein the glycine at position 120 of the growth hormone sequence is substituted with another a ino acid. In one embodiment, the glycine at position 120 of the growth hormone sequence is substituted with an amino acid residue other than alanine.
  • the growth hormone antagonist as described herein comprises a growth hormone sequence with a Glyl20 substitution and one or more mutations described in Lowman and Wells, 1993 (Journal of molecular biology 234.3 (1993): 564-578).
  • the invention includes a homologue of a growth hormone antagonist.
  • the invention includes a homologue of a human growth hormone antagonist.
  • homologues e.g., polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a growth hormone or human growth hormone antagonist as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.
  • NCBI National Center of Biotechnology Information
  • “signal sequence” and“signal peptide” are used interchangeably herein having all the same qualities and meanings.
  • “sequence” when in reference to a polynucleotide molecule can refer to a coding portion.
  • an engineered hGHA comprising at least one CTP as described herein has enhanced in vivo biological activity compared the same hGHA without at least one CTP.
  • the enhanced biological activity stems from the longer half-life of the engineered hGHA while maintaining at least some biological activity.
  • the enhanced biological activity stems from enhanced biological activity resulting from the CTP modification.
  • the enhanced biological activity stems from both a longer half-life and from enhanced functionality of the CTP-modified hGHA.
  • the CTP-modified hGHA includes a signal peptide. In another embodiment, the CTP-modified hGHA does not comprise a signal peptide.
  • cgCTPs Chorionic gonadotrophin carboxy terminal peptides
  • the present invention provides a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs).
  • the CTP sequence comprises: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 1). In another embodiment, the CTP sequence comprises: SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 2). In another embodiment, the CTP sequence comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
  • the carboxy terminal peptide (CTP) peptide of the present invention comprises the amino acid sequence from amino acid 112 to position 145 of human chorionic gonadotrophin.
  • the human chorionic gonadotrophin carboxy terminal peptide of the present is referred to as either CTP or cgCTP.
  • the CTP sequence of the present invention comprises the amino acid sequence from amino acid 118 to position 145 of human chorionic gonadotropin, as set forth in SEQ ID NO: 2.
  • the CTP sequence also commences from any position between positions 112-118 and terminates at position 145 of human chorionic gonadotrophin.
  • the CTP sequence peptide is 28, 29, 30, 31, 32, 33 or 34 amino acids long and commences at position 112, 113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.
  • the cgCTP of the compositions and methods of the present invention is truncated.
  • the truncated CTP comprises SSSSKAPPPSLP (SEQ ID NO: 3).
  • the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 3.
  • the truncated CTP comprises the first 11 amino acids of SEQ ID NO: 3.
  • the truncated CTP comprises the first 14 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 13 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 12 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 11 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 10 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 9 amino acids of SEQ ID NO: 2. In one embodiment, the truncated CTP comprises the first 8 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3.
  • the truncated CTP comprises the first 7 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the truncated CTP comprises the first 6 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the truncated CTP comprises the first 5 amino acids of SEQ ID NO: 2 or SEQ ID NO: 3.
  • the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1-5 conservative amino acid substitutions as described in U.S. Pat. No. 5,712,122, which is incorporated herein by reference in its entirety.
  • the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 1 conservative amino acid substitution.
  • the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 2 conservative amino acid substitutions.
  • the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 3 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 4 conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of chorionic gonadotrophin CTP which differs from the native CTP by 5 conservative amino acid substitutions.
  • the CTP peptide amino acid sequence of the present invention is at least 70% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 80% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 85% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 90% homologous to the native CTP amino acid sequence or a peptide thereof.
  • the CTP peptide amino acid sequence of the present invention is at least 95% homologous to the native CTP amino acid sequence or a peptide thereof. In another embodiment, the CTP peptide amino acid sequence of the present invention is at least 98% homologous to the native CTP amino acid sequence or a peptide thereof.
  • the polypeptide comprising a growth hormone antagonist comprises a single cgCTP at the amino terminus of said growth hormone antagonist. In another embodiment, comprises two cgCTPs at the amino terminus of said growth hormone antagonist. In another embodiment, comprises three cgCTPs at the amino terminus of said growth hormone antagonist. In another embodiment, comprises four cgCTPs at the amino terminus of said growth hormone antagonist. In another embodiment, comprises five cgCTPs at the amino terminus of said growth hormone antagonist.
  • the polypeptide comprising a growth hormone antagonist comprises a single cgCTP at the carboxy terminus of said growth hormone antagonist. In another embodiment, comprises two cgCTPs at the carboxy terminus of said growth hormone antagonist. In another embodiment, comprises three cgCTPs at the carboxy terminus of said growth hormone antagonist. In another embodiment, comprises four cgCTPs at the carboxy terminus of said growth hormone antagonist. In another embodiment, comprises five cgCTPs at the carboxy terminus of said growth hormone antagonist.
  • the polypeptide comprising a growth hormone antagonist comprises a single cgCTP at the amino terminus and a single cgCTP at the carboxy terminus. In another embodiment, the polypeptide comprising a growth hormone antagonist comprises a single cgCTP at the amino terminus and three cgCTPs at the carboxy terminus. In another embodiment, the polypeptide comprising a growth hormone antagonist comprises a single cgCTP at the amino terminus and two cgCTPs at the carboxy terminus.
  • the wild type human growth hormone comprises the following amino acid sequence:
  • the amino acid sequence of the CTP-modified human growth hormone (hGFl) antagonist Variant MOD1201-1 comprises the following amino acid sequence:
  • the amino acid sequence of the CTP-modified human growth hormone (hGH) antagonist Variant MOD1201-2 (CTP-mut hGHA-CTP-CTP-CTP, G120K + 6 mutations) comprises the following amino acid sequence:
  • the amino acid sequence of the CTP-modified human growth hormone (hGH) antagonist Variant MOD 1201-3 comprises the following amino acid sequence:
  • the amino acid sequence of the CTP-modified human growth hormone (hGH) antagonist Variant MOD1201-4 (CTP-mut hGHA-CTP-CTP, G120K + 8 mutations), also described as MOD-12014, 12014, or 1201-4, comprises the following amino acid sequence:
  • the MOD- 1201-4 variant has the following point mutations as shown in Table 1, when comparing the amino acid sequence of the mutated hGH antagonist to the wild type hGH antagonist.
  • Table 1 Point Mutations of hGH antagonist in MOD-1201-4 as Compared to Wild Type
  • the amino acid sequence of the CTP-modified human growth hormone (hGH) antagonist Variant MOD1201-5 (CTP-mut hGHA-CTP-CTP, G120K + 15 mutations) comprises the following amino acid sequence:
  • SSSSKAPPPSLPSPSRLPGPSDTPILPQFPTIPLSRLADNAWLRADRLNQLAFDTYQEFEEAYI PKEQIHSFWWNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFAN SLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKN YGLLYCFNKDMSKVSTYLRTVQCRSVEGSCGFSSSSKAPPPSLPSPSRLPGPSDTPILPQSSS SKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 9).
  • the CTP-modified hGH antagonist is a recombinant protein. In another embodiment, the CTP-modified hGH antagonist is a recombinant glycoprotein. In another embodiment, the CTP-modified hGH antagonist comprises a signal peptide. In another embodiment, a recombinant CTP-modified hGH antagonist does not comprise a signal peptide. In one embodiment, the CTP-modified hGH antagonist includes a signal peptide. In another embodiment, the CTP- modified hGH antagonist does not include a signal peptide.
  • the signal peptide of the CTP-modified hGH antagonist comprises the following amino acid sequence:
  • amino acid sequence of the precursor polypeptide of Variant is amino acid sequence of the amino acid sequence of the precursor polypeptide of Variant
  • MOD1201-4 comprises the following amino acid sequence: MATGSRTSLLLAFGLLCLPWLQEGSASSSSKAPPPSLPSPSRLPGPSDTPILPQFPTIPLSRLF DNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKS NLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGS PRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTFLRTVQCRSVEGSCGFS SSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 11).
  • the signal peptides are cleaved from the precursor engineered CTP-modified hGFl antagonist resulting in the mature engineered CTP-modified hGFl antagonist lacking a signal peptide.
  • amino acid sequence of the CTP-modified hGFl lacking a signal peptide comprises any one of SEQ ID NO: 4 through 9.
  • the amino acid sequence of the hGFl antagonist (hGFIA) variant containing at least 6 amino acid substitutions at any residues corresponding to residues 1-119 and 121-191 of human growth hormone, numbered from the N-terminus, as compared to the wild type hGFl, comprises the following amino acid sequence:
  • the amino acid sequence of the hGFl antagonist (hGFIA) variant containing at least 8 amino acid substitutions at any residues corresponding to residues 1-119 and 121-191 of human growth hormone, numbered from the N-terminus, as compared to the wild type hGFl, comprises the following amino acid sequence:
  • the amino acid sequence of the hGH antagonist (hGHA) variant containing at least 15 amino acid substitutions at any residues corresponding to residues 1-119 and 121-191 of human growth hormone, numbered from the N-terminus, as compared to the wild type hGH, comprises the following amino acid sequence:
  • the CTP sequence of the present invention comprises at least one glycosylation site. In one embodiment, the CTP sequence of the present invention comprises 2 glycosylation sites. In one embodiment, the CTP sequence of the present invention comprises 3 glycosylation sites.
  • the CTP sequence of the present invention comprises 4 glycosylation sites.
  • one or more of the chorionic gonadotropin CTP amino acid sequences is fully glycosylated.
  • one or more of the chorionic gonadotropin CTP amino acid sequences is partially glycosylated.
  • partially glycosylated indicates that one of the CTP glycosylation sites is glycosylated.
  • two of the CTP glycosylation sites are glycosylated.
  • three of the CTP glycosylation sites are glycosylated.
  • the present invention provides a polynucleotide encoding a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • cgCTPs chorionic gonadotrophin carboxy terminal peptides
  • an expression vector comprising a polynucleotide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • cgCTPs chorionic gonadotrophin carboxy terminal peptides
  • the GH receptor antagonist variants of the present invention are synthesized using a polynucleotide molecule encoding a polypeptide of the present invention.
  • the polynucleotide molecule encoding the GH receptor antagonist of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence).
  • a cis-regulatory sequence e.g., promoter sequence
  • the cis-regulatory sequence is suitable for directing constitutive expression of an GH receptor antagonist of the present invention.
  • the cis-regulatory sequence is suitable for directing tissue-specific expression of the GH receptor antagonist peptides of the present invention.
  • the cis-regulatory sequence is suitable for directing inducible expression of the GH receptor antagonist variants of the present invention.
  • Variant MOD1201-4 comprises the following nucleic acid sequence: GCGGCCGCCATGGCAACTGGATCACGTACAAGCCTGTTGCTCGCTTTCGGATTGCTCT
  • tissue-specific promoters suitable for use with the present invention include sequences which are functional in one or more specific cell populations. Examples include, but are not limited to, promoters such as albumin that is liver-specific [Pinkert et al., (1987) Genes Dev. 1 :268-277], lymphoid-specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas- specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • Inducible promoters suitable for use with the present invention include, for example, the tetracycline-inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398- 405).
  • the phrase“a polynucleotide molecule” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • a composition comprising the polypeptide, polynucleotide, expression vector, or a combination thereof.
  • a method for extending the biological half-life of an hGHA variant by attaching 9-fluorenylmethoxycarbonyl (Fmoc), a maleimide moiety of Fmoc (MAL-Fmoc), 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), a maleimide moiety of FMS (MAL- FMS), 2-methoxy-9-fluorenylmethoxycarbonyl (MeOFmoc), or NRFmoc at one or more amino acid positions of the hGHA variant.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • MAL-Fmoc 2-sulfo-9-fluorenylmethoxycarbonyl
  • MAL- FMS 2-methoxy-9-fluorenylmethoxycarbonyl
  • MeOFmoc 2-methoxy-9-fluorenylmethoxycarbonyl
  • a method for extending the biological half-life of an hGHA variant by attaching 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), MAL-FMS, 2-methoxy-9- fluorenylmethoxycarbonyl (MeOFmoc), or NRFmoc to the amino terminus of the hGHA variant.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • FMS MAL-Fmoc 2-sulfo-9-fluorenylmethoxycarbonyl
  • MeOFmoc 2-methoxy-9- fluorenylmethoxycarbonyl
  • a method for reducing the dosing frequency of an hGHA variant by attaching 9-fluorenylmethoxycarbonyl (Fmoc), a maleimide moiety of Fmoc (MAL-Fmoc), 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), a maleimide moiety of FMS (MAL- FMS), MeOFmoc, or NRFmoc at one or more amino acid positions of the hGHA variant.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • MAL-Fmoc 2-sulfo-9-fluorenylmethoxycarbonyl
  • MAL- FMS 2-sulfo-9-fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • NRFmoc NRFmoc
  • a method for reducing the dosing frequency of an hGHA variant by attaching 9-fluorenylmethoxycarbonyl (Fmoc), MAL-FMoc, 2-sulfo-9- fluorenylmethoxycarbonyl (FMS), MAL-FMS, MeOFmoc, or NRFmoc to the amino terminus, of the hGHA variant.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • MAL-FMoc 2-sulfo-9- fluorenylmethoxycarbonyl
  • FMS 2-sulfo-9- fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • NRFmoc NRFmoc
  • a method for improving the biological efficacy of hGHA variant by attaching 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9- fluorenylmethoxycarbonyl (FMS), MAL-FMS, MeOFmoc, or NRFmoc at one or more amino acid positions of the hGHA variant.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • FMS 2-sulfo-9- fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • NRFmoc NRFmoc
  • provided herein is a method for extending the biological half-life of an hGHA variant by attaching 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), MAL-FMS, MeOFmoc, or NRFmoc to the amino terminus of the hGHA variant.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • MAL-Fmoc 2-sulfo-9-fluorenylmethoxycarbonyl
  • FMS 2-sulfo-9-fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • NRFmoc NRFmoc
  • a method for improving the biological efficacy and/or extending the biological half-life of an hGHA variant by attaching 9- fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), MAL-FMS, MeOFmoc, or NRFmoc to the amino terminus of the hGHA variant.
