Pegylated growth hormone antagonists

Abstract

To provide compositions for use in treatment of diseases responsive to the use of human growth hormone receptor antagonists or other receptor antagonists.SOLUTION: The present invention provides a composition that is a human growth hormone receptor antagonist, comprising: a human growth hormone receptor antagonist G120K, wherein one or two amino acids of the human growth hormone receptor antagonist G120K have been mutated to cysteine; and a polyethylene glycol molecule conjugated to each substituted cysteine in the human growth hormone receptor antagonist G120K mutant.SELECTED DRAWING: None

Description

[Technical field]

[0001] A sequence listing in computer readable format (CRF) is stored. The sequence listing is an ASCII text (.txt) file entitled SEQIDNOS_1_24_ST25.txt, created on December 10, 2018, and is 33KB in size. The sequence listing is incorporated by reference as if fully set forth herein. [Background technology]

[0002] The invention relates generally to compositions for use as receptor antagonists, and more specifically to novel human growth hormone antagonists that have the potential to be highly effective therapeutic agents.

[0003] 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 mitogen that acts specifically on certain cells. It is a single-chain polypeptide of 191 amino acids that is synthesized, stored, and secreted by somatotropic cells in the lateral wings of the anterior pituitary gland. Acromegaly is a syndrome that occurs when the anterior pituitary gland secretes too much growth hormone (hGH) after the epiphyseal plate closes during puberty. If too much hGH is produced before the epiphyseal plate closes, gigantism (or gigantism) results. Many diseases can increase the pituitary's production of hGH, but the most common are tumors called pituitary adenomas, which are derived from different types of cells (somatotrophs). Acromegaly most commonly affects middle-aged adults and, if untreated, can lead to severe disfigurement, morbidity and premature death. Due to its nature and slow progression, the disease can be difficult to diagnose in its early stages and often goes unnoticed for years, until noticeable changes in appearance, especially of the face, occur.

[0004] A receptor is a protein molecule that is usually embedded in the cell membrane surface and receives chemical signals from outside the cell. When such a chemical signal binds to a receptor, the cell or tissue responds in some way, for example, by changing the electrical activity of the cell. In this sense, a receptor is a protein molecule that recognizes and responds to endogenous chemical signals. Agonists, such as human growth hormone, are chemical compositions that bind to receptors and activate them to cause a biological response. Whereas agonists produce an effect, antagonists block the action of agonists, and inverse agonists produce the opposite effect of agonists. Receptor antagonists are a type of receptor ligand or drug that, when bound to a receptor, does not itself produce a biological response but blocks or attenuates the response mediated by agonists. These compositions are sometimes called blockers, and examples include alpha blockers, beta blockers, and calcium channel blockers. Pharmacologically, antagonists have affinity but no efficacy for their cognate receptors, and by binding to them, they block the interaction and inhibit the function of the agonist or inverse agonist at the receptor. Antagonists mediate their effects by binding to the receptor's active site (orthosteric) or other sites (allosteric). They may also interact at specific binding sites that are not normally involved in the biological regulation of the receptor's activity. The activity of antagonists can change reversibly or irreversibly depending on the lifetime of the antagonist-receptor complex, which also depends on the nature of the antagonist-receptor binding. The majority of antagonists exert their potency by competing with endogenous ligands or substrates for structurally defined binding sites on the receptor. By definition, antagonists do not show potency to activate the bound receptor, and antagonists do not retain the ability to activate the receptor. However, once bound, antagonists inhibit the function of agonists, inverse agonists, and partial agonists.

[0005] Growth hormone receptor antagonists such as pegvisomant (sold under the trademark SOMAVERT®) are used to treat acromegaly. Such compositions are used when the pituitary tumor causing acromegaly cannot be controlled by surgery or radiation and when the use of somatostatin analogues has been unsuccessful. Pegvisomant is usually delivered as a powder that is mixed with water and injected subcutaneously.

[0006] PEGylation is the process of attaching polyethylene glycol (PEG) polymer chains, both covalently and non-covalently, to molecules or macrostructures, such as drugs, peptides, antibody fragments, or therapeutic proteins. PEGylation is typically achieved by incubating reactive derivatives of PEG with target molecules, resulting in changes in physicochemical properties, such as changes in molecular size and molecular charge. These physical and chemical changes can increase systemic retention of therapeutics, affect the binding affinity of therapeutic moieties to cellular receptors, and alter absorption and distribution patterns. Covalent attachment of PEG to drugs or therapeutic proteins can "mask" the drug from the host's immune system (i.e., reduce immunogenicity or antigenicity) and increase the hydrodynamic size (i.e., size in solution) of the drug, thereby decreasing renal clearance and increasing circulation time. PEGylation can also impart water solubility to hydrophobic drugs and proteins.

