NOVEL GROWTH HORMONE RECEPTOR ANTAGONIST AND FUSION PROTEIN THEREOF

Abstract

The present invention relates to a growth hormone receptor antagonist comprising a growth hormone variant which is modified by substitution of one or more amino acids of growth hormone. Further, the growth hormone receptor antagonist of the present invention may further comprise a long-acting carrier which is fused to the growth hormone variant. The growth hormone receptor antagonist may have strong binding potency to growth hormone receptor and may exhibit a long-lasting antagonistic action.

Claims

1. A growth hormone receptor antagonist comprising a growth hormone variant in which one or more amino acids in an amino acid sequence of growth hormone are substituted with another amino acid.

2. The growth hormone receptor antagonist of claim 1, wherein the substitution with another amino acid comprises substitution of the 46.sup.th amino acid from the N-terminus with lysine, substitution of the 120.sup.th amino acid from the N-terminus with lysine or arginine, or a combination thereof.

3. The growth hormone receptor antagonist of claim 2, wherein the substitution with another amino acid further comprises substitution at any one or more positions selected from the group consisting of the 18.sup.th amino acid, the 21.sup.st amino acid, the 54.sup.th amino acid, the 64.sup.th amino acid, the 167.sup.th amino acid, the 168.sup.th amino acid, the 171.sup.st amino acid, the 172.sup.nd amino acid, the 174.sup.th amino acid, the 176.sup.th amino acid, and the 179.sup.th amino acid from the N-terminus.

4. The growth hormone receptor antagonist of claim 2, wherein the substitution with another amino acid further comprises substitution of the 174.sup.th amino acid from the N-terminus with serine, substitution of the 21.sup.st amino acid from the N-terminus with asparagine, or a combination thereof.

5. The growth hormone receptor antagonist of claim 1, wherein the substitution with another amino acid comprises any one or more substitutions selected from the group consisting of substitution of the 18.sup.th amino acid from the N-terminus with aspartic acid, substitution of the 21.sup.st amino acid from the N-terminus with asparagine, substitution of the 46.sup.th amino acid from the N-terminus with lysine, substitution of the 54.sup.th amino acid from the N-terminus with proline, substitution of the 64.sup.th amino acid from the N-terminus with lysine, substitution of the 120.sup.th amino acid from the N-terminus with lysine or arginine, substitution of the 167.sup.th amino acid from the N-terminus with asparagine, substitution of the 168.sup.th amino acid from the N-terminus with alanine, substitution of the 171.sup.st amino acid from the N-terminus with serine, substitution of the 172.sup.nd amino acid from the N-terminus with arginine, substitution of the 174.sup.th amino acid from the N-terminus with serine, substitution of the 176.sup.th from the N-terminus with tyrosine, and substitution of the 179.sup.th amino acid from the N-terminus with threonine.

6. The growth hormone receptor antagonist of claim 1, wherein a long-acting carrier is linked to the growth hormone variant.

7. The growth hormone receptor antagonist of claim 6, wherein the long-acting carrier is fused to the N-terminus or the C-terminus of the growth hormone variant.

8. The growth hormone receptor antagonist of claim 6, wherein the long-acting carrier is selected from the group consisting of polyethylene glycol, fatty acids, albumin or fragments thereof, albumin-binding substances, alpha-1 antitrypsin or variants thereof, immunoglobulin Fc or fragments thereof, a polymer of repeating units of a specific amino acid sequence, antibodies or fragments thereof, FcRn-binding substances, in vivo connective tissues or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and polymers.

9. The growth hormone receptor antagonist of claim 8, wherein the long-acting carrier is an alpha-1 antitrypsin or a variant thereof.

10. The growth hormone receptor antagonist of claim 9, wherein the alpha-1 antitrypsin variant comprises substitution of one or more amino acids with another amino acid, and the substitution comprises substitution at one or more positions among the 1.sup.st and 25.sup.th amino acids from the N-terminus.