  • Fmoc 9- fluorenylmethoxycarbonyl
  • MAL-Fmoc 2-sulfo-9-fluorenylmethoxycarbonyl
  • FMS 2-sulfo-9-fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • NRFmoc NRFmoc
  • Fmoc-Osu linker attaching an Fmoc-Osu linker to the hGHA variant via any of the free amines potentially located at the N-terminus.
  • Fmoc-Osu structure is described below in Formula I.
  • the Fmoc-Osu linker is sulfonated.
  • Fmoc-Osu is a mono- functional linker.
  • the Fmoc-Osu linker is covalently bound to a hGHA variant via a carbamate bond.
  • other potential interactions e.g, hydrophobic interactions between the linker moieties and other bio-molecules are non-co valent-based.
  • MAL-Fmoc-OR of this invention is presented by the following structure.
  • MAL-FMS-OR of this invention is presented by the following structure.
  • MeOFmoc of this invention is presented by the following structure.
  • NRFmoc of this invention is presented by the following structure.
  • the maleimide moiety MAL-FMS-NF1S of this invention is presented by the following structure.
  • the MAL-FMS-NHS is prepared by mixing MAL-Fmoc-NHS with trifluoroacetic acid and chlorosulfonic acid, wherein said MAL-Fmoc-NHS is dissolved in neat trifluoroacetic acid, and an excess of said chlorosulfonic acid dissolved in neat trifluoroacetic acid is added to the reaction mixture.
  • the maleimide moiety MAL-Fmoc-NHS of this invention is presented by the following structure.
  • the invention provides a composition comprising or consisting of hGHA variants linked to one or more polyethylene glycol polymers (PEG) via a reversible linker, such as 9-fluorenylmethoxycarbonyl (Fmoc), a maleimide moiety of Fmoc (MAL-Fmoc), 2-sulfo-9- fluorenylmethoxycarbonyl (FMS), a maleimide moiety of FMS (MAL-FMS), or MeOFmoc.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • MAL-Fmoc maleimide moiety of Fmoc
  • FMS 2-sulfo-9- fluorenylmethoxycarbonyl
  • MAL-FMS 2-sulfo-9- fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • the invention provides a composition comprising or consisting of an hGHA variant, a polyethylene glycol polymer (PEG polymer) and 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), MAL-FMS, or MeOFmoc.
  • PEG polymer polyethylene glycol polymer
  • Fmoc 9-fluorenylmethoxycarbonyl
  • FMS 2-sulfo-9-fluorenylmethoxycarbonyl
  • MeOFmoc MeOFmoc
  • the invention provides a composition comprising or consisting of hGHA variants linked to one or more polyethylene glycol polymers (PEG) via an irreversible linker, such as 2,5-dioxopyrrolidin-l-yl-3-(2-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propanamido)-9H-fluoren-9-yl)propanoate (NRFmoc).
  • PEG polyethylene glycol polymers
  • a method for extending the serum half-life of peptides is based on the reversible attachment of a polyethylene glycol (PEG) chain to the peptide through a chemical linker (called FMS, MAL-FMS, Fmoc, MAL-Fmoc, or MeOFmoc) resulting in the slow release of the native peptide into the bloodstream.
  • FMS, MAL-FMS, Fmoc, MAL-Fmoc, or MeOFmoc linker leads to a specific rate of peptide release.
  • the hGHA variant upon administration of the PEGylated hGHA variant composition of the present invention into a subject, the hGHA variant is released into a biological fluid in the subject as a result of chemical hydrolysis of said FMS, MAL-FMS, Fmoc, or MAL-Fmoc linker from said composition.
  • chemically hydrolyzing said FMS, MAL-FMS, Fmoc, or MAL-Fmoc extends the circulating time of said hGHA variant in said biological fluid.
  • the present invention provides a composition comprising an hGHA variant peptide, and a polyethylene glycol (PEG) polymer conjugated to the amino terminus of the hGHA variant peptide via a 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9- fluorenylmethoxycarbonyl (FMS), MAL-FMS, or MeOFmoc linker.
  • PEG polyethylene glycol
  • the invention relates to a composition consisting of an hGHA variant, a polyethylene glycol polymer (PEG polymer), and a 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9- fluorenylmethoxycarbonyl (FMS), MAL-FMS, or MeOFmoc linker, wherein said PEG polymer is attached to a position on said hGHA variant’s amino acid sequence via Fmoc, MAL-Fmoc, FMS, MAL-FMS, or MeOFmoc.
  • PEG polymer polyethylene glycol polymer
  • Fmoc 9-fluorenylmethoxycarbonyl
  • FMS 2-sulfo-9- fluorenylmethoxycarbonyl
  • MeOFmoc linker wherein said PEG polymer is attached to a position on said hGHA variant’s amino acid sequence via Fmoc, MAL-F
  • the present invention provides a heterologous composition
  • a heterologous composition comprising an hGHA variant peptide attached to a polyethylene glycol (PEG) polymer via a Fmoc, MAL-Fmoc, FMS, MAL-FMS, or MeOFmoc linker at the amino terminus of the hGHA variant peptide.
  • PEG polyethylene glycol
  • a long-acting hGHA variant is a pegylated hGHA variant. In another embodiment, a long-acting hGHA variant is a reversed pegylated hGHA variant.
  • a long- acting hGHA variant is hGHA variant linked to PEG via Fmoc, MAL- Fmoc, FMS, MAL-FMS, or MeOFmoc.
  • the long-acting hGHA variant is linked to Fmoc, MAL-Fmoc, FMS, MAL-FMS, or MeOFmoc via its amino (N’) terminus.
  • the invention provides a composition comprising or consisting of an hGHA variant reversibly PEGylated via a MAL-Fmoc or MAL-FMS linker.
  • the hGHA variant reversibly PEGylated via a MAL-Fmoc or MAL-FMS linker can be further conjugated to another molecule in addition to the PEG.
  • the additional conjugated molecule is a thiol-containing molecule.
  • the additional conjugated molecule is a SH active group or an amine, hydrazine, or hydrazide.
  • the additional conjugated molecule is Cys or cysteamine.
  • the hGHA variants provided herein have 9- fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), MAL-FMS, or MeOFmoc attached to one or more amino acid positions of the hGHA variant.
  • the hGHA variants provided herein have 9-fluorenylmethoxycarbonyl (Fmoc), MAL-Fmoc, 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), MAL-FMS, or MeOFmoc attached to the amino terminus of the hGHA variant.
  • a reverse pegylated hGHA variant is a composition wherein hGHA variant is linked to PEG via a reversible linker.
  • a reverse pegylated hGHA variant releases a free hGHA variant upon exposure to a natural to basic environment.
  • a reverse pegylated hGHA variant releases a free hGHA variant upon exposure to blood or plasma.
  • a long-acting hGHA variant comprises PEG and hGHA variant that are not linked directly to each other, as in standard pegylation procedures, but rather both residues are linked to different positions of Fmoc, MAL-Fmoc, FMS, or MAL-FMS which are highly sensitive to pH conditions and are removable under regular physiological conditions.
  • regular physiological conditions include a physiologic environment such as the blood or plasma.
  • the maleimide moiety linkers of the present invention are hydrogenated.
  • the maleimide moiety linkers have one or more maleimide groups replaced with a succinimide group.
  • the linkers containing a succinimide group have the following structure:
  • a method of reducing the dosing frequency of an hGHA variant, due to the improved efficacy of a long acting hGHA variant as described herein is a method of reducing the dosing frequency and/or increasing the efficacy of the hGHA or hGHA variant, consisting of the step of conjugating at least one linker said Fmoc, MAL-Fmoc, FMS, MAL-FMS, MeOFmoc, or NRFmoc or combination thereof to the hGHA or hGHA variant at the N terminal, and further reducing the maleimide functional group using, but not limit to thiol- containing molecules (e.g, cysteine and cysteamine), amine-containing molecules, and hydrogenation.
  • thiol- containing molecules e.g, cysteine and cysteamine
  • reacting the thiol-containing molecule with the hGHA variant results in the reduction of the MAL-linker-hGHA such as maleimide hydrogenation, and / or the coupling of the thiol-containing molecule to the linker- hGHA.
  • PEG is linear. In another embodiment, PEG is branched. In another embodiment, PEG has a molecular weight in the range of 1 to 200 Da. In another embodiment, PEG has a molecular weight in the range of 200 to 200,000 Da. In another embodiment, PEG has a molecular weight in the range of 5,000 to 80,000 Da. In another embodiment, PEG has a molecular weight in the range of 5,000 to 40,000 Da. In another embodiment, PEG has a molecular weight in the range of 20,000 Da to 40,000 Da. In one embodiment, PEG20 refers to a PEG with an average molecular weight of 20,000 Da.
  • PEG5 refers to a PEG with an average molecular weight of 5,000 Da.
  • PEG30 refers to a PEG with an average molecular weight of 30,000 Da.
  • PEG40 refers to a PEG with an average molecular weight of 40,000 Da.
  • PEG has a molecular weight of about 2,000 Da. In another embodiment, PEG has a molecular weight of about 1 ,000 Da. In another embodiment, PEG has a molecular weight of about 5000 Da. In another embodiment, PEG has a molecular weight of about 100 Da. In another embodiment, PEG has a molecular weight in the range of 1 to 500 Da. In another embodiment, PEG has a molecular weight in the range of 500 to 1,000 Da. In another embodiment, PEG has a molecular weight in the range of 1 ,000 to 2,000 Da. In another embodiment, PEG has a molecular weight in the range of 2,000 to 5,000 Da.
  • the polyethylene glycol is a branched PEG represented as
  • (PEG)m-R-SH in which R represents a central core moiety and m represents the number of branching arms.
  • the PEG is represented as (PEG)m-R-SH with only one available connection to the polypeptide.
  • the number of branching arms (m) can range from two to a hundred or more.
  • the hydroxyl groups are subject to chemical modification.
  • the branched PEG has an average molecular weight of 20 KD or 40 KD and is represented as (PEG)2-R-SH.
  • the branched PEG is represented as (PEQ2-R-SH and has the following chemical structure:
  • the PEG is a multi-arm PEG represented as (PEG)4-R-SH.
  • the PEG is a multi-arm PEG represented as (PEG)4-R-SH in which each PEG arm has a molecular weight of 20KD or 40 KD.
  • the PEG is a multi-arm PEG represented by the following chemical structure:
  • the PEG is a multi-arm PEG represented by formula 1 above and each PEG arm has an average molecular weight of 20 KD or 40 KD.
  • branched PEGs are represented as R(PEG-OH) m in which R represents a central core moiety such as pentaerythritol or glycerol, and m represents the number of branching arms.
  • the number of branching arms (m) can range from two to a hundred or more.
  • the hydroxyl groups are subject to chemical modification.
  • branched PEG molecules are described in U.S. Pat. No. 6,113,906, No. 5,919,455, No. 5,643,575, and No. 5,681,567, which are hereby incorporated by reference in their entirety.
  • the PEGylating agent is usually used in its mono-methoxylated form where only one hydroxyl group at one terminus of the PEG molecule is available for conjugation.
  • a bifunctional form of PEG where both termini are available for conjugation may be used if, for example, it is desired to obtain a conjugate with two peptide or protein residues covalently attached to a single PEG moiety.
  • the long acting hGHA variant of the present invention is presented by the following structure:
  • hGHA is any hGH antagonist or hGH antagonist variant described through this application.
  • this structure is referred to as MAL-FMS-hGHA.
  • the long acting hGF!A variant of the present invention is presented by the following structure:
  • hGF!A is any hGFl antagonist or hGFl antagonist variant described through this application.
  • this structure is referred to as PEG30-Fmoc- hGF!A.
  • the long acting hGF!A variant of the present invention is presented by the following structure:
  • hGF!A is any hGFl antagonist or hGFl antagonist variant described through this application.
  • this structure is referred to as PEG30-NRF- hGFIA.
  • the long acting hGFIA variant of the present invention is presented by the following structure:
  • hGHA is any hGH antagonist or hGH antagonist variant described through this application.
  • this structure is referred to as PEG30-MeOF- hGFIA.
  • the long acting hGFIA variant of the present invention is presented by the following structure:
  • hGFIA is any hGFl antagonist or hGFl antagonist variant described through this application.
  • this structure is referred to as PEG30-FMS- hGFIA.
  • a "pharmaceutical composition” or a “pharmaceutical formulation” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition or a“pharmaceutical formulation” is to facilitate administration of a compound to an organism.