[0007] PEGylation, by increasing molecular weight, can confer several important pharmacological advantages compared to unmodified molecules, such as (i) improved drug solubility, (ii) reduced dosing frequency without compromising efficacy and potentially reducing toxicity, (iii) increased circulatory life, (iv) improved drug stability, and (v) enhanced protection from proteolytic degradation. PEGylated drugs also offer commercial advantages, such as (i) opportunities for new delivery formats and dosing regimens, and (ii) extended patent life for already approved drugs. PEG is a particularly attractive polymer for conjugation, and specific properties of the PEG moiety that are relevant for 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.

[0008] The addition of high molecular weight polyethylene glycol (PEG) to proteins has previously been shown to increase the in vivo half-life of these proteins by a size-dependent reduction in excretion by the kidney. The addition of PEG also reduces protein immunogenicity and reduces aggregation and protease cleavage (Pasut and Vronese, 2012; and Parveen and Sahoo, 2006). Several known PEGylated proteins, including hormones, cytokines, antibody fragments, and enzymes, are USFDA approved for therapeutic use (Pasut, and Veronese, 2012; Alconcel et al, 2011; and Kling, 2013). Thus, further development of PEGylated therapeutics is currently required, especially for use in the treatment of diseases that respond to the use of human growth hormone (hGH) receptor antagonists or other receptor antagonists. Summary of the Invention [Means for solving the problem]

[0009] The following provides a summary of certain exemplary embodiments of the present invention. This summary is not an extensive overview and is not intended to identify key or critical aspects or elements of the present invention or to define its scope. However, it should be understood that the use of indefinite articles in the language used to describe and claim the present invention is not intended to limit the described system in any way. Rather, the use of "a" or "an" should be interpreted to mean "at least one" or "one or more". As will be appreciated by those skilled in the art, the single-letter amino acid abbreviations used herein follow the IUPAC format.

[0010] According to one aspect of the present invention, a first composition or compound is provided that functions as a human growth hormone receptor antagonist, comprising human growth hormone receptor antagonist G120K, wherein one amino acid of said human growth hormone receptor antagonist G120K is mutated to cysteine, or two amino acids of said human growth hormone receptor antagonist G120K are mutated to cysteine, said one amino acid mutated to cysteine ​​is selected from the group consisting of N99, T142 and H151, and said 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 ​​of the human growth hormone receptor antagonist G120K mutant.

[0011] According to another aspect of the present invention, there is provided a second composition or compound that functions as a human growth hormone receptor antagonist, the human growth hormone receptor antagonist being a human growth hormone receptor antagonist G120K, in which one amino acid of the human growth hormone receptor antagonist G120K is mutated to cysteine, or two amino acids of the human growth hormone receptor antagonist G120K are mutated to cysteine, the one amino acid mutated to cysteine ​​being selected from the group consisting of N99, T142, and H151, and the two amino acids mutated to cysteine ​​being selected from the group consisting of N99 / T142, N99 / H151, and T142 / H151. and a polyethylene glycol molecule conjugated to each substituted cysteine ​​of the human growth hormone receptor antagonist G120K mutant, wherein the polyethylene glycol molecule conjugated to the one amino acid mutated to cysteine ​​is a polydisperse 40 kDa branched polyethylene glycol molecule and the polyethylene glycol molecules conjugated to the two amino acids mutated to cysteine ​​are two 40 kDa branched polyethylene glycol molecules or two 4.5 kDa branched polyethylene glycol molecules each containing three carboxylate anions.

[0012] In yet another embodiment of the present invention, there is provided a third composition or compound that functions as a human growth hormone receptor antagonist, comprising human growth hormone receptor antagonist G120K, where two amino acids of human growth hormone receptor antagonist G120K are mutated to cysteines, and the two amino acids mutated to cysteines are selected from the group consisting of N99 / T142, N99 / H151, and T142 / H151, and a polyethylene glycol molecule conjugated to each substituted cysteine ​​of the human growth hormone receptor antagonist G120K variant.

[0013] Additional features and aspects of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the illustrative embodiments. As will be understood by those of ordinary skill in the art, further embodiments of the present invention are possible without departing from the scope and spirit of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Exemplary embodiments of the present invention are described below. Although the following detailed description contains many specific details for purposes of explanation, those skilled in the art will appreciate that many variations and modifications to the following details are within the scope of the present invention. Thus, the following embodiments of the present invention are described without any loss of generality to, and without imposing limitations on, the claimed invention.

[0015] The present invention provides novel human growth hormone (hGH) antagonists primarily for use as therapeutic agents. The hGH antagonists of the present invention are typically made by mutating one or more selected amino acids of a known hGH antagonist, hGH G120K, to cysteine, and then conjugating the cysteine ​​to a chemically activated polyethylene glycol molecule. The position of the substituted cysteine ​​is selected to minimize loss of receptor binding activity after conjugation with polyethylene glycol. The type and number of polyethylene glycol modifiers of the present invention are selected to produce an antagonist with a long in vivo half-life.

