Classifications

• • 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
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• a sequence listing in computer readable form (CRF) is on file.
• the sequence listing is in an ASCII text (.txt) file entitled SEQIDNOS_l_24_ST25.txt created on December 10, 2018 and is 33 KB in size.
• the sequence listing is incorporated by reference as if fully recited herein.
• the described invention relates in general to compositions for use as receptor antagonists, and more specifically to novel human growth hormone antagonists that have the potential to be highly effective therapeutics.
• Human growth hormone also known as somatotropin or somatropin, is a peptide hormone that stimulates growth, cell reproduction, and regeneration in humans and other animals.
• Growth hormone is a type of mitogen that is specific only to certain kinds of cells and is a 191- amino acid, single-chain polypeptide that is synthesized, stored, and secreted by somatotropic cells within the lateral wings of the anterior pituitary gland.
• Acromegaly is a syndrome that results when the anterior pituitary gland produces excess growth hormone (hGH) after epiphyseal plate closure at puberty. If hGH is produced in excess prior to epiphyseal plate closure, the result is gigantism (or giantism).
• a number of disorders may increase the pituitary's hGH output, although most commonly it involves a tumor called pituitary adenoma, derived from a distinct type of cell (somatotrophs).
• somatotrophs most commonly affects adults in middle age and can result in severe disfigurement, complicating conditions, and premature death if untreated. Because of its pathogenesis and slow progression, the disease is hard to diagnose in the early stages and is frequently missed for years until changes in external features, especially of the face, become noticeable.
• a receptor is a protein molecule usually found embedded within the plasma membrane surface of a cell that receives chemical signals from outside the cell. When such chemical signals bind to a receptor, they cause some form of cellular/tissue response such as, for example, a change in the electrical activity of the cell. In this sense, a receptor is a protein molecule that recognizes and responds to endogenous chemical signals.
• An agonist such as human growth hormone, is a chemical composition that binds to a receptor and activates the receptor to produce a biological response. Whereas an agonist causes an action, an antagonist blocks the action of the agonist and an inverse agonist causes an action opposite to that of the agonist.
• a receptor antagonist is a type of receptor ligand or drug that blocks or dampens agonist-mediated responses rather than provoking a biological response itself upon binding to a receptor.
• These compositions are sometimes called blockers and examples include alpha blockers, beta blockers, and calcium channel blockers.
• antagonists In pharmacology, antagonists have affinity but no efficacy for their cognate receptors, and binding will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors.
• Antagonists mediate their effects by binding to the active (orthosteric) site or to other (allosteric) sites on receptors, or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity.
• Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex, which, in turn, depends on the nature of antagonist-receptor binding.
• the majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally defined binding sites on receptors.
• antagonists display no efficacy to activate the receptors they bind and antagonists do not maintain the ability to activate a receptor. Once bound, however, antagonists inhibit the function of agonists, inverse agonists, and partial agonists.
• Growth hormone receptor antagonists such as the product pegvisomant (sold under the trademark SOMA VERT ® ) are used in the treatment of acromegaly. Such compositions are used if the tumor of the pituitary gland causing the acromegaly cannot be controlled with surgery or radiation and the use of somatostatin analogues is unsuccessful.
• Pegvisomant is typically delivered as a powder that is mixed with water and injected under the skin.
• PEGylation is the process of both covalent and non-covalent amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures, such as drugs, peptides, antibody fragments, or therapeutic proteins. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule and produces alterations in physiochemical properties, including changes in molecular size and molecular charge. These physical and chemical changes increase systemic retention of the therapeutic agent and can influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.
• PEG polyethylene glycol
• the covalent attachment of PEG to a drug or therapeutic protein can also "mask" the agent from the host's immune system (i.e., reducing immunogenicity and antigenicity), and increase the hydrodynamic size (i.e., size in solution) of the agent which prolongs its circulatory time by reducing renal clearance.
• PEGylation can also provide water solubility to hydrophobic drugs and proteins.
• PEGylation by increasing the molecular weight of a molecule, can impart several significant pharmacological advantages over the unmodified form of the molecule, such as: (i) improved drug solubility; (ii) reduced dosage frequency, without diminished efficacy and with potentially reduced toxicity; (iii) extended circulating life; (iv) increased drug stability; and (v) enhanced protection from proteolytic degradation.
• PEGylated drugs also include the following commercial advantages: (i) opportunities for new delivery formats and dosing regimens; and (ii) extended patent life of previously approved drugs.
• PEG is a particularly attractive polymer for conjugation and the specific characteristics of PEG moieties relevant to pharmaceutical applications include: (i) water solubility; (ii) high mobility in solution; (iii) lack of toxicity and low immunogenicity; and (v) altered distribution in the body.
• PEGs polyethylene glycols
• the addition of PEGs also lowers the immunogenicity of the proteins and decreases aggregation and protease cleavage (Pasut and Vronese, 2012; and Parveen and Sahoo, 2006).
• Multiple known PEGylated proteins have been approved by the USFDA for therapeutic use, including hormones, cytokines, antibody fragments, and enzymes (Pasut, and Veronese, 2012; Alconcel et al., 2011; and Kling, 2013).
• hGH human growth hormone
• a first composition or compound that functions as a human growth hormone receptor antagonist includes human growth hormone receptor antagonist G120K, wherein one amino acid of human growth hormone receptor antagonist G120K has been mutated to cysteine or wherein two amino acids of human growth hormone receptor antagonist G120K have been mutated to cysteine, and wherein the one amino acid mutated to cysteine is selected from the group consisting of N99, T142, and H151, and wherein the two amino acids mutated to cysteine are selected from the group consisting of N99/T142, N99/H151, and T142/H151; and a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K mutant.
• a second composition or compound that functions as a human growth hormone receptor antagonist includes human growth hormone receptor antagonist G120K, wherein one amino acid of human growth hormone receptor antagonist G120K has been mutated to cysteine or wherein two amino acids of human growth hormone receptor antagonist G120K have been mutated to cysteine, and wherein the one amino acid mutated to cysteine is selected from the group consisting of N99, T142, and H151, and wherein the two amino acids mutated to cysteine are selected from the group consisting of N99/T142, N99/H151, and T142/H151; and a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K mutant, wherein the polyethylene glycol molecule conjugated to the one amino acid mutated to cysteine is a polydispersed 40 kDa branched polyethylene glycol molecule; and wherein the polyethylene glycol molecules
• a third composition or compound that functions as a human growth hormone receptor antagonist includes human growth hormone receptor antagonist G120K, wherein two amino acids of human growth hormone receptor antagonist G120K have been mutated to cysteine, and wherein the two amino acids mutated to cysteine are selected from the group consisting of N99/T142, N99/H151, and T142/H151; and a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K mutant.
• the present invention provides novel human growth hormone (hGH) antagonists for use primarily as therapeutics.
• the hGH antagonists of this invention are typically made by mutating one or more selected amino acids of hGH G120K, a known hGH antagonist, to cysteines and then conjugating the cysteines to chemically activated polyethylene glycol molecules.
• the positions of the substituted cysteines have been selected for minimal loss in receptor binding activity after conjugation with polyethylene glycol.
• the specific type and number of polyethylene glycol modifiers of this invention have been selected to produce antagonists with increased in-vivo half-lives.
• PEGylated proteins Two important variables in the preparation of PEGylated proteins in accordance with this invention are: (i) the amino acid position used for PEG attachment; and (ii) the size and type of the conjugated PEG.
• Initial research with similar compositions was done using random attachment of relatively small PEGs (e.g., about 5 kDa) to multiple lysines on the surfaces of proteins. This procedure successfully increased the in vivo half-lives of the proteins, but resulted in large decreases in the affinity of the proteins for their receptors. More recent experimental approaches have added PEG molecules to specific amino acid sites on proteins.
• Two common methods used for site specific PEGylation are: (i) addition of PEG to the N-terminal amine of proteins by way of low pH reductive amination; and (ii) addition of PEG to the thiol groups of cysteines that are either native to the protein or engineered into specific positions.
• Other methods include PEG addition to unnatural amino acids; PEG addition to proteins C-termini by way of intern fusion proteins; and PEG addition to accessible glutamines by way of transaminase catalysis (Pasut and Veronese, 2012).
• PEG polyethylene glycol
• a first type of PEG is prepared by polymerization and is by nature polydispersed, in that there is a distribution of molecular weight products around the average molecular weight.