  • a “pharmaceutical composition” or a “pharmaceutical formulation” provides the pharmaceutical dosage form of a drug.
  • “Pharmaceutical compositions” or“pharmaceutical formulations” in certain embodiments include slow release technologies, transdermal patches, or any known dosage form in the art.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • compositions, formulations and methods of the present invention comprising the elements or steps as described herein may, in another embodiment, consist of those elements or steps, or in another embodiment, consist essentially of those elements or steps.
  • the term“comprise” refers to the inclusion of the indicated active agent, such as the CTP-modified hGHA, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry.
  • the term“consisting essentially of’ refers to a composition, whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc.
  • the term“consisting essentially of’ may refer to components which facilitate the release of the active ingredient.
  • the term“consisting” refers to a composition, which contains the active ingredient and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical compositions and pharmaceutical formulations are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation.
  • liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical compositions and pharmaceutical formulations are administered intravenously, and are thus formulated in a form suitable for intravenous administration.
  • the pharmaceutical compositions and pharmaceutical formulations are administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration.
  • the pharmaceutical compositions and pharmaceutical formulations are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.
  • the pharmaceutical compositions and pharmaceutical formulations are administered topically to body surfaces, and are thus formulated in a form suitable for topical administration.
  • suitable topical formulations include gels, ointments, creams, lotions, drops and the like.
  • the compounds of the present invention are combined with an additional appropriate therapeutic agent or agents, prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.
  • the long-acting somatostatin analogues (LA-SSA) described herein are constrained peptides of 6 to 10 alpha-amino acids, of which particular examples include octreotide, lanreotide (of sequence NH2-(D)Naph Cys-Tyr-(D)Trp-Lys-Val-Cy3 ⁇ 4-Thr- €ONli2 and its cyclic derivative of sequence NH2-(D)Naph-Cys-Tyr-(D)Phe-Lys-Vid-Cys-Thr-CONH ? both having a Cys-Cys intramolecular disulphide crosslink) and vapreotide.
  • octreotide is a synthetic octa-peptide with sequence D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-o! (2-7 disulphide bridge).
  • octreotide is administered as an acetate salt.
  • somatostatin analogues include pharmaceutically acceptable salts and derivatives thereof.
  • octreotide will be the most common salt, octreotide acetate, but the free molecule, or any other biologically acceptable salt, such as hydrochloride, pamoate, citrate etc. is also encompassed by the term, unless context prohibits.
  • the pharmaceutical composition disclosed herein comprises an hGHA variant and a somatostatin or long-acting somatostatin analogue (LA-SSA). In another embodiment, the pharmaceutical composition disclosed herein comprises an hGHA variant and dopamine. In another embodiment, the pharmaceutical composition disclosed herein comprises an hGHA variant, a somatostatin or LA-SSA, and dopamine.
  • LA-SSA long-acting somatostatin analogue
  • the pharmaceutical composition disclosed herein comprises a CTP-modified hGHA variant and a somatostatin or long-acting somatostatin analogue (LA-SSA).
  • the pharmaceutical composition disclosed herein comprises a CTP-modified hGHA variant and dopamine.
  • the pharmaceutical composition disclosed herein comprises a CTP-modified hGHA variant, a somatostatin or LA-SSA, and dopamine.
  • the pharmaceutical composition disclosed herein comprises a
  • the pharmaceutical composition disclosed herein comprises a PEGylated or reversibly PEGylated hGHA variant and a somatostatin or long-acting somatostatin analogue (LA-SSA).
  • the pharmaceutical composition disclosed herein comprises a PEGylated or reversibly PEGylated hGHA variant and dopamine.
  • the pharmaceutical composition disclosed herein comprises a PEGylated or reversibly PEGylated hGHA variant, a somatostatin or LA-SSA, and dopamine.
  • compositions and pharmaceutical formulations of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions and pharmaceutical formulations for use in accordance with the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically.
  • formulation is dependent upon the route of administration chosen. Manufacturing
  • the invention provided herein relates to a method of manufacturing a human chorionic gonadotropin peptide (CTP)-modified human growth hormone antagonist (ahGH) polypeptide, the method comprising the steps of: a. stably transfecting a predetermined number of cells with an expression vector comprising a coding portion encoding said CTP-modified human growth hormone antagonist, wherein said transfected cell expresses said CTP-modified ahGH; b. obtaining cell clones that overexpress said CTP-modified ahGH; c. expanding said clones in solution to a predetermined scale, as shown in Table 2 below; d. harvesting said solution containing said clones; e.
  • CTP human chorionic gonadotropin peptide
  • ahGH human growth hormone antagonist
  • the clarified harvest is held up to 72 hours at 5X1.
  • the clones are propagated in medium to form a research cell hank (RCB).
  • RCB research cell hank
  • the cell clones are expanded in solution through a series of sub-cultivation steps up to production bioreactor level.
  • the solution containing said subcultivated clones is seeded in a bioreactor ( Figure 10).
  • the cell clones are expanded further or up-scaled by serially culturing said cell in increasing sizes of the bioreactor until a desired scale is reached.
  • the clarified harvest obtained is tested for bioburden, bacterial endotoxin, specific protein content, residual DNA, viruses, virus-like particles, and Mycoplasma.
  • At least 50% of the purified human chorionic gonadotropin carboxy terminal peptide (CTP)-modified human growth hormone antagonist (ahGH) polypeptide from said clarified harvest comprises a high glycosylation form of CTP-modified ahHG.
  • purification comprises sequentially performing steps comprising passing said clarified harvest through anion exchange column, an hydrophobic interaction column, and an anion exchange column; inactivating viruses present in the clarified harvest or in the eluate collect following column chromatography, or any combination thereof, wherein inactivating viruses comprises incubation in a low pH buffer toxic to said viruses or nanofilitration, or any combination thereof; and wherein the anion exchange eluate undergoes an ultrafiltration/diafiltration step ( Figure 10).
  • the purification of the clarified harvest is accomplished by sequentially performing the steps comprising:
  • concentration and diafiltration is accomplished by sequentially passing said clarified harvest solution through a membrane; ii. obtaining said clarified harvest obtained following step and inactivating viruses present in said clarified harvest by incubating in a low pH toxic to the viruses and passing said clarified harvest solution through an anion exchange column;
  • concentration and diafiltration is accomplished by sequentially passing said clarified harvest solution through a hydrophobic interaction column and an anion exchange column; iv. obtaining said clarified harvest solution following step and inactivating said clarified harvest solution from viruses by nanofiltration;
  • the first anion exchange column purifies the highly glycosylated form of said CTP-modified ahGH.
  • viruses present in the clarified harvest due to contamination are inactivated in the clarified harvest.
  • the viruses are inactivated using a low pH buffer on the range of 2-4 (See Table 2).
  • the hydrophobic column purifies the highly glycosylated form of said CTP-modified ahGH by removing the HCP/DNA impurities, dimer and LMWF with a refinement of the monomer and main peak (See Table 2).
  • the third chromatography step of anion exchange purifies the highly glycosylated form of said CTP-modified ahGH by removing the HCP impurities ( Figure 10).
  • viruses are inactivated by using nanofiltration. It will be appreciated by the skilled artisan that any filter known in the art for inactivating viruses (removing viruses) may be applied in methods of the present invention. Such methods are followed by confirmation of viral clearance from the clarified harvest using methods known in the art.
  • the manufacturing method achieves at least a 30% recovery rate of highly glycosylated CTP-modified ahGH.
  • a composition comprises a manufactured CTP-modified ahGH, and a pharmaceutically acceptable carrier.
  • a human chorionic gonadotropin carboxy terminal peptide (CTP)-modified human growth hormone antagonist (ahGH) polypeptide comprising two CTP molecules attached in tandem to the C-terminal end of ahGH and one CTP molecule attached to the N-terminal end of ahGH, wherein said CTP-modified ahGH polypeptide is in a substantially pure and active form, said CTP-modified ahGH polypeptide comprising: (a) a high sialic acid content; (b) a low oxidized form; (c) a high glycosylation form.
  • CTP carboxy terminal peptide
  • ahGH human growth hormone antagonist
  • the high sialic acid content consists of at least 13 mol/m l.
  • the substantially pure and active form comprises at least 60% of a high glycosylation form of said active CTP-modified ahGH.
  • the low percentage of oxidized form consists of less than 2%.
  • the purity of said substantially pure and active CTP-modified ahGH polypeptide is at least 90%. In a further related aspect, the purity percentage is not less than 94% (See Table 3).
  • Table 3 Critical Quality Attributes of MOD-12014 in lOmM Histidine, 10 mM Citrate, 150 mM NaCl pH 6.6
  • composition comprising the CTP-modified ahGH comprising: (a) a high sialic acid content; (b) a low oxidized form; (c) a high glycosylation form; or any combination thereof, wherein said CTP-modified ahGH comprises the amino acid sequence set forth in SEQ ID NO: and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating acromegaly in a subject comprising the step of administering to said subject a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • administering is via a subcutaneous route.
  • administering is via the intravenous route.
  • the present invention provides a method of treating acromegaly in a subject comprising the step of administering to said subject a polypeptide comprising a PEGylated or reversibly PEGylated hGHA variant as described herein.
  • a method of treating acromegaly in a subject comprising the step of administering a hGH antagonist variant in combination with a somatostatin or LA-SSA.
  • a method of treating acromegaly in a subject comprising the step of administering a hGH antagonist variant in combination with dopamine.
  • a method of treating acromegaly in a subject comprising the step of administering a hGH antagonist variant in combination with a somatostatin or LA-SSA and dopamine.
  • disclosed herein is a method of reducing IGF-l levels in a subject by at least 20% by administering the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein. In another embodiment, disclosed herein is a method of reducing IGF-l levels in a subject by at least 25% by administering the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein.
  • disclosed herein is a method of reducing IGF-l levels in a subject by at least 30% by administering the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein. In another embodiment, disclosed herein is a method of reducing IGF-l levels in a subject by at least 35% by administering the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein.
  • a method of normalizing IGF-l levels in a subject by administering the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein.
  • a method of normalizing IGF-l levels in a subject by co-administering (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA.
  • a method of normalizing IGF-l levels in a subject by co administering (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) dopamine.
  • a method of normalizing IGF-l levels in a subject by co-administering (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA and dopamine.
  • the co-administration of i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA occurs after neurosurgery.
  • the co-administration of i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA and dopamine occurs after neurosurgery.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA occurs after neurosurgery.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) dopamine occurs after neurosurgery.
  • the co administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA and dopamine occurs after neurosurgery.
  • the administration of the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described results in a subject’s IGF-l levels being within normal SDS.
  • the administration of the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described results in a subject’s IGF-l levels being within normal SDS below 1.3.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA results in a subject’s IGF-l levels being within normal SDS.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) dopamine results in a subject’s IGF-l levels being within normal SDS.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA and dopamine results in a subject’s IGF-l levels being within normal SDS.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA results in a subject’s IGF-l levels being within normal SDS below 1.3.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) dopamine results in a subject’s IGF-l levels being within normal SDS below 1.3.
  • the co-administration of (i) the hGHA variant, CTP-modified hGHA variant, or PEGylated or reversibly PEGylated hGHA variant described herein with (ii) somatostatin or LA-SSA and dopamine results in a subject’s IGF-l levels being within normal SDS below 1.3.
  • acromegaly is a disorder which results from excess growth hormone (GH) after the growth plates have closed.
  • the initial symptom is typically enlargement of the hands and feet. There may also be enlargement of the forehead, jaw, and nose.
  • Other symptoms may include water retention, joint pain, thicker skin, deepening of the voice, headaches, and problems with vision.
  • Complications of the disease may include type 2 diabetes, sleep apnea, carpal tunnel syndrome, and high blood pressure.
  • GH hypersecretion in acromegaly can also be associated with menstrual disturbances and reduced fertility.
  • the internal organs can also grow in size, which may lead, for example, to an enlarged heart and eventually to heart disease if not recognized and treated.
  • methods of the present invention are for treating the diseases, disorders, and symptoms associated with long-term excess GH, as described herein.
  • the present invention provides a method of treating the overexpression of growth hormone in a subject comprising the step of administering to said subject a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • cgCTPs chorionic gonadotrophin carboxy terminal peptides
  • the present invention provides a method of treating the overexpression of growth hormone in a subject comprising the step of administering to said subject a PEGylated or reversibly PEGylated hGHA variant as described herein.
  • the subject is a child or teenager.
  • the present invention provides a method of treating gigantism in a subject comprising the step of administering to said subject a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • cgCTPs chorionic gonadotrophin carboxy terminal peptides
  • the present invention provides a method of treating a benign pituitary adenoma in a subject comprising the step of administering to said subject a polypeptide comprising a growth hormone antagonist and at least two chorionic gonadotrophin carboxy terminal peptides (cgCTPs), wherein the first cgCTP of said at least two cgCTPs is attached to the amino terminus of said growth hormone antagonist, and the second cgCTP of said at least two cgCTPs is attached to the carboxy terminus of said growth hormone antagonist.
  • cgCTPs chorionic gonadotrophin carboxy terminal peptides
  • the present invention provides a method of treating a benign pituitary adenoma in a subject comprising the step of administering to said subject a PEGylated or reversibly PEGylated hGHA variant as described herein.
  • the subject is human.
  • the subject is a non-human primate.