[0016] There are two important variables in the preparation of PEGylated proteins according to the present invention: (i) the amino acid position used for PEG attachment; and (ii) the size and type of PEG conjugated. Early work with similar compositions was performed using a method to randomly attach relatively small PEG (e.g., about 5 kDa) to multiple lysines on the surface of the protein. This method was successful in increasing the in vivo half-life of the protein, but it greatly reduced the affinity of the protein for its receptor. More recent experiments use a method to attach PEG molecules to specific amino acid sites on the protein. Two common methods used for site-specific PEGylation are (i) attachment of PEG to the N-terminal amine of the protein by low pH reductive amination, and (ii) attachment of PEG to the thiol group of a cysteine ​​that is naturally present or engineered into the protein at a specific position. Other methods include attachment of PEG to unnatural amino acids, attachment of PEG to the C-terminus of a protein using an intein fusion protein, and attachment of PEG to accessible glutamines using transaminase catalysis (Pasut and Veronese, 2012).

[0017] In the present invention, two or more different types of polyethylene glycol (PEG) molecules are utilized. The first type of PEG is prepared by polymerization and is polydisperse in nature in that there is a distribution of molecular weight products around an average molecular weight. The second class of polyethylene glycol is discrete PEG (dPEG®; Quanta BioDesign). Such dPEG® are single PEG molecules prepared organically in a stepwise manner such that each dPEG® species is a pure single compound with a specific structure and molecular weight (Povosky et al., 2013). Different types of PEG are produced in both linear and branched structures. For large polydisperse PEGs, the addition of branched PEG to a protein can result in less loss of binding affinity and a greater increase in half-life than the addition of linear PEG of the same molecular weight (Zhang et al. 2012). Branched dPEG® has been shown to increase protein half-life, with negatively charged dPEG® being particularly effective (Ding et al., 2013).

[0018] PEGylated Growth Hormone Antagonists Conversion of hGH from a growth agonist to a growth antagonist requires only a single amino acid change at position 120 of hGH from the native glycine to any amino acid except alanine (Chen et al., 1994). However, this molecule cannot be used as a treatment for hypergrowth conditions (such as acromegaly) due to its short half-life in vivo. Researchers have addressed this issue by adding polyethylene glycol molecules to the hGH antagonist hGH G120K to reduce renal clearance. SOMAVERT®, an FDA-approved drug for the treatment of acromegaly, contains 4-6 linear PEG molecules, each with a molecular weight of 5000 daltons. The addition of PEG randomly attached to surface lysines (van der Lely and Kopchick, 2006) extends the in vivo half-life of the antagonist from less than 1 hour to approximately 72 hours (Finn, 2009). However, the affinity of the PEGylated antagonist for the membrane-bound receptor is reduced by approximately 30-fold compared to the unPEGylated molecule (Ross et al., 2001). Despite the reduced receptor affinity, SOMAVERT® is an effective treatment for acromegaly, but large doses of 5-30 mg per day are typically prescribed.

[0019] Loss of receptor binding following the addition of multiple low molecular weight PEGs (~5 kDa) to random lysines on a protein was common to early protein-PEG conjugates (Parveen and Sahoo, 2006). However, more recently, the addition of a single high molecular weight PEG to a specific amino acid position of the target protein has produced conjugates with higher receptor binding activity (Pasut and Veronese, 2012). For growth hormone antagonists, linear PEGs of 20 kDa or 40 kDa were added to the N-terminus of the unPEGylated precursor of SOMAVERT® (B2036) using reductive alkylation at low pH (Wu et al., 2013). Addition of 20 kDa and 40 kDa PEGs reduced the affinity of the antagonist for the soluble hGH receptor by ~50% and ~95%, respectively. The ability of these molecules to inhibit insulin growth factor-1 (IGF-1) production in rats was tested. The 40 kDa PEG conjugate was inactive, whereas the 20 kDa conjugate reduced IGF-1 production by 30-40%.

[0020] PEGylated Growth Hormone Insight into the predicted effects of different PEGylation strategies on the activity and half-life of growth hormone antagonists can be gained by analyzing the results from the PEGylation of human growth hormone (hGH) (Finn, 2009). Cox and colleagues (2007) mutated the threonine at position 3 of human growth hormone to a cysteine ​​(T3C hGH) and conjugated the cysteine ​​to a 20 kDa linear PEG. The activity of hGH, as measured in a proliferation assay, decreased approximately fourfold, while the half-life increased approximately eightfold. Similar increases in half-life occurred following enzyme-catalyzed addition of 20 kDa PEG to Gln141 and chemical addition of 20 kDa PEG to the N-terminus (Freitas et al., 2013). Addition of a 30 kDa linear PEG to different amino acid positions mutated to chemically active non-native amino acids resulted in an approximately 10-fold loss of proliferative activity and an approximately 10-fold increase in half-life in the optimal case (Cho et al., 2011). Addition of a linear 43 kDa PEG (Gln mutated to Cysteine) at position 141 increased the half-life by ~30-fold, and addition of a branched 40 kDa PEG to the N-terminus of hGH reportedly increased the half-life by approximately 20-fold (Rasmussen et al., 2010).

[0021] PEGylated cytokines Site-specific addition of PEG to cytokines with molecular weights similar to hGH shows that a single PEG causes a significant increase in half-life. Rosendahl et al. (2005) reported that the addition of 10 kDa, 20 kDa, or 40 kDa PEGs after mutation of position 5 of interferon α-2 to cysteine ​​reduced in vitro bioactivity by 2-3 fold. On the other hand, the half-life was increased by 14-fold, 23-fold, and 40-fold with 10 kDa, 20 kDa, and 40 kDa PEGs, respectively. Similar results were seen when PEGs of different molecular weights were added to human granulocyte-macrophage colony-stimulating factor (Doherty et al., 2005). Bell et al. (2008) found that the addition of 20 kDa or 40 kDa PEGs to position 111 (M111C) of interferon α reduced receptor binding activity by 3-fold. Furthermore, the half-lives of 20 kDa and 40 kDa substituted interferons were increased 25- and 39-fold, respectively, whereas site-specific attachment of 40 kDa PEG to interferon-β-1b only increased the half-life by approximately three-fold (Lee et al., 2013).

[0022] Qiu et al. (2013) found that substitution of position 22 of human thyroid stimulating hormone with linear, bi-branched, and tri-branched 40 kDa PEG reduced receptor binding activity by 5-, 14-, and 11-fold, respectively. A comparison of the half-lives of these different conjugates was not reported. Fam et al. (2014) investigated the effect of adding 10 kDa, 20 kDa, and 40 kDa PEG to position 103 of interferon-γ and found that PEG did not significantly affect the in vitro activity of the cytokine, but increased the half-life by approximately 30-fold in all cases.

[0023] Antibody fragments The addition of PEG to antibody fragments to increase their residence time in the body has been extensively studied. Lee et al. (1999) conjugated a single-chain antibody fragment (scFv MAb; 26 kDa) to six different PEG polymers ranging in MW from 2 to 20 kDa. These conjugates showed longer half-lives compared to the unPEGylated parent. Increasing the length of the PEG polymer was found to be more effective at extending the half-life than increasing the total amount of PEG. Li et al. (2010) showed that the addition of two discrete linear PEG units conjugated to random lysines on the diabody increased blood residence time compared to unPEGylated or polydisperse PEGylated products. Chapman et al. (2002) showed that the half-life of an antibody Fab' fragment (approximately 50 kDa) was directly related to the size and number of site-specific PEGs.

[0024] Lee et al. (2013) found that a linear random increase in conjugated PEG mass (4-20 kDa) in scFv-PEG conjugation effectively increased half-life approximately linearly with mass. These researchers found that a single 20 kDa PEG was more effective than four 5,000 PEGs and concluded that PEG length was more important than PEG mass. In a study of a Fab' antibody fragment of approximately 50 kDa, site-specific addition of 4.4 kDa branched discrete PEG (dPEG) increased half-life approximately two-fold compared to unconjugated Fab' (Ding et al., 2013).

[0025] Selection of the amino acid position of hGH G120K to be mutated to cysteine In various embodiments of the present invention, PEGylated versions of antagonist hGH G120K are prepared by attaching PEG to cysteine ​​residues that are engineered into the antagonist sequence.The positions of the antagonist selected for mutation to cysteine ​​were selected using the X-ray structure of the complex of hGH with the hGH receptor dimer (hGHR2; Sundstrom et al., 1996).The structure of hGH when bound to hGHR2 is nearly identical to that of hGH antagonist hGH G120R when bound to the same receptor (Sundstrom et al., 1996).

[0026] Based on the crystal structure of hGH-hGHR2, two criteria were used to select amino acids for cysteine ​​mutation: i) the solvent accessibility of the amino acid, and (ii) the substitution of cysteine ​​for the selected amino acid should be close to an energetically neutral process. The solvent accessibility of each amino acid in hGH-hGHR2 was determined by the modeling program Swiss PDB Viewer (Guex and Peitsch, 1997) and the method of PoPMuSIC (Dehouck et al., 2009; Dehouck et al., 2011). The energetic cost of substituting cysteine ​​at each amino acid position was determined using the Prediction of Protein Mutant Stability Changes (PoPMuSIC) program (http: / / babylone.ulb.ac.be / popmusic / ).

[0027] The amino acids selected for their solvent (e.g., water) accessibility and mutation energy are listed in seven spatially separated domains in Table 1, “All Selected Positions”. However, water accessibility is not necessarily the only selection criterion. For PEGylated antagonists to bind to the target receptor, the amino acids must be accessible to the much larger PEG molecule. The X-ray structure of hGH-hGHR2 was examined to confirm whether the side chains of the selected amino acids point towards the solvent or towards the hGH-hGHR2 protein complex. The amino acid positions with side chains pointing towards the solvent are the most desirable candidates for PEG substitution and are listed below in Table 1, “Final Selection”. An additional position, H151, was also selected for cysteine ​​mutation. This position is part of a section of loop 3 that is not revealed in the crystal structure. The general location of this missing segment points away from the hGH-hGHR2 protein complex.

[0028] [Table 1]

[0029] Selection of PEG for conjugation to the hGH G120K mutant In the present invention, two different classes of polyethylene glycol (PEG) molecules are utilized. The first class of PEGs are prepared by polymerization and have been used to modify proteins to increase their in vivo half-life (Kling, 2013). This type of PEG is polydisperse in nature, meaning that there is a distribution of molecular weight products around an 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). Each of these PEGs contains a maleimide group for conjugation to the free sulfhydryl groups of mutant proteins. The second class of polyethylene glycol is the "discrete" PEG (dPEG®; Quanta BioDesign). These dPEGs are pure single PEG molecules prepared using organic chemistry in a stepwise manner such that each dPEG species is a pure single compound with a specific structure and molecular weight (Povosky et al., 2013). The dPEGs used in the present invention typically contain maleimide groups for coupling to free thiols and include: a tri-arm molecule with a molecular weight of 4473 daltons and a carboxylate anion at the end of each arm (Quanta BioDesign #10451, MAL-dPEGA); a neutral tri-arm molecule with a molecular weight of 4299 daltons (Quanta BioDesign #4229, MAL-dPEGB); a neutral 9-arm molecule with a molecular weight of 8324 (Quanta Biodesign #10484, MAL-dPEGE); and a neutral 9-arm molecule with a molecular weight of 15,592 (Quanta Biodesign #11487, MAL-dPEGF). A tri-branched 4473 Da molecule was conjugated to an antibody fragment and its effect on blood clearance in mice was examined (Ding et al., 2013). Although added dPEG® increased the molecular weight of a 50 kDa protein by only about 8%, the "area under the curve" (AUC) for blood clearance increased by about 2.5-fold compared to the AUC for the unPEGylated protein.

[0030] Purification and PEGylation of the hGH G120K mutant Cell destruction 250 mL of growth medium containing the expressed mutants was centrifuged and the resulting cell pellet was suspended in 10 mL of PBS and mixed with 0.05 mL of EDTA-free protease inhibitor cocktail (Sigma P8849). The solution was chilled in ice-water and sonicated for 4 min with 30-s bursts. After each sonication, the sample was cooled in the ice-water mixture until it was below 4°C. The sonicated suspension was then centrifuged at 25,000Xg for 30 min at 4°C and the supernatant was collected and stored on ice.

[0031] Affinity purification The sonicated supernatant was adjusted to 0.3 M sodium chloride and 5 mM imidazole was added with 150 mM imidazole, pH 7. The sample was applied to a gravity-flow column with a plugged outlet packed with 5 mL of TALON® (Clontech) Immobilized Metal Affinity Resin (IMAC). The resin was equilibrated with 0.05 M sodium phosphate buffer, pH 7.0, containing 5 mM imidazole and 0.3 M sodium chloride prior to loading of the supernatant. The top of the column was then also plugged and the column was mixed end-to-end for 30 minutes at room temperature. The column was then drained and washed with five aliquots of at least 5 mL of equilibration buffer. Washing was continued until the A(280) nm of the eluate no longer decreased. The column was then eluted with equilibration buffer containing 150 mM imidazole at pH 7, and the product-containing fractions were brought to 5 mM EDTA by adding a 100 mM solution of disodium EDTA adjusted to pH 7.

[0032] Cleavage of His-Tag by TEV protease The IMAC-purified mutants were concentrated to 2 mg / mL by molecular filtration, and 0.5 mL of the solution was added to 0.05 mL of a solution containing 15 mM reduced glutathione + 1.5 mM oxidized glutathione. An aliquot of 0.04 mg of TEV protease (TurboTev, Accelagen) was then added, and the solution was incubated at room temperature for 2 hours, followed by overnight incubation at 4°C. The imidazole-containing buffer was exchanged on a spin column into a buffer containing 0.05 M sodium phosphate, pH 7.0 and 0.3 M sodium chloride.

[0033] PEGylation and purification The desalted variants were PEGylated by bringing the solution to 0.5 mM maleimide-PEG and reacting for 2 h at room temperature followed by overnight incubation at 4°C. The PEGylated variants were then applied to a gravity-flow IMAC column containing 1 mL of TALON® resin equilibrated with spin column buffer, and the column was washed with 5 CV of the same buffer. The TALON® flow-through and wash contained the product, which was concentrated to 0.3 mL in a centrifugal concentrator and purified by size-exclusion chromatography using a Superdex 200 Increase 10 / 300GL column (GE Healthcare) equilibrated with 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 absorbance at A(280) nm and purity by SDS-PAGE. The addition of one dPEGB to the single-cysteine ​​variant and two dPEGB to the double-cysteine ​​variant was confirmed by MALDI mass spectrometry.

[0034] Competitive ELISA assay of relative affinity for the hGH receptor For the competitive ELISA, biotinylated hGH had to be prepared, which was prepared using Biotin-dPEG12-NHS (Quanta BioDesign) using standard methods (Hermanson, 2008). Microtiter plates (Corning 96-well plates, half area, polystyrene) were coated with 0.05 mL of a 0.125 μg / mL solution of hGH receptor (R&D Systems, 1210-GR-50; cloned as a chimera with an antibody Fc region) in 0.05 M sodium carbonate buffer at pH 9. After washing three times with 0.125 mL PBS, 0.05% Tween 20 (Wash Buffer), the plates were blocked by incubation with 2% BSA in PBS for 1 h.

[0035] A preliminary ELISA assay was performed to determine the concentration of biotin-hGH to be used in the completed ELISA. Plates coated with hGH receptors and blocked were incubated with different concentrations of biotinylated hGH dissolved in PBS, 0.1% BSA, 0.05% Tween 20 (Dilution Buffer) for 1 hour at RT. The plates were then washed 3 times with Wash Buffer and incubated with 0.5ug / mL Streptavidin-HRP (Pierce, 21130) dissolved in Dilution Buffer for 1 hour at room temperature. The plates were washed again 3 times and developed by adding 0.05mL TMB (KPL). After 2-20 minutes of incubation at room temperature, the plates were quenched by adding 0.1mL 1M HCl and read at 450nm wavelength on a plate reader.

[0036] The concentration of biotin-hGH was chosen so that the assay response was in the linear range (approximately 1 OD unit) of a plot of biotin-hGH versus A(450) nm. Competitive assays were performed by preparing solutions containing selected concentrations of biotin-hGH and various concentrations of hGH, hGH G120K mutant, or PEGylated hGH G120K mutant in polypropylene 96-well plates. 96-well immunoassay plates were coated with hHG receptor and blocked as described above, then incubated for 1 h at RT with solutions containing selected concentrations of biotin-hGH and different concentrations of inhibitor. Plates were then washed and treated with Streptavidin-HRP and TMB as described above.

[0037] To determine the relative affinity of the variants for the hGH receptor, the concentration of recombinant hGH that gives 50% inhibition of the assay response (IC 50 ) was used as a standard. Each assay plate contained a range of concentrations of hGH standards and the variants being tested to determine the relative IC 50 values ​​were determined. Two polydisperse 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 20% and 50% of the inhibitory activity of G120K, respectively. N99C-ME400MA and N99C-GL2-400MA had 20% and 2% of the inhibitory activity of G120K.

[0038] The PEGylated hGH antagonist hGH G120K-T142C-GL2-400MA was prepared and purified using the procedures described herein. GL2-400MA is a 40 kDa bi-branched PEG containing a maleimide group that was reacted with the inserted cysteine ​​of hGH G120K-T142C. This molecule, which is predicted to have a long half-life in serum (Zhang et al. 2012), retained 50% of the hGH receptor binding activity of unmodified hGH. The molecule hGH G120K-H151C-GL2-400MA disclosed herein was also shown to retain 50% of the hGH receptor binding activity of unmodified hGH.

[0039] The binding affinity of different dPEG® conjugated variants of the present invention to that of hGH is shown in Table 2 below. Seven single variants and three double variants were conjugated to a single three-arm molecule dPEG® (dPEGA) with a molecular weight of 4473 daltons, and the molecule was purified as described. As shown in Table 2, some of these single variants were also conjugated to three other dPEGs. Three double cysteine ​​variants were also prepared and conjugated to different dPEGs, as shown in Table 2.

[0040] [Table 2] 1 Receptor binding activity was determined using a competitive ELISA in which recombinant receptor was bound to plates, and the concentration of each sample required to inhibit 50% (I50) binding of biotin-hGH to the coated plates was determined. Table entries show the I50 of hGH defined as 100%. 50 I 50 The numbers are rounded up to one significant digit. For most variants, the competitive ELISA was performed only once, resulting in an estimated relative standard deviation of 25%. Entries marked NT were not tested in this assay. 2 dPEGA is a tri-arm molecule with a molecular weight of 4473 daltons and a carboxylate anion at the end of each arm; dPEGB is a neutral tri-arm molecule with a molecular weight of 4299 daltons; dPEGE is a neutral nona-arm molecule with a molecular weight of 8324; and dPEGF is a neutral nona-arm molecule with a molecular weight of 15,592. 3 These reactions did not proceed to doubly PEGylated products.

[0041] Western blot assay for the ability of PEGylated variants to inhibit hGH-stimulated phosphorylation of STAT 5 The ability of the PEGylated variants of the invention to inhibit stimulation of Stat5 Protein phosphorylation by hGH was measured in a cell-based assay. IM9 cells were incubated in RPMI medium for 2 hours. The cells were then resuspended in fresh RPMI medium at 1 million cells / mL and treated with either hGH, PEGylated hGH variants, or hGH+PEGylated variants at concentrations ranging from 0 to 5000 ng / mL. The treated cells were then incubated for 15 minutes at 37°C in a 5% CO2 incubator. The cells were then spun down, lysed in a buffer containing 1% Triton X-100 and sodium orthovanadate, and loaded onto an SDS PAGE gel. The gel was run under standard conditions, and the proteins were electrophoretically transferred to a PVDF membrane. The membrane was blocked and then incubated overnight at 4°C with a mixture of rabbit anti-Stat5 Protein and rabbit β anti-actin antibodies (positive cell control). The membrane was then washed and incubated with HRP-conjugated goat anti-rabbit antibody at room temperature for 1 hour. Finally, the bands were visualized using Pierce Supersignal West chemiluminescent substrate. The qualitative results of the PEGylated variants are shown below in Table 3. The relative ability of the hGH G120K PEGylated double mutants of the present invention to inhibit stimulation of STAT5 phosphorylation by hGH, as measured by Western blot assay, is shown below in Table 4.

[0042] [Table 3] 1 The Western assay qualitatively measures the ability of hGH antagonists to inhibit hGH-stimulated phosphorylation of STAT5. Inhibition is expressed relative to the inhibition obtained with the parent antagonist, hGH G120K. In all cases, the relative ability of the PEGylated antagonists to inhibit STAT5 phosphorylation was between ~20% and ~100% compared to that of hGH G120K. The variation between duplicate runs was too large to make this a quantitative assay. Entries marked NT were not tested in this assay. 2 dPEGA is a tri-arm molecule with a molecular weight of 4473 daltons and a carboxylate anion at the end of each arm; dPEGB is a neutral tri-arm molecule with a molecular weight of 4299 daltons; dPEGE is a neutral nona-arm molecule with a molecular weight of 8324; and dPEGF is a neutral nona-arm molecule with a molecular weight of 15,592.

[0043] [Table 4] 1 This Western blot assay measures the ability of PEGylated hGH antagonists to inhibit the stimulation of STAT5 phosphorylation by hGH. Percentage of inhibition is expressed relative to that obtained with the parent antagonist hGH G120K. Quantitation was obtained from the intensity of the phosphorylated STAT5 band on the Western blot. 2 dPEGA is a three-branched molecule with a molecular weight of 4473 daltons, and each branch has a carboxylate anion at its end.

[0044] As shown by the above disclosure, the compositions of the present invention provide novel human growth hormone receptor antagonists useful for therapeutic applications. For reference purposes, SEQ ID NO:1 provides the DNA sequence of human growth hormone WThGH, and SEQ ID NO:2 provides the amino acid sequence of 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 of human growth hormone receptor antagonist G120K, and SEQ ID NO:4 provides the amino acid sequence of human growth hormone receptor antagonist G120K (mature form). As mentioned above, the single-letter amino acid abbreviations used herein follow the IUPAC format.

[0045] A first human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, wherein the amino acid T3 is mutated to cysteine, and a polyethylene glycol molecule is conjugated to the cysteine ​​mutation. SEQ ID NO: 5 provides the DNA sequence of human growth hormone antagonist G120K-T3C, and SEQ ID NO: 6 provides the amino acid sequence of human growth hormone antagonist G120K-T3C.

[0046] A second human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, with amino acid E39 mutated to cysteine, and a polyethylene glycol molecule conjugated to the cysteine ​​mutation. SEQ ID NO:7 provides the DNA sequence of human growth hormone antagonist G120K-E39C, and SEQ ID NO:8 provides the amino acid sequence of human growth hormone antagonist G120K-E39C.

[0047] A third human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, where amino acid P48 is mutated to cysteine, and a polyethylene glycol molecule is conjugated to the cysteine ​​mutation. SEQ ID NO:9 provides the DNA sequence of human growth hormone antagonist G120K-P48C, and SEQ ID NO:10 provides the amino acid sequence of human growth hormone antagonist G120K-P48C.

[0048] A fourth human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, with amino acid Q69 mutated to cysteine, and a polyethylene glycol molecule conjugated to the cysteine ​​mutation. SEQ ID NO:11 provides the DNA sequence of human growth hormone antagonist G120K-Q69C, and SEQ ID NO:12 provides the amino acid sequence of human growth hormone antagonist G120K-Q69C.

[0049] A fifth human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, with amino acid N99 mutated to cysteine, and a polyethylene glycol molecule conjugated to the cysteine ​​mutation. SEQ ID NO:13 provides the DNA sequence of human growth hormone antagonist G120K-N99C, and SEQ ID NO:14 provides the amino acid sequence of human growth hormone antagonist G120K-N99C.

[0050] A sixth human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, where amino acid T142 is mutated to cysteine, and a polyethylene glycol molecule is conjugated to the cysteine ​​mutation. SEQ ID NO:15 provides the DNA sequence of human growth hormone antagonist G120K-T142C, and SEQ ID NO:16 provides the amino acid sequence of human growth hormone antagonist G120K-T142C.

[0051] A seventh human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, where amino acid H151 is mutated to cysteine, and a polyethylene glycol molecule is conjugated to the cysteine ​​mutation. SEQ ID NO: 17 provides the DNA sequence of human growth hormone antagonist G120K-H151C, and SEQ ID NO: 18 provides the amino acid sequence of human growth hormone antagonist G120K-H151C.

[0052] An eighth human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, with amino acids N99 and H151 mutated to cysteine, and a polyethylene glycol molecule conjugated to each cysteine ​​mutation. SEQ ID NO:19 provides the DNA sequence of human growth hormone antagonist G120K-N99C-dPEGX-H151C, and SEQ ID NO:20 provides the amino acid sequence of human growth hormone antagonist G120K-N99C-dPEGX-H151C.

[0053] A ninth human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, with amino acids T142 and N99 mutated to cysteine, and a polyethylene glycol molecule conjugated to each cysteine ​​mutation. SEQ ID NO:21 provides the DNA sequence of human growth hormone antagonist G120K-T142C-dPEGX-N99C, and SEQ ID NO:22 provides the amino acid sequence of human growth hormone antagonist G120K-T142C-dPEGX-N99C.

[0054] A tenth human growth hormone antagonist according to an exemplary embodiment of the present invention comprises human growth hormone antagonist G120K, with amino acids T142 and H151 mutated to cysteine, and a polyethylene glycol molecule conjugated to each cysteine ​​mutation. SEQ ID NO:23 provides the DNA sequence of human growth hormone antagonist G120K-T142C-dPEGX-H151C, and SEQ ID NO:24 provides the amino acid sequence of human growth hormone antagonist G120K-T142C-dPEGX-H151C.

[0055] While the present invention has been illustrated by a description of exemplary embodiments thereof and the embodiments have been described in certain detail, it is not intended to restrict or in any way limit the scope of the appended claims to such details. Additional advantages and modifications will readily occur to those skilled in the art. Thus, the invention in its broader aspects is not limited to any of the specific details, representative apparatus and methods, and / or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

Claims

1. A human growth hormone receptor antagonist, a) a human growth hormone receptor antagonist G120K having the amino acid sequence of SEQ ID NO:4 (DNA sequence of SEQ ID NO:3); b) a polyethylene glycol molecule conjugated to each substituted cysteine ​​of the human growth hormone receptor antagonist G120K mutant, the human growth hormone receptor antagonist G120K in which two amino acids are mutated to cysteine, the two amino acids being T142 and H151; the polyethylene glycol molecules conjugated to the two amino acids mutated to cysteine ​​are two 4.5 kDa branched polyethylene glycols each containing three carboxylate anions, the polyethylene glycol molecules being prepared by stepwise organic chemistry and being substantially pure single compounds; A human growth hormone receptor antagonist, which inhibits stimulation of STAT-5 phosphorylation by 50% under quantitative comparison.

2. 2. The human growth hormone receptor antagonist of claim 1, wherein the polyethylene glycol molecule contains a maleimide group for conjugation to free sulfhydryl groups.

3. The human growth hormone receptor antagonist of claim 1, wherein the human growth hormone receptor antagonist is encoded by a DNA molecule having a sequence consisting of SEQ ID NO: 23, and the human growth hormone receptor antagonist has an amino acid sequence consisting of SEQ ID NO:

24.

4. 2. The human growth hormone receptor antagonist of claim 1, wherein an effective amount of the human growth hormone receptor antagonist is used to treat a disease or condition that responds to the human growth hormone receptor antagonist.

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