11. The growth hormone receptor antagonist of claim 9, wherein the alpha-1 antitrypsin variant comprises any one or more substitutions selected from the group consisting of substitution of the 9.sup.th amino acid from the N-terminus with asparagine, substitution of the 232.sup.nd amino acid from the N-terminus with serine, substitution of the 37.sup.th amino acid from the N-terminus with asparagine, and substitution of the 359.sup.th amino acid from the N-terminus with threonine.

12. The growth hormone receptor antagonist of claim 6, wherein the growth hormone variant is fused with the long-acting carrier directly or via a linker.

13. The growth hormone receptor antagonist of claim 12, wherein the linker is a peptidyl linker or a non-peptidyl linker.

14. The growth hormone receptor antagonist of claim 13, wherein the non-peptidyl linker is polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), a lipid polymer, chitins, hyaluronic acid, or a combination thereof.

15. The growth hormone receptor antagonist of claim 13, wherein the peptidyl linker comprises two or more amino acids.

16. A pharmaceutical composition for preventing or treating a disease caused by human growth hormone, comprising the growth hormone receptor antagonist of claim 1.

17. The pharmaceutical composition of claim 16, wherein the disease is selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, lung inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner's syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).

18. A method of preparing a growth hormone receptor antagonist, the method comprising the step of culturing cells comprising a polynucleotide encoding the growth hormone variant in which one or more amino acids in an amino acid sequence of growth hormone are substituted with another amino acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 shows a structure of a complex of growth hormone and growth hormone receptor (PDB id: 3HHR);

[0053] FIG. 2 shows hGH efficacy on hGH, hGH-A1, and hGH-A2;

[0054] FIG. 3 shows chromatography purification of hGHR-A3 which is a growth hormone receptor antagonist of the present invention, in which FIG. 3A shows a chromatogram of second ion-exchange column chromatography for purification of hGHR-A3, FIG. 3B shows the result of 10% SDS-polyacrylamide gel electrophoresis of each purification fraction, and FIG. 3C shows purity of purified hGHR-A3, as analyzed by size-exclusion HPLC;

[0055] FIG. 4 shows the results of hGH receptor binding assay and hGH competitive inhibition assay for hGH-A7, hGH-A9, hGH-A10, hGH-A11 and Pegvisomant;

[0056] FIG. 5 shows a plot of the results of relative binding potency and hGH competitive efficacy of hGH-A1 to hGH-A11 and Pegvisomant of Comparative Example; and

[0057] FIG. 6 shows a diagram illustrating a process of preparing the growth hormone receptor antagonist of the present invention.

BEST MODE FOR THE INVENTION

[0058] Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, the following Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited by these Examples.

Preparation Example

Preparation of Growth Hormone Receptor Antagonist

[0059] 1. Cloning, Transfection, and Cell Culture

[0060] cDNA clones of hGH-NexP were prepared according to a known method. The hGH receptor antagonists (hGHRA-NexP) were prepared by site-directed mutagenesis of hGH-NexP gene. Afterwards, the mutations were confirmed by DNA sequencing. CHO (Chinese hamster ovary)-K1 cells were transiently transfected with a plasmid containing a nucleotide sequence of each hGHRA-NexP clone. The transfected cells were grown in IMDM medium (Iscove's Modified Dulbecco's Medium) supplemented with 10% FBS in a 5% CO.sub.2 humidified incubator for 7 days.

[0061] 2. Purification of hGHRA-NexP

[0062] hGHRA-NexP variants were purified from the transiently transfected CHO-K1 cells by a series of column chromatography. The culture supernatant was diluted with an equal volume of buffer A (20 mM sodium phosphate, pH 8.0), and applied to an ion-exchange column equilibrated with buffer A. After a washing step with buffer A, the proteins were eluted with a linear gradient of NaCl in buffer A. Then the fractions containing hGHRA-NexP were directly loaded onto an affinity column equilibrated with buffer B (20 mM Tris-HCl, 100 mM NaCl, pH 7.5), and the fraction was eluted with a gradient of MgCl.sub.2 in buffer B. After adjusting pH and conductivity of the elution pool, they were loaded onto a second ion exchange column that was equilibrated with buffer C (20 mM sodium phosphate, 80 mM NaCl, pH 8.0). The protein fraction was concentrated with a Vivaspin 20 concentrator (Sartorius), and dialyzed against phosphate buffer saline (PBS).

[0063] 3. SE-HPLC Analysis

[0064] To determine purity of hGHRA-NexP, size-exclusion high-performance liquid chromatography (SE-HPLC) was performed. The protein solution was loaded onto a TSKgel G3000SWXL column (Tosoh), and a chromatogram was obtained in a running buffer (50 mM sodium phosphate, 150 mM NaCl, 0.05% sodium azide, pH 6.8) at a flow rate of 0.5 mL/min. An area percentage (%) of a main peak of the chromatogram was calculated.

Example

Examples of Growth Hormone Receptor Antagonist

[0065] An object of the present invention is to prepare growth hormone receptor antagonists by using site-directed mutagenesis on hGH sequence and a NexP technology to provide for long-acting property, and Example 1 (hGH-A1: G120K) and Example 2 (hGH-A2: G120R), each in which an amino acid at position 120 of hGH was substituted, were prepared. Further, for the above object, additional mutation was introduced into site 1 of hGH-A1 in the hGH sequence to prepare Examples 3 to 7. Specific amino acids substituted in Examples are shown in Table 3 below. In Example 9, Q46K mutation was introduced to induce cation-n interaction through introduction of lysine.

TABLE-US-00003 TABLE 3 Name Mutations Example 1 hGH-A1 G120K Example 2 hGH-A2 G120R Example 3 hGH-A3 H21N/G120K/E174S Example 4 hGH-A4 H18D/H21N/G120K/E174S/I179T Example 5 hGH-A5 H21N/G120K/E174S/F176Y Example 6 hGH-A6 H21N/F54P/R64K/G120K/E174S/F176Y Examples 7 hGH-A7, H21N/F54P/R64K/G120K/R167N/ and 10 hGH-A10 D171S/E174S/F176Y Example 8 hGH-A8 H18D/H21N/G120K/R167N/K168A/ D171S/K172R/E174S/I179T Examples 9 hGH-A9, H18D/H21N/Q46K/G120K/E174S/I179T and 11 hGH-A11

[0066] In Table 3, hGH-A1 to hGH-A9 were obtained by fusing NexP to the C-terminus of the variant, and hGH-A10 and hGH-A11 were obtained by fusing NexP to the N-terminus of the variant.

[0067] The hGHRA-NexP proteins were prepared by transfection of CHO-K1 cell and a series of column chromatography according to Preparation Example described above. Representatively, a chromatogram of the second ion exchange column chromatography is shown in FIG. 3A. Pure proteins were obtained from chromatography, and purified proteins were migrated in the position between 100 kDa and 70 kDa (FIG. 3B). After pooling the fractions from fraction number 7 to 14, the solutions were dialyzed against phosphate buffer saline (PBS). It was confirmed that the protein showed high purity of 95%, as shown in the main peak of SE-HPLC chromatogram (FIG. 3C).

Experimental Example

Analysis of hGH Efficacy and Receptor Binding Potency

[0068] 1. hGH Efficacy Analysis Method

[0069] For this analysis, hGH receptor gene was introduced into the chromosome of HEK293F cells containing a luciferase gene that could be induced by hGH receptor signaling. The prepared cell line was named as hGHR/Luc/HEK293F. Serial dilutions of hGH and hGHRA-NexP were added to a 96-well white plate containing hGHR/Luc/HEK293F, respectively. This plate was incubated in a 5% CO.sub.2 incubator at 37 C. for 24 hours. After incubation, 100 L of luciferase assay reagent (Steady-Glo luciferase assay system, Promega) was added to each well, and the plate was wrapped for protection against light. After 5 minutes at room temperature, a multi-mode micropiate reader (SpectraMax M5, Molecular Devices) was used to analyze luminescence from wells.

[0070] The results of measuring hGH, hGH-A1, and hGH-A2 are shown in FIG. 2. It was confirmed that hGH-A1 and hGH-A2 showed no efficacy, unlike hGH.

[0071] 2. hGH Receptor Binding Potency Analysis

[0072] Binding potency of hGRA-NexP to hGH receptor was evaluated by affinity analysis using a recombinant hGH receptor Fc chimera. A microplate was coated with the hGH receptor chimera at 25 C. overnight, and washed with TPBS buffer (PBS buffer containing 0.05% Tween-20), and samples (Examples 1 to 11 and Pegvisomant) were loaded on each well. The samples were washed three times, and conjugated with anti-hGH-polyclonal antibody-biotin. After additional washing step, 3,3,5,5-tetramethylbenzidine (TMB) was added to each well to allow TMB reaction. Absorbance signals from the reaction were recorded at 450 nm to 650 nm.

[0073] The results of measuring binding profiles of hGH-A7 ( ), hGH-A9 (), and Pegvisomant () are shown in FIG. 4A. Further, the results of measuring binding profiles of hGH-A10 (.box-tangle-solidup.), hGH-A11 (), and Pegvisomant () are shown in FIG. 4C.

[0074] hGH-A7 and hGH-A9 were found to have high binding potency to hGH receptor, as compared with Pegvisomant. Further, hGH-A10 and hGH-A11 were found to have remarkably high binding potency to hGH receptor, as compared with Pegvisomant.

[0075] 3. hGH Competitive Inhibitory Potency Analysis

[0076] Inhibitory potency of hGHRA-NexP on downstream signaling was analyzed by hGH competitive analysis. Serial dilutions of Examples 1 to 11 and Pegvisomant were added to each well of a 96-well, plate containing hGHR/Luc/HEK293F, respectively. This plate was incubated in a 5% CO.sub.2 incubator at 37 C. for 24 hours. After incubation, 100 L of luciferase assay reagent (Steady-Glo luciferase assay system, Promega) was added to each well, and the plate was covered and protected from light. After 5 minutes at room temperature, a multi-mode microplate reader (SpectraMax M5, Molecular Devices) was used to analyze luminescence from wells.

[0077] The results of measuring binding profiles of hGH-A7 (), hGH-A9 (), and Pegvisomant () are shown in FIG. 4B. Further, the results of measuring binding profiles of hGH-A10 (.box-tangle-solidup.), hGH-A11 (), and Pegvisomant () are shown in FIG. 4D.

[0078] hGH-A7 and hGH-A9 were found to have high hCH competitive inhibitory potency, as compared with Pegvisomant. Further, hGH-A10 and hGH-A11 were found to have remarkably high hGH competitive inhibitory potency, as compared with Pegvisomant.

[0079] The results of measuring the relative binding potency and hGH competitive inhibitory potency of hGH-A1to hGH-A11 and Comparative Example Pegvisomant are shown in Table 4, below. These results were plotted in FIG. 5.

TABLE-US-00004 TABLE 4 Test sample Relative binding potency Relative inhibitory potency Pegvisomant 1.000 1.000 hGH-A1 0.226 0.074 hGH-A2 0.234 0.069 hGH-A3 0.925 0.957 hGH-A4 2.305 1.222 hGH-A5 0.767 0.769 hGH-A6 3.373 1.424 hGH-A7 4.733 1.841 hGH-A8 4.642 1.418 hGH-A9 4.579 1.887 hGH-A10 6.085 17.70 hGH-A11 9.949 17.34

[0080] As shown in Table 4, hGH-A4, hGH-A6, hGH-A7, hGH-A8, hGH-A9, hGH-A10, and hGH-A11 showed high hGH receptor binding potency and competitive inhibitory potency, as compared with the known Pegvisomant. In particular, hGH-A10 and hGH-A11, each prepared by fusing NexP to the N-terminus of the variant, showed remarkably high competitive inhibitory potency.

[0081] Based on the above description, it will be understood by those skilled in the art that the present invention may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the disclosure is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.