  • the subject is murine, which in one embodiment is a mouse, and, in another embodiment is a rat.
  • the subject is canine, feline, bovine, equine, laprine or porcine.
  • the subject is mammalian.
  • hGHA hGH antagonist
  • cassettes number and position (C- or N- terminal) of the CTP atta chments The number of CTP cassettes may dictate the long acting property of the product, while the C or N-terminal position of the CTP can affect the protein’ s physiochemical characteristics and biological activity.
  • C- or N-terminal position of the CTP can affect the protein’ s physiochemical characteristics and biological activity.
  • two to four CTPs were added to the various hGHA variants.
  • the N terminal signal peptide (SP) is needed in order to allow CTP-modified hGHA secretion following their cleavage and translocate the protein.
  • SP N terminal signal peptide
  • Table 6 lists measurement of IC50 values in the five variants and in Somavert®.
  • variants exhibited a significantly higher receptor affinity than the commercial protein Somavert®.
  • variant 5 (containing 15 mutations) exhibited the highest affinity, likely due to the high number of mutations, which increase the binding affinity to the receptor.
  • variants 1 and 4 which differ in the number of mutations (6 vs. 8) and CTP copies, resulted unexpectedly with similar IC50.
  • two or three copies of CTP in the C- terminus (variant 3 and 2, respectively), resulted as well with similar affinity to the receptor (4 CTP copies did not reduce the affinity).
  • variant 1 which possessed the same number of mutations as variant 2 & 3, but only one CTP copy in the C-terminus, resulted with 2-fold higher receptor affinity.
  • EXAMPLE 3 COMPARATIVE HALF-LIFE
  • variant 2 and 5 showed the highest half-life and concentrations measurements in each timepoint, compared to the other three variants, even though variant 2 has 4 CTP copies and variant 5 has only 3. These results may suggest that the high affinity of variant 5, which is probably due to the 15 aa substitutions, may contribute as well to the longevity of the protein.
  • variant 1 which contains only two CTP copies, unexpectedly showed slightly higher half-life than variant 3, which shares the same number of AA substitutions as variant 1 but has 3 CTP copies.
  • EXAMPLE 4 COMPARATIVE PHARMACOLOGY EFFECT IN MICE
  • MOD-1201 For a preliminary evaluation of MOD-1201, a mouse model was used in which an IGF-1 decrease was observed after treatment with Somavert® (JW van Neck et al, 2000)e. Various injection dosages and frequencies of Somavert® and MOD-1201 variants were tested for ability to reduce IGF-1 levels. MOD-1201 variants and Somavert® were injected on days 1 and 4 at a dose of 1818 nmol/kg (See Table 8). The dose level was in moles due to different molecular weights and hGHA content in each one of the injected materials. IGF-1 levels were measured in serum samples taken before the first injection on day 1 of the experiment (pre-dose) and again on the fifth day, 24 hours after the second injection.
  • variants 2, 3 and 4 showed a significant and similar IGF-1 reduction ( ⁇ 45%) compared to the pre-dose levels, even though each one of the variants differ in the number of CTP copies and AA substitutions.
  • the 4 CTP copies in variant 2 did not cause lower potency compared to variant 3, which possesses only three CTP copies.
  • variant #5 showed the most pronounce IGF-l reduction (about 70%), likely due to the 15 AA substitutions in site 1, which is responsible for the increased affinity to the GHR.
  • Variant #1 appeared to be the least active variant with 20% IGF-l reduction, although it has slightly higher longevity and similar in-vitro affinity as variant 4.
  • Two Somavert® injections appeared to be ineffective, with only a 15% reduction in IGF-l levels, compared to MOD-1201 variants.
  • Table 8 Average % reduction in IGF-l levels of the five variants and Somavert® 24 hours after injection at 1818 nmol/kg
  • PBMCs peripheral blood mononuclear cells
  • Table 10 In-vitro study results for the predisposition of a human immune response as a result of exposure to MOD-1201 variants.
  • RIs between 0 and 0.5 are considered to be a low immune response that is unlikely to cause a human immune response (See Table 11). In general, all four variants showed low immunogenic response. However, even though variants 4 possess two additional AA substitutions compare to variant 3, both resulted with the same immunogenic response (RI ⁇ 0.2). Surprisingly, variant 2 showed higher RI than variant 3 and 4, even though it has the same AA substitutions as variant 3. In addition, RI higher than 0.5 was observed for variant 5, which suggest that it may have a slightly higher potential to cause for an immunogenicity response, which in line with the high number of AA substitutions in its hGHA sequence.
  • Table 11 RI values as an indicator of human immune response development.
  • MOD-1201 variant 4 and IGF-l levels were quantitated following single SC injection at 3 dose levels, 15, 30, and 50mg/kg (See Figures 4 and 5).
  • PK parameters were calculated for each dose group PK profile (See Table 12). Percentage decrease in IGF-l level was calculated compare to pre-dose level for each animal per timepoint and averaged for each group (See Table 13).
  • Table 12 PK parameters for single SC MOD-12014 injection at 3 dose levels
  • EXAMPLE 7 PHARMACOLOGY ASSESSMENT OF MOD- 1201 VARIANTS 2 AND 4 IN RABBITS
  • variants 2 and 4 had the highest potential to act as an effective, long-acting hGH antagonists. Therefore, a head to head comparison of those two most promising MOD- 1201 variants were evaluated in a rabbit model, which is considered as the common model for hGHAs.
  • each of the two variants was administrated at two dose levels, ⁇ 7.5 and -15 mg/kg (corresponding to 239 and 477nmol/kg), on days 1, 4, and 7, while Somavert® was injected at 3.5mg/kg at the same regimen.
  • Blood samples were drawn daily from each rabbit up to day 10 (72 hours post last injection) and the serum levels of MOD-1201 and IGF-l were quantitated. Percentage IGF-l decrease from pre-dose level was calculated for each animal at each timepoint and averaged for all animals group (See Table l4and Figure 6).
  • variant 4 showed higher efficacy response, which obtained from the higher reductions in IGF-l throughout the study (Table 14, Figure 6). This result was unexpected due to the similar efficacy that was obtained in the mice studies (Tables 8 and 9) and the higher longevity for variant 2 as was measured in rats (Table 7). Moreover, variant 4 showed a pronounced IGF-l decrease (-50%) following single injection and even greater response after the third injection (-60%). Based on the above, variant 4 was selected as the most promised MOD-1201 variant (MOD-12014) and further development work was be performed to better understand its efficacy potential in humans.
  • MOD-12014 at 25mg/kg showed a pronounce reduction in IGF-l (below 30% reduction) that lasted for 5 days as compare to the l5mg/kg and 7mg/kg doses, which lasted for 4 and 3 days, respectively.
  • F1GF1 antagonists including PEGylated versions, REV-PEG versions and the F1GF1 antagonists variants without the CTP or PEGylated modifications.
  • Chimers are produced using the cDNA sequence of the growth hormone antagonist (ahGFl) with 3 or 4 copies of CTP at the N terminal and/or C-terminal of the protein and expressed in DG44 CHO (Chinese Hamster Ovary) cells which are DHFR negative.
  • DG44 CHO Choinese Hamster Ovary
  • Each component in the vector is important for optimal expression of the target protein and minimizing undesirable effects in the growth of CHO DG44 cells.
  • the rationale is based on the amplification of DITFR (dihydrofolate reductase) with different concentrations of methotrexate (MTX).
  • DITFR dihydrofolate reductase
  • MTX methotrexate
  • Each component was chosen carefully, and even the distance between the genes was optimized to produce cells that secrete the protein at high concentration.
  • the process is characterized by repeatability and the ability to move to larger production and process cycles in the Fed batch. Optimal process development will allow for a more effective procedure and produce a higher stability protein for administration to humans.
  • All variants are expressed as described above and form a pool of cells that express the protein.
  • the growth hormone antagonist is an hGH analog that has been structurally modified to act as a dedicated GH receptor antagonist.
  • ahGH is an hGH analog that has been structurally modified to act as a dedicated GH receptor antagonist.
  • one mutation is required in the Glycine amino acid residue at position 120 (Glyl20), where Glycine is substituted with another amino acid (except alanine).
  • This ahGH binds to the GH receptor and prevents the signal from being passed from the GH receptor to the cell ( Figure 9).
  • the complex moves into the cell.
  • the antagonist binds to the same receptor, a dimer forms and passes into the cell without being able to transmit a specific signal. Therefore, the antagonist does not inhibit the dimerization but inhibits the function of the receptor itself.
  • each CTP there are between 4-6 populated O-glycosylation sites.
  • CHO cells are able to express only one core (GalGalNac), whose terminal antenna consists of Sialic acid-laden glycan.
  • Sialic acid affects the loading of the molecule as well as the pi. Therefore, it is important to produce a protein with CTP with high sialic acid that contributes to the population of O-glycine chains, and to stabilize the protein in order to obtain a highly charged protein with maximum homogeneity.

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Abstract

Des compositions qui comprennent des variants d'antagoniste de l'hormone de croissance humaine (hGH), des polypeptides comprenant au moins un peptide carboxy-terminal (CTP) de gonadotrophine chorionique fixé au terminus carboxy d'un antagoniste de l'hormone de croissance humaine (hGH) et au moins un CTP fixé au terminus amino d'un antagoniste de hGH, des variants de hGHA liés à un ou plusieurs polymères de polyéthylène glycol (PEG) par l'intermédiaire de lieurs réversibles ou non réversibles. L'invention concerne des polynucléotides codant pour ceux-ci. L'invention concerne en outre des compositions pharmaceutiques et des formulations pharmaceutiques comprenant les polypeptides et les polynucléotides selon l'invention, ainsi que leurs procédés d'utilisation et de production.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020123450A1 (fr) 2018-12-11 2020-06-18 Brody Richard S Antagonistes d'hormone de croissance pégylés

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264166A1 (fr) 1986-04-09 1988-04-20 Genzyme Corporation Animaux transformés génétiquement sécrétant une protéine désirée dans le lait
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
WO1997011178A1 (fr) * 1995-09-21 1997-03-27 Genentech, Inc. Variants de l'hormone de croissance humaine
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5681567A (en) 1995-05-15 1997-10-28 Enzon, Inc. Method of preparing polyalkylene oxide carboxylic acids
US5712122A (en) 1989-02-21 1998-01-27 Washington University Carboxy terminal peptide-extended proteins
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
WO2003090785A1 (fr) * 2002-04-26 2003-11-06 Pharmacia & Upjohn Company Llc Techniques et compositions permettant de traiter, de prevenir ou de retarder la survenue d'un neoplasme
US7585837B2 (en) 2003-04-08 2009-09-08 Yeda Research And Development Co. Ltd. Reversible pegylated drugs
US20100081614A1 (en) * 2006-02-03 2010-04-01 Fuad Fares Long-acting growth hormone and methods of producing same
US20170007711A1 (en) * 2015-07-07 2017-01-12 Richard S. Brody Pegylated growth hormone antagonists

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264166A1 (fr) 1986-04-09 1988-04-20 Genzyme Corporation Animaux transformés génétiquement sécrétant une protéine désirée dans le lait
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US5712122A (en) 1989-02-21 1998-01-27 Washington University Carboxy terminal peptide-extended proteins
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US6113906A (en) 1993-10-27 2000-09-05 Enzon, Inc. Water-soluble non-antigenic polymer linkable to biologically active material
US5681567A (en) 1995-05-15 1997-10-28 Enzon, Inc. Method of preparing polyalkylene oxide carboxylic acids
WO1997011178A1 (fr) * 1995-09-21 1997-03-27 Genentech, Inc. Variants de l'hormone de croissance humaine
WO2003090785A1 (fr) * 2002-04-26 2003-11-06 Pharmacia & Upjohn Company Llc Techniques et compositions permettant de traiter, de prevenir ou de retarder la survenue d'un neoplasme
US7585837B2 (en) 2003-04-08 2009-09-08 Yeda Research And Development Co. Ltd. Reversible pegylated drugs
US20100081614A1 (en) * 2006-02-03 2010-04-01 Fuad Fares Long-acting growth hormone and methods of producing same
US20170007711A1 (en) * 2015-07-07 2017-01-12 Richard S. Brody Pegylated growth hormone antagonists

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BANERJI ET AL., CELL, 1983, pages 33729 - 740
BYRNE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 5473 - 5477
CALAME ET AL., ADV. IMMUNOL., vol. 43, 1988, pages 235 - 275
EDLUNCH ET AL., SCIENCE, vol. 230, 1985, pages 912 - 916
LOWMAN, HENRY B.JAMES A. WELLS: "Affinity maturation of human growth hormone by monovalent phage display", JOURNAL OF MOLECULAR BIOLOGY, vol. 234.3, 1993, pages 564 - 578
LOWMANWELLS, JOURNAL OF MOLECULAR BIOLOGY, vol. 234.3, 1993, pages 564 - 578
PINKERT ET AL., GENES DEV., vol. 1, 1987, pages 268 - 277
SROUR, M.A. ET AL., THROMB. HAEMOST., vol. 90, 2003, pages 398 - 405
WINOTO ET AL., EMBO J., vol. 8, 1989, pages 729 - 733

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020123450A1 (fr) 2018-12-11 2020-06-18 Brody Richard S Antagonistes d'hormone de croissance pégylés

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