• a second class of polyethylene glycols are discrete PEGs (dPEG ® s; Quanta BioDesign).
• dPEG ® s are single PEG molecules that are prepared by step-wise, organic chemistry so that each dPEG ® species is a pure single compound with a specific structure and molecular weight (Povosky et al., 2013).
• the different types of PEGs have been produced as both linear and branched structures.
• Branched dPEG ® s have been shown to increase protein half-lives and a negatively charged dPEG ® has been shown to be particularly efficacious (Ding et al., 2013).
• PEGylated versions of the antagonist hGH G120K were prepared by attaching PEGs to cysteine residues that have been incorporated into the antagonist sequence through genetic engineering.
• the antagonist positions selected for mutation to cysteine were selected using the X-ray structure of the complex of hGH with an hGH receptor dimer (hGHRi; Sundstrom el al., 1996).
• the structure of hGH when bound to I1GHR2 is almost identical to the structure of the hGH antagonist hGH G120R when the antagonist is bound to the same receptor (Sundstrom et al., 1996).
• the amino acids selected by solvent (e.g., water) accessibility and mutation energy considerations are listed below in Table 1 under“All Selected Positions” in seven spatially separate domains.
• solvent e.g., water
• being accessible to water is not necessarily a sole criteria for selection; the amino acids need to be accessible to the much larger PEG molecules in order for the PEGylated antagonists to bind to a target receptor.
• the X-ray structure of hGH-hGHR2 was inspected to determine if the side chains of the selected amino acids are directed towards solvent or towards the hGH-hGHR2 protein complex.
• Amino acid positions whose side chains point into the solvent are the most desirable candidates for PEG substitution and are listed below in Table 1 under“Final Selection”.
• PEG polyethylene glycol
• the first class of PEGs was prepared by polymerization and has been used to modify proteins in order to increase their in vivo half-lives (Kling, 2013).
• This type of PEG is by nature polydispersed, meaning that there is a distribution of molecular weight products around the average molecular weight.
• the PEGs include a 20 kDa linear PEG (Layson Bio, MPEG-MAL- 20,000), a 40 kDa branched PEG (NOF, Sunbright GL2-400MA), and a linear 40 kDa PEG (NOF, Sunbright ME-400MA).
• PEGS each contain a maleimide group for conjugation to the free sulfhydryl groups of the mutant proteins.
• the second class of polyethylene glycols are“discrete” PEGs (dPEG ® s; Quanta BioDesign). These dPEG ® s are pure single PEG molecules that are prepared using step-wise, organic chemistry so that each dPEG ® species is a pure single compound with a specific structure and molecular weight (Povosky et al., 2013).
• the dPEGs used in this invention which typically contain a maleimide group for coupling to free thiols, include the following: a tri-branched molecule with a molecular weight of 4473 Daltons and a carboxylate anion at the terminus of each branch (Quanta BioDesign #10451, MAL-dPEGA); a neutral tri- branched molecule with a molecular weight of 4299 Daltons (Quanta BioDesign #4229, MAL- dPEGB); a neutral 9-branched molecule with a molecular weight of 8324 (Quanta Biodesign #10484; MAL-dPEGE); and a neutral 9-branched molecule with a molecular weight of 15,592 (Quanta Biodesign #11487; MAL-dPEGF).
• a tri-branched molecule with a molecular weight of 4473 Daltons and a carboxylate anion at the terminus of each branch Quanta BioDesign #10451, MAL-dPEGA
• the tri-branched 4473 Da molecule has been conjugated to an antibody fragment and its effect on blood clearance in mice has been investigated (Ding et al., 2013). While the added dPEG ® increased the molecular weight of a 50 kDa protein molecular weight by only about 8%, the“area under the curve” (AUC) for blood clearance increased by a factor of about 2.5 over the AUC for the unPEGylated protein.
• AUC area under the curve
• a cell pellet obtained from centrifugation of 250 mL of growth medium containing the expressed mutant was suspended in 10 mL PBS and combined with 0.05 mL of a protease inhibitor cocktail without EDTA (Sigma P8849). The solution was cooled in an ice water mixture and sonicated for four minutes in 30 second bursts. After each sonication, the sample was cooled in the ice-water mixture until the temperature was below 4°C. The sonicated suspension was then centrifuged at 4°C and 25,000 X g for 30 minutes and the supernatant was collected and kept on ice.
• a protease inhibitor cocktail without EDTA Sigma P8849
• the sonicated supernatant was adjusted to 0.3 M sodium chloride and made 5 mM imidazole by addition of a pH 7 solution of 150 mM imidazole.
• the sample was then applied to a gravity flow column having a stoppered outlet packed with 5 mL of TALON ® (Clontech) immobilized metal affinity resin (IMAC).
• IMAC immobilized metal affinity resin
• the resin was equilibrated in 0.05 M sodium phosphate buffer, pH 7.0 containing 5 mM imidazole and 0.3 M sodium chloride prior to addition of the supernatant.
• the top of the column was then also stoppered and the column mixed end-over-end at room temperature for 30 minutes.
• the IMAC purified mutant was concentrated by molecular filtration to 2 mg/mL and 0.5 mL of the solution was combined with 0.05 mL of a solution containing 15 mM reduced glutathione + 1.5 mM oxidized glutathione. An aliquot of 0.04 mg TEV Protease (TurboTev, Accelagen) was then added and the solution incubated for two hours at room temperature followed by overnight incubation at 4°C. The imidazole containing buffer was then exchanged on a spin column for a buffer containing 0.05 M sodium phosphate, pH 7.0 and 0.3 M sodium chloride.
• the desalted mutant was PEGylated by making the solution 0.5 mM maleimide-
• the PEGylated mutant was then applied to a gravity flow IMAC column containing 1 mL TALON ® resin equilibrated in the spin column buffer and the column was washed with 5 CVs of the same buffer.
• the TALON ® flow through and wash contained the product, which was then concentrated by a centrifugal concentrator to 0.3 mL and purified by size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in 0.05 M Tris Buffer, pH 8, containing 0.15 M sodium chloride and 10% glycerol.
• the product fractions were combined and analyzed for protein concentration by absorption at A(280) nm and for purity by SDS-PAGE.
• the addition of a single dPEGB to the single cysteine mutants and two dPEGBs to the double cysteine mutants was confirmed by MALDI mass spectrometry.
• a Competition ELISA required the preparation of biotinylated hGH, which was prepared by standard methods (Hermanson, 2008) using Biotin-dPEG12-NHS (Quanta BioDesign).
• Microtiter plates Coming 96 well plates, half-area, polystyrene) were coated with 0.05 mL of 0.125 mg/mL solutions of the hGH receptor (R&D Systems, 1210-GR-50; cloned as a chimira with an antibody Fc region) in a 0.05 M sodium carbonate buffer at pH 9.6 and incubated either at 37°C for one hour or overnight at 4°C. After washing the plate three times, with three minute incubations between washes, with of 0.125 mL PBS, 0.05% Tween 20 (Wash Buffer), the plates were blocked for one hour by incubation with 2% BSA in PBS.
• Preliminary ELISA assays were performed to determine the concentration of biotin-hGH to use in the completion ELISA. Plates coated with the hGH receptor and blocked were incubated for one hour at RT with different concentrations of biotinylated hGH dissolved in PBS, 0.1% BSA, 0.05% Tween 20 (Dilution Buffer). The plates were then washed 3X with Wash Buffer and incubated for 1 hour at room temperature with 0.5 ug/mL Strep tavidin-HRP (Pierce, 21130) in Dilution Buffer. The plates were again washed 3 times and developed by the addition of 0.05 mL TMB (KPL). After incubation for 2-20 minutes at room temperature, the plates were then quenched by the addition of 0.1 mL 1 M HC1 and read on a plate reader at 450 nm.
• KPL 0.05 mL TMB
• a concentration of biotin-hGH was selected such that the assay response was in the linear range of the plot of biotin-hGH versus A(450) nm (approximately 1 OD unit).
• Competition assays were performed by the preparation of solutions in polypropylene 96 well plates that contained the selected concentration of biotin-hGH and varying concentrations of hGH, hGH G120K mutant, or pegylated hGH G120K mutant.
• Ninety-six well immunoassay plates were coated with the hHG receptor, blocked as described above, and then incubated for 1 hour at RT with the solutions containing the selected concentration of biotin-hGH and different concentrations of the inhibitors. The plates were then washed and treated with Strep tavidin-HRP and TMB as described above.
• the concentration of recombinant hGH that gave a 50% inhibition of the assay response (IC50) was used as the standard to determine the relative affinities of the mutants for the hGH receptor.
• Each assay plate contained a series of concentrations of both the hGH standard and the mutants to be tested and the relative IC50 values were determined.
• Two polydispersed PEGs (ME400MA and GL2-400MA) were conjugated to the free thiols of hGH G120K-H151C and hGH G120K-N99C.
• G120K-H151C-ME400MA and G120K-H151C-GL2-400MA had, respectively, 20% and 50% of the inhibitory activity of G120K.
• N99C-ME400MA and N99C-GL2-400MA had 20% and 2% of the inhibitory activity of G120K.
• the pegylated hGH antagonist hGH G120K-T142C-GL2-400MA was been prepared and purified using the procedures described herein.
• GL2-400MA is a 40 kDa two- branched PEG containing a maleimide group that was reacted with the inserted cysteine of hGH G120K-T142C. This molecule, which is expected to have a long serum half-life (see Zhang et ah, 2012), retained 50% of the hGH receptor binding activity of unmodified hGH.
• the molecule hGH G120K-H151C-GL2-400MA which is also disclosed herein, was shown to also retain 50% of the hGH receptor binding activity of unmodified hGH.
• the binding affinities of the different dPEG ® conjugated mutants of the present invention relative to that of hGH are shown in Table 2, below. Seven single mutants and three double mutants were conjugated to a single tri-branched molecule dPEG ® with a molecular weight of 4473 Daltons (dPEGA) and the molecule was purified as described. As shown in Table 2, certain of these single mutants were also conjugated to three other dPEGs. Three double cysteine mutants were also prepared and conjugated to different dPEGs, as shown in Table 2.
• the receptor binding activities were determined using a competitive ELISA where the recombinant receptor was bound to a plate and the concentration of each sample needed to inhibit the binding of biotin-hGH to the coated plate by 50% (150) was determined.
• the Table entries show the Isos relative to that of hGH, which is defined as 100%, and are rounded to a single significant figure. Only a single competitive ELISA was run for most of the mutants and the estimated relative standard deviation is 25%. Entries marked NT were not tested in this assay.
• dPEGA is a tri-branched molecule with a molecular weight of 4473 Daltons and a carboxylate anion at the terminus of each branch;
• dPEGB is a neutral tri-branched molecule with a molecular weight of 4299 Daltons, dPEGE is a neutral 9-branched molecule with a molecular weight of 8324; and
• dPEGF is a neutral 9-branched molecule with a molecular weight of 15,592.
• IM9 cells were incubated in RPMI media for two hours. The cells were then resuspended in fresh RPMI media at 1 million cells per mL and treated with either hGH, pegylated hGH mutants, or hGH + pegylated mutants at concentrations from 0 to 5000 ng/mL. The treated cells were then incubated for 15 minutes at 37°C in a 5% carbon dioxide incubator.
• the cells were then spun down, lysed in a buffer containing 1% Triton X-100 and sodium orthovanadate, and loaded on an SDS PAGE gel.
• the gel was run under standard conditions and the proteins then transferred electrophoretically to a PVDF membrane.
• the membrane was blocked and then incubated overnight at 4°C with a mixture of rabbit anti-Stat5 Protein antibody and rabbit anti- -actin antibody (positive cell control).
• the membrane was then washed and incubated with a HRP conjugated goat anti-rabbit antibody for one hour at room temperature. Finally, the bands were visualized using Pierce Supersignal West chemiluminescent substrate.
• Qualitative results for the PEGylated mutants are given in Table 3, below.
• the relative abilities of the hGH G120K pegylated double mutants of the present invention to inhibit stimulation of STAT 5 phosphorylation by hGH as measured by Western blot assay are presented in Table 4, below.
• the Western assay qualitatively measures the abilities of hGH antagonists to inhibit the hGH stimulation of STAT 5 phosphorylation.
• the inhibition is expressed as relative to the inhibition obtained with the parent antagonist hGH G120K.
• the relative abilities of the pegylated antagonists to inhibit STAT 5 phosphorylation was between -20% and -100% that of hGH G120K.
• the variation between duplicate runs was too great to make this a quantitative assay. Entries marked NT were not tested in this assay.
• dPEGA is a tri-branched molecule with a molecular weight of 4473 Daltons and a carboxylate anion at the terminus of each branch;
• dPEGB is a neutral tri-branched molecule with a molecular weight of 4299 Daltons, dPEGE is a neutral 9-branched molecule with a molecular weight of 8324; and
• dPEGF is a neutral 9-branched molecule with a molecular weight of 15,592.
• This Western Blot assay measures the abilities of pegylated hGH antagonists to inhibit the hGH stimulation of STAT 5 phosphorylation. The inhibition is expressed as relative to the inhibition obtained with the parent antagonist hGH G120K. The quantification was obtained from the intensities of the phosphorylated STAT 5 band on the Western Blots.
• 4 dPEGA is a tri-branched molecule with a molecular weight of 4473 Daltons and a
• compositions of the present invention provide novel human growth hormone receptor antagonists that are useful in therapeutic applications.
• SEQ ID NO: 1 provides the DNA sequence for human growth hormone WThGH and SEQ ID NO: 2 and provides the amino acid sequence for human growth hormone WThGH (mature form).
• Human growth hormone receptor antagonist G120K is the parent receptor antagonist for the compositions of the present invention, and for reference purposes, SEQ ID NO: 3 provides the DNA sequence for human growth hormone receptor antagonist G120K and SEQ ID NO: 4 provides the amino acid sequence for human growth hormone receptor antagonist G120K (mature form).
• the single letter amino acid abbreviations used herein follow the IUPAC format.
• a first human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid T3 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 5 provides the DNA sequence for human growth hormone antagonist G120K-T3C and SEQ ID NO: 6 provides the amino acid for sequence human growth hormone antagonist G120K-T3C.
• a second human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid E39 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 7 provides the DNA sequence for human growth hormone antagonist G120K-E39C and SEQ ID NO: 8 provides the amino acid sequence for human growth hormone antagonist G120K-E39C.
• a third human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid P48 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 9 provides the DNA sequence for human growth hormone antagonist G120K-P48C and SEQ ID NO: 10 provides the amino acid sequence for human growth hormone antagonist G120K-P48C.
• a fourth human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid Q69 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 11 provides the DNA sequence for human growth hormone antagonist G120K-Q69C and SEQ ID NO: 12 provides the amino acid sequence for human growth hormone antagonist G120K-Q69C.
• a fifth human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid N99 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 13 provides the DNA sequence for human growth hormone antagonist G120K-N99C and SEQ ID NO: 14 provides the amino acid sequence for human growth hormone antagonist G120K-N99C.
• a sixth human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid T142 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 15 provides the DNA sequence for human growth hormone antagonist G120K-T142C and SEQ ID NO: 16 provides the amino acid sequence for human growth hormone antagonist G120K-T142C.
• a seventh human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acid H151 has been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to the cysteine mutation.
• SEQ ID NO: 17 provides the DNA sequence for human growth hormone antagonist G120K-H151C and SEQ ID NO: 18 provides the amino acid sequence for human growth hormone antagonist G120K-H151C.
• An eighth human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acids N99 and H151 have been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to each cysteine mutation.
• SEQ ID NO: 19 provides the DNA sequence for human growth hormone antagonist G120K-N99C-dPEGX-H151C and SEQ ID NO: 20 provides the amino acid sequence for human growth hormone antagonist G120K-N99C- dPEGX-H151C.
• a ninth human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acids T142 and N99 have been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to each cysteine mutation.
• SEQ ID NO: 21 provides the DNA sequence for human growth hormone antagonist G120K-T142C-dPEGX-N99C and SEQ ID NO: 22 provides the amino acid sequence for human growth hormone antagonist G120K-T142C- dPEGX-N99C.
• a tenth human growth hormone antagonist in accordance with an exemplary embodiment of the present invention includes human growth hormone antagonist G120K, wherein amino acids T142 and H151 have been mutated to cysteine, and wherein a polyethylene glycol molecule has been conjugated to each cysteine mutation.
• SEQ ID NO: 23 provides the DNA sequence for human growth hormone antagonist G120K-T142C-dPEGX-H151C and SEQ ID NO: 24 provides the amino acid sequence for human growth hormone antagonist G120K-T142C- dPEGX-H151C.

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• 2019
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• 2024
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