WO2019125002A1 - 신규한 성장 호르몬 수용체 길항제 및 이의 융합 단백질 - Google Patents
신규한 성장 호르몬 수용체 길항제 및 이의 융합 단백질 Download PDFInfo
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- WO2019125002A1 WO2019125002A1 PCT/KR2018/016311 KR2018016311W WO2019125002A1 WO 2019125002 A1 WO2019125002 A1 WO 2019125002A1 KR 2018016311 W KR2018016311 W KR 2018016311W WO 2019125002 A1 WO2019125002 A1 WO 2019125002A1
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- amino acid
- growth hormone
- substitution
- receptor antagonist
- hgh
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- 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
- A61K47/62—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 a protein, peptide or polyamino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
Definitions
- the present invention relates to a growth hormone receptor antagonist comprising a growth hormone variant in which at least one amino acid in the amino acid sequence of a growth hormone is replaced by another amino acid.
- Growth hormone is an endocrine hormone that promotes growth.
- Human growth hormone (hGH) is secreted from the pituitary gland and acts on various tissues including the liver. It binds with the extracellular domain of the human growth hormone receptor (hGHR) belonging to the class I cytokine receptor superfamily that forms dimers in complex with hGH (JF Bazan, Haemopoietic receptors and helical cytokines, Immunol. ) 350-354.), Subsequent signal transduction increases the expression of insulin-like growth factor I (IGF-1) (FF Casanueva, Physiology of growth hormone secretion and action, Endocrinol. Metab. ) 483-517 .; H.
- IGF-1 insulin-like growth factor I
- hGHRA hGH receptor antagonist
- HGHRA which can last a long time in the human body, can improve the quality of life of patients by reducing the number of doses.
- Pegvisomant is commercially available and commercially available as a pegylated version of hGH antagonist (MO Thorner, CJ Strasburger, Z. Wu, M. Straume, M. Bidlingmaier, SS Pezzoli, K. Zib, JC Scarlett, WF Bennett, Growth hormone GH) receptor blockade with a PEG-modified GH (B2036-PEG) lowers serum insulin-like growth factor-I but does not acutely stimulate serum GH, J. Clin. Endocrinol Metab 84 (1999) 2098-2103.
- Pegvisomant is to increase the molecular size by attaching polyethylene glycol polymer to the therapeutic protein so that it remains in the bloodstream without being filtered in the kidney (RJ Ross, KC Leung, M. Maamra, W.
- pegvisomant Another aspect of pegylation is to protect the protein from proteolytic enzymes (S. Jevsevar, M. Kunststoffelj, VG Porekar, PEGylation of therapeutic proteins, Biotechnol. J. 5 (2010) 113-128. Doi: 10.1002 / biot .200900218.).
- pegylation of therapeutic proteins may be limited in its usefulness.
- the pegylation process requires a series of chemical reactions, which are not cost effective and the reaction products are generally not homogeneous. As a result, additional purification steps, which are generally difficult to achieve, are required.
- Pegylated proteins also appear to be safe when using small size polyethylene glycols (eg, 5 kDa), but animal studies have shown that renal vacuolation as well as the appearance of antibodies to pegylation As you can see there is still a safety issue.
- the pegylated protein tends to have a lower binding capacity to protein receptors than the original protein (RP Garay, R. El-Gewely, JK Armstrong, G. Garratty, P.
- the present inventors have designed a novel growth hormone receptor antagonist hGHRA which exhibits a long-lasting and potent inhibitory activity on growth hormone receptors, and characterized the same in vitro, thereby completing the present invention.
- One object of the present invention is to provide a growth hormone receptor antagonist comprising a growth hormone variant in which at least one amino acid in the amino acid sequence of the growth hormone is replaced by another amino acid.
- the present invention provides a growth hormone receptor antagonist comprising a growth hormone mutant in which at least one amino acid is substituted with another amino acid in the amino acid sequence of the growth hormone.
- 'growth hormone' refers to a peptide hormone that promotes body growth secreted by the pituitary gland, which has other metabolic control functions besides body growth.
- Growth hormone may be specifically human growth hormone (hGH), and hGH is made up of 191 amino acids as is well known.
- " growth hormone variant "
- growth hormone variant " means that at least one amino acid is replaced by another amino acid in the amino acid sequence of a growth hormone. That is, a growth hormone having one or more amino acid substitutions.
- the substitution may include substitution of the 120th amino acid in the amino acid sequence of the growth hormone (more specifically, lysine or arginine). It may also include substitution of the 46th amino acid (more specifically, substituted with lysine).
- substitution may include substitution of the 174th amino acid in the amino acid sequence of the growth hormone (more specifically, substitution with serine), and may include substitution of the 21st amino acid (more specifically, substitution with asparagine) have.
- the substitution includes substitution of at least one amino acid selected from the group consisting of 18th amino acid, 21st amino acid, 46th amino acid, 54th amino acid, 64th amino acid, 120th amino acid, 167th amino acid, 168th amino acid, 171th amino acid, 172th amino acid, 174 Th amino acid, the 176th amino acid, and the 179 th amino acid.
- the substitution may include any one or more substitutions selected from the amino acid sequence of growth hormone, H18D, H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K172R, E174S, F176Y, have.
- the mutant may be selected from the group consisting of phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation. And the like.
- the growth hormone mutant may be a protein having the sequence numbers as shown in Table 1 below.
- the growth hormone mutants of SEQ ID NOS: 1 to 9 are the same as the sequences of the growth hormone mutants contained in Examples 1 to 9 described later.
- Examples 10 and 11 include growth hormone mutants of SEQ ID NO: 7 and SEQ ID NO: 9, respectively.
- GHR Growth Hormone Receptor
- GHR Growth Hormone Receptor
- IGF insulin like growth factor
- " growth hormone receptor antagonist " refers to an agent that antagonizes the binding of growth hormone to growth hormone receptors and inhibits the adverse effects of growth hormone receptors on growth hormone binding do.
- the growth hormone receptor antagonist may be a growth hormone variant that has high binding affinity to the growth hormone receptor and can competitively antagonize the efficacy of the growth hormone.
- the growth hormone receptor antagonist may comprise a sustained carrier that is fused to a growth hormone variant.
- " sustained carrier " as used herein means a substance capable of increasing in vivo half-life. Fusing a persistent carrier known to be capable of increasing various in vivo half lives of the growth hormone mutant according to the present invention can be used as a sustained preparation having an increased in vivo half life while antagonizing growth hormone receptors It is expected.
- sustained carriers which are not limited in this context include various carriers capable of reducing renal clearance, in particular polyethylene glycol, fatty acids, albumin or a fragment thereof, albumin-binding substances, alpha-1 antitrypsin or variants thereof , Immunoglobulin Fc or a fragment thereof, a repeat unit polymer of a specific amino acid sequence, an antibody or fragment thereof, an FcRn binding substance, an in vivo connective tissue or a derivative thereof, a nucleotide, a fibronectin, a transferrin, a saccharide, May be used.
- carriers capable of reducing renal clearance in particular polyethylene glycol, fatty acids, albumin or a fragment thereof, albumin-binding substances, alpha-1 antitrypsin or variants thereof , Immunoglobulin Fc or a fragment thereof, a repeat unit polymer of a specific amino acid sequence, an antibody or fragment thereof, an FcRn binding substance, an in vivo connective tissue or a derivative thereof, a nucleo
- alpha-1 antitrypsin or variants thereof may be used as a sustained carrier.
- Alpha-1 antitrypsin or its variants are known from the prior published patents KR 10-2013-0136883 A and KR 10-2013-0029713 A. Specifically, A1AT is one of the most abundant proteins with a concentration of 1.5-3.5 grams per liter in human plasma, mainly synthesized in hepatocytes and secreted into the blood. Alpha-1 antitrypsin variants are designed to have additional mutations in A1AT to increase glycosylation and eliminate its intrinsic activity. Alpha-1 antitrypsin variants can be fused to the target protein to extend the half-life of the target protein.
- A1AT in human plasma has already been used as a treatment for patients with A1AT deficiency due to emphysema and pulmonary disease, and the weekly dose is very high at 60 mg per kg body weight. Serious side effects have not been reported yet and indicate the safety of A1AT as a treatment.
- alpha-1 antitrypsin mutants are fused to growth hormone variants to provide long lasting hGHRA.
- the alpha-1 antitrypsin mutant may comprise at least one substitution of at least one of the amino acids at positions 1 to 25 in the sequence of alpha-1 antitrypsin, wherein the growth hormone mutant is selected from the group consisting of H18D, One or more substitutions selected from H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K172R, E174S, F176Y and I179T.
- the alpha-1 antitrypsin mutant may have the amino acid sequence of SEQ ID NO: 10 in Table 2 below.
- growth hormone receptor antagonists in which alpha-1 antitrypsin mutants are fused to growth hormone mutants, have a higher binding affinity to growth hormone receptors as compared to pegylated growth hormone mutants, It can be strongly antagonistic.
- the sustained carrier may be one fused to the N-terminal or C-terminal end of the growth hormone mutant.
- a growth hormone receptor antagonist fused with a sustained carrier at its N-terminus is fused with the C-terminal, and thus the binding strength to the growth hormone receptor is remarkably high or the potency of the growth hormone can be strongly antagonized have.
- hGH-A10 and hGH-A11 as compared to hGH-A7 and hGH-A9 have growth hormone variants of the same amino acid sequence, but the fusion of the N- (See C and D in Fig. 4, Fig. 5, and Table 4).
- the persistent carrier herein may be fused directly with the persistent carrier, or may be fused via a linker.
- the linker may be used without limitation as long as it is used for the covalent bond between the sustained carrier and the growth hormone mutant and does not affect the activity.
- a non-limiting example is GGGGS, which may be a linker with varying lengths (2X, 3X, 4X, etc.).
- the present invention provides a pharmaceutical composition for preventing or treating a disease caused by human growth hormone comprising a growth hormone receptor antagonist.
- a pharmaceutical composition for preventing or treating a disease caused by human growth hormone comprising a growth hormone receptor antagonist.
- the terms used herein are as described above.
- " a disease caused by human growth hormone " refers to a disease caused by a cause such as the pituitary gland failing to regulate growth hormone secretion and over secretion. Examples include, but are not limited to, hypertrophy, hyperactivity, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, GHD, idiopathic short stature, Turner's syndrome, Prader-Willi syndrome, small for gestational age ), And chronic renal failure (CRI).
- CRI chronic renal failure
- the diseases caused by human growth hormone include diseases caused by an increase in the secretion of IGF (insulin like growth factor) -1 due to excessive growth hormone action.
- the Examples inhibit the binding of growth hormone and its receptor, thus confirming that it can be used for the prevention or treatment of diseases caused by human growth hormone (See Experimental Examples and Table 4).
- a method for producing a cell comprising a polynucleotide encoding a growth hormone mutant in which at least one amino acid is substituted with another amino acid in the amino acid sequence of a growth hormone, Wherein the growth hormone receptor antagonist is a growth hormone receptor antagonist.
- the terms used herein are as described above.
- the cell may be a cell in which an expression vector for a growth hormone mutant has been transfected.
- CHO Choinese hamster ovary
- K1 may be used.
- the present invention provides a method of treating a disease caused by human growth hormone, comprising the step of administering to a subject a pharmaceutical composition comprising the growth hormone receptor antagonist Prevention or treatment.
- a method of treating a disease caused by human growth hormone comprising the step of administering to a subject a pharmaceutical composition comprising the growth hormone receptor antagonist Prevention or treatment.
- the terms used herein are as described above.
- administering means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the route of administration of the composition of the present invention is not limited to a variety of oral or parenteral routes Lt; / RTI >
- the preparation according to the present invention can be manufactured into various formulations depending on the intended administration mode.
- Administration can be effected prophylactically or therