Novel growth hormone receptor antagonists and fusion proteins thereof
阅读说明：本技术 新型生长激素受体拮抗剂及其融合蛋白 (Novel growth hormone receptor antagonists and fusion proteins thereof ) 是由 朴淳宰 高在亨 柳善儿 尹相勋 于 2018-12-20 设计创作，主要内容包括：本发明涉及一种生长激素受体拮抗剂,其包含通过生长激素的一个或多个氨基酸的取代来修饰的生长激素变体。此外,本发明的生长激素受体拮抗剂还可包括与生长激素变体融合的长效载体。生长激素受体拮抗剂可以具有与生长激素受体的强结合效力,并可以表现出长效拮抗作用。(The present invention relates to a growth hormone receptor antagonist comprising a growth hormone variant modified by substitution of one or more amino acids of the growth hormone. In addition, the growth hormone receptor antagonists of the present invention may also include a long acting carrier fused to the growth hormone variant. The growth hormone receptor antagonist may have a strong binding potency to a growth hormone receptor and may exhibit a long-lasting antagonistic effect.)
1. A growth hormone receptor antagonist comprising a growth hormone variant wherein one or more amino acids in the 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 a substitution of the 46 th amino acid from the N-terminus with lysine, a substitution of the 120 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 a substitution at any one or more positions selected from the group consisting of 18 th amino acid, 21 st amino acid, 54 th amino acid, 64 th amino acid, 167 th amino acid, 168 th amino acid, 171 th amino acid, 172 th amino acid, 174 th amino acid, 176 th amino acid and 179 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 a substitution of the 174 th amino acid from the N-terminus with serine, a substitution of the 21 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 th amino acid from the N-terminus with aspartic acid, substitution of the 21 st amino acid from the N-terminus with aspartic acid, substitution of the 46 th amino acid from the N-terminus with lysine, substitution of the 54 th amino acid from the N-terminus with proline, substitution of the 64 th amino acid from the N-terminus with lysine, substitution of the 120 th amino acid from the N-terminus with lysine or arginine, substitution of the 167 th amino acid from the N-terminus with asparagine, the 168 th amino acid from the N-terminus is substituted with alanine, the 171 th amino acid from the N-terminus is substituted with serine, the 172 th amino acid from the N-terminus is substituted with arginine, the 174 th amino acid from the N-terminus is substituted with serine, the 176 th amino acid from the N-terminus is substituted with tyrosine, and the 179 th amino acid from the N-terminus is substituted with threonine.
6. The growth hormone receptor antagonist of any one of claims 1 to 5, 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 C-terminus of the growth hormone variant.
8. The growth hormone receptor antagonist of claim 6 or 7, wherein the long acting carrier is selected from the group consisting of polyethylene glycol, fatty acids, albumin or a fragment thereof, albumin binding substances, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or a fragment thereof, polymers of specific amino acid sequence repeats, antibodies or fragments thereof, FcRn binding substances, connective tissue or derivatives thereof in vivo, nucleotides, fibronectin, transferrin, sugars and polymers.
9. The growth hormone receptor antagonist of claim 7, wherein the long acting carrier is alpha-1 antitrypsin or a variant thereof.
10. The growth hormone receptor antagonist of claim 9, wherein the alpha-1 antitrypsin variant comprises a substitution of one or more amino acids with another amino acid, and the substitution comprises a substitution at one or more of amino acid 1 and amino acid 25 from the N-terminus.
11. The growth hormone receptor antagonist of claim 9 or 10, wherein the alpha-1 antitrypsin variant comprises any one or more substitutions selected from the group consisting of: the 9 th amino acid from the N-terminus was substituted with asparagine, the 232 th amino acid from the N-terminus was substituted with serine, the 37 th amino acid from the N-terminus was substituted with asparagine, and the 359 th amino acid from the N-terminus was substituted with threonine.
12. The growth hormone receptor antagonist of any one of claims 6 to 11, wherein the growth hormone variant is fused to the long acting carrier directly or through a connector.
13. A growth hormone receptor antagonist according to claim 12, wherein the connector is a peptidyl or non-peptidyl connector.
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, a polyoxyethylated polyol, polyvinyl alcohol, a polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), a lipopolymer, chitin, hyaluronic acid, and combinations thereof.
15. A growth hormone receptor antagonist according to claim 13, wherein the peptidyl connector comprises two or more amino acids.
16. A pharmaceutical composition for preventing or treating diseases caused by human growth hormone, comprising the growth hormone receptor antagonist according to any one of claims 1 to 15.
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 making a growth hormone receptor antagonist, the method comprising the step of culturing cells comprising a polynucleotide encoding the growth hormone variant, wherein one or more amino acids in the amino acid sequence of growth hormone are substituted with another amino acid.
1. Field of the invention
The present invention relates to a growth hormone receptor antagonist comprising a growth hormone variant wherein one or more amino acids in the amino acid sequence of the growth hormone is substituted with another amino acid.
Background
Disclosure of Invention
It is an object of the present invention to provide a growth hormone receptor antagonist comprising a growth hormone variant wherein one or more amino acids in the amino acid sequence of the growth hormone is substituted with another amino acid.
It is another object of the present invention to provide a pharmaceutical composition for preventing or treating diseases caused by human growth hormone, which comprises a growth hormone receptor antagonist.
It is a further object of the present invention to provide a method for preparing growth hormone receptor antagonists.
Drawings
FIG. 1 shows the structure of a complex of growth hormone with growth hormone receptor (PDB id:3 HHR);
FIG. 2 shows the efficacy of hGH on hGH, hGH-A1 and hGH-A2;
FIG. 3 is a chromatographic purification of the growth hormone receptor antagonist hGHR-A3 of the present invention, wherein FIG. 3A is a two-step ion exchange column chromatography of purified hGHR-A3, FIG. 3B is the results of 10% SDS-polyacrylamide gel electrophoresis of each purified fraction, and FIG. 3C shows the purity of purified hGHR-A3, as analyzed by size exclusion HPLC;
FIG. 4 shows the results of hGH receptor binding assay and hGH competition inhibition assay of hGH-A7, hGH-A9, hGH-A10, hGH-A11 and pegvisomant;
FIG. 5 is a graph showing the relative binding potency and competition efficacy results for hGH-A1 with hGH-A11 and pegvisomant in comparative examples; and
FIG. 6 shows a schematic diagram illustrating the process of preparation of the growth hormone receptor of the present invention.
Best mode for carrying out the invention
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 examples: preparation of growth hormone receptor antagonists
1. Cloning, transfection and cell culture
cDNA clones of hGH-NexP were prepared according to known methods. Preparation of hGH receptor antagonist (hGH RA-Ne) by site-directed mutagenesis of hGH-NexP GenexP). Subsequently, DNA sequencing confirmed the mutation. CHO (Chinese hamster ovary) -K1 cells were transiently transfected with a plasmid containing the nucleotide sequence of each hGHRA-NexP clone. Transfected cells at 5% CO2The cultures were grown in a humidified incubator for 7 days in IMDM Medium (Iscove's Modified Dulbecco's Medium) supplemented with 10% FBS.
Purification of hGHRA-NexP
The hGHRA-NexP variant was purified from transiently transfected CHO-K1 cells by a series of column chromatography. The culture supernatant was diluted with an equal volume of buffer A (20mM sodium phosphate, pH 8.0) and applied to an ion exchange column equilibrated with buffer A. After the washing step with buffer A, the proteins were eluted with a linear gradient of NaCl in buffer A. The hGHRA-NexP containing fractions were then loaded directly onto an affinity column equilibrated with buffer B (20mM Tris-HCl, 100mM NaCl, pH 7.5) and with MgCl in buffer B2The fraction was eluted with a gradient. After adjusting the pH and conductivity of the elution pool, it was loaded onto a second ion exchange column, which was equilibrated with buffer C (20mM sodium phosphate, 80mM NaCl, pH 8.0). The protein fraction was concentrated using a
SE-HPLC analysis
To determine the purity of hGHRA-NexP, size exclusion high performance liquid chromatography (SE-HPLC) was used. The protein solution was loaded onto a TSKgel G3000SWXL column (Tosoh) and chromatograms were obtained in flow buffer (50mM sodium phosphate, 150mM sodium chloride, 0.05% sodium azide, pH6.8) at a flow rate of 0.5 mL/min. The area percentage (%) of the main peak of the chromatogram was calculated.
Detailed Description
Hereinafter, the present invention will be described in more detail.
Also, each of the descriptions and embodiments disclosed herein may be applied to the other descriptions and embodiments as well. That is, all combinations of the various elements disclosed herein are within the scope of the invention. Furthermore, the scope of the present invention is not limited by the specific description set forth below.
Further, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalent embodiments should be construed as falling within the scope of the present invention.
In order to achieve the above objects, one aspect of the present invention provides a growth hormone receptor antagonist comprising a growth hormone variant in which one or more amino acids in the amino acid sequence of growth hormone are substituted with another amino acid.
As used herein, the term "growth hormone" refers to a peptide hormone secreted by the pituitary gland, which has a metabolic regulation function in addition to a function of stimulating the growth of the body. The growth hormone may be in particular human growth hormone (hGH), and it is well known that hGH consists of 191 amino acids.
As used herein, the term "growth hormone variant" refers to the substitution of one or more amino acids in the amino acid sequence of growth hormone with another amino acid. In other words, it refers to growth hormone with one or more amino acid substitutions.
Specifically, the substitution may include a substitution of the 120 th amino acid in the amino acid sequence of growth hormone (more specifically, a substitution with lysine or arginine). In addition, the substitution may include a substitution of the 46 th amino acid (more specifically, a substitution with lysine).
In addition, the substitution may include a substitution of 174 th amino acid (more specifically, a substitution with serine) and a substitution of 21 st amino acid (more specifically, a substitution with asparagine) in the amino acid sequence of growth hormone.
Specifically, the substitution may include substitution at one or more positions selected from the group consisting of 18 th amino acid, 21 st amino acid, 46 th amino acid, 54 th amino acid, 64 th amino acid, 120 th amino acid, 167 th amino acid, 168 th amino acid, 171 st amino acid, 172 th amino acid, 174 th amino acid, 176 th amino acid, and 179 th amino acid in the amino acid sequence.
More specifically, the substitution may include one or more substitutions selected from the group consisting of H18D, H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K17R, E174S, F176Y, and I179T in the amino acid sequence of growth hormone.
Variants may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, if desired.
For non-limiting example in the present invention, the growth hormone variant may be a protein having SEQ id No. as shown in table 1 below. The growth hormone variants of
[ Table 1]
As used herein, the term "Growth Hormone Receptor (GHR)" refers to a receptor to which growth hormone binds to transmit a signal to a cell. Growth hormone receptors have a structure that penetrates the cell membrane once. STAT dimers regulate transcription of various genes in the nucleus through the JAK/STAT pathway when the receptor is activated. Growth hormone receptors are present in tissues throughout the body, including liver, muscle, fat, kidney, early embryo, fetal tissue, and the like.
When growth hormone binds to the receptor, subsequent signaling leads to increased secretion of IGF (insulin-like growth factor) -1. hGH hypersecretion and the resulting rise in IGF-1 can cause acromegaly, a chronic disease with hand and foot swelling as a typical symptom.
As used herein, the term "growth hormone receptor antagonist" refers to an agent that antagonizes the binding of growth hormone to growth hormone receptors, thereby inhibiting the side effects caused by the excessive binding of growth hormone to growth hormone receptors.
In particular, the growth hormone receptor antagonist may be a growth hormone variant having high binding potency to growth hormone receptors and being capable of competitively antagonizing growth hormone efficacy.
In addition, the growth hormone receptor antagonist may comprise a long acting carrier fused to a growth hormone variant.
As used herein, the term "long acting carrier" refers to a substance capable of increasing half-life in vivo. When various long-acting carriers known to increase half-life in vivo are fused with the growth hormone variants according to the invention, it is expected that they will be used as long-acting agents with increased half-life in vivo when antagonizing growth hormone receptors.
In the present invention, non-limiting examples of the long-acting carrier may include various carriers capable of reducing renal clearance, specifically, any one or more selected from the group consisting of polyethylene glycol, fatty acid, albumin or a fragment thereof, albumin binding substance, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or a fragment thereof, polymer of a specific amino acid sequence repeat unit, antibody or a fragment thereof, FcRn binding substance, in vivo connective tissue or a derivative thereof, nucleotide, fibronectin, transferrin, sugar, and polymer.
More specifically, alpha-1 antitrypsin (A1AT) or variants thereof may be used as long acting carriers.
Alpha-1 antitrypsin or variants thereof are disclosed in the patent publications KR10-2013-0136883A and KR 10-2013-0029713A. Specifically, A1AT is one of the most abundant proteins in human plasma, and its concentration is 1.5-3.5 g per liter, and it is mainly synthesized in hepatocytes and secreted into blood. The alpha-1 antitrypsin variant was designed by adding an additional mutation in A1AT to increase glycosylation and eliminate its intrinsic activity. The alpha-1 antitrypsin variant may be fused to the target protein to extend the half-life of the target protein.
One of the major advantages of the alpha-1 antitrypsin variant technology is non-immunogenicity. In particular, human A1AT, a source of plasma, has been used as a drug for treating patients with emphysema, a lung disease caused by A1AT deficiency. Its weekly dose is up to 60 mg/kg body weight. Serious side effects have not been reported, indicating the safety of A1AT as a therapeutic agent.
In the present invention, a non-limiting example of a growth hormone receptor antagonist can provide a long-acting hGHRA obtained by fusing an alpha-1 antitrypsin variant to a growth hormone variant. The alpha-1 antitrypsin variant may comprise a substitution of one or more of
More specifically, the alpha-1 antitrypsin variant may have, for example, the amino acid sequence of
[ Table 2]
A growth hormone receptor antagonist obtained by fusing an alpha-1 antitrypsin variant with a growth hormone variant to increase the plasma half-life may have a high binding potency to the growth hormone receptor and may strongly antagonize the efficacy of growth hormone, as compared to a pegylated growth hormone variant.
In particular, examples 4, 6, 7, 8, 9, 10 and 11, which are non-limiting examples of growth hormone receptor antagonists of the present invention, show high binding ability to hGH receptor and high inhibitory potency compared to the known growth hormone receptor antagonist pegvisomant (see fig. 4 and 5, table 4).
In the growth hormone receptor antagonist obtained by fusing the long-acting carrier to the growth hormone variant, the long-acting carrier may be fused to the N-terminus or C-terminus of the growth hormone variant.
In particular, a growth hormone receptor antagonist obtained by fusing a long-acting carrier to the N-terminus of a specific growth hormone variant may have a remarkably high binding potency to a growth hormone receptor or may strongly antagonize the efficacy of growth hormone, as compared to a growth hormone receptor antagonist obtained by fusing a long-acting carrier to the C-terminus thereof. For the non-limiting example of the present invention, even though hGH-a10 and hGH-a11 have growth hormone variants with the same amino acid sequence as hGH-a7 and hGH-a9, respectively, those fused to the N-terminus of the variants were demonstrated to strongly antagonize the efficacy of growth hormone (see fig. 4C and 4D, fig. 5 and table 4).
In the present invention, the long-acting carrier may be fused with the long-acting carrier directly or through a connector.
Any connector can be used without limitation as long as it is used in a covalent bond between a long-acting carrier and a growth hormone variant and does not affect activity. Specifically, the connector may be a non-peptidyl connector of 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), lipopolymer, chitin, hyaluronic acid, and a combination thereof, and may be a peptidyl connector in which two or more amino acids are connected. Non-limiting examples may include GGGGS and connectors with variable lengths (2X, 3X, 4X, etc.).
In order to accomplish the above objects, another aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases caused by human growth hormone, the pharmaceutical composition comprising a growth hormone receptor antagonist. The terminology used herein is the same as described above.
As used herein, the term "diseases caused by human growth hormone" refers to diseases caused by hypersecretion of growth hormone due to abnormal control of pituitary gland and the like. For example, the disease may include 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, Chronic Renal Insufficiency (CRI), and the like.
In addition, diseases caused by human growth hormone include diseases caused by increased secretion of IGF (insulin-like growth factor) -1 due to excessive action of growth hormone.
For non-limiting illustration in the present invention, examples (hGH-A1 through hGH-A11) inhibit the binding of growth hormone to its receptor, and thus are useful for preventing or treating diseases caused by human growth hormone (see experimental examples and Table 4).
In order to accomplish the above object, another aspect of the present invention provides a method for preparing a growth hormone receptor antagonist, comprising the step of culturing a cell comprising a polynucleotide encoding a growth hormone variant in which one or more amino acids in the amino acid sequence of growth hormone are substituted with another amino acid. The terminology used herein is the same as described above.
These cells may be cells transfected with an expression vector for a growth hormone variant, non-limiting examples of which may include CHO (Chinese hamster ovary) -K1 and the like.
In order to achieve the above objects, another aspect of the present invention provides a method for preventing or treating a disease caused by human growth hormone, the method comprising the step of administering to a subject a pharmaceutical composition comprising a growth hormone receptor antagonist. The terminology used herein is the same as described above.
As used herein, the term "administering" refers to introducing a pharmaceutical composition of the present invention into a patient by any suitable method, and administration of the composition of the present invention can be carried out by various routes such as oral or parenteral routes as long as the composition of the present invention can reach a desired tissue. The agent according to the present invention can be prepared into various formulations according to the desired administration mode.
Administration can be effected prophylactically or therapeutically.
The frequency of administration of the agent of the present invention is not particularly limited, but may be administered once a day or in divided doses.
The subject to be administered with the agent according to the present invention may refer to all animals including humans. Such animals may include mammals such as, but not limited to, cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. that are in need of treatment for similar symptoms as humans.
In order to achieve the above objects, another aspect of the present invention provides a use of a pharmaceutical composition comprising a growth hormone receptor antagonist for preventing or treating diseases caused by human growth hormone. The terminology used herein is the same as described above.
Technical effects of the invention
The growth hormone receptor antagonist according to the present invention may have a strong binding potency to a growth hormone receptor and may exhibit a long-lasting antagonistic effect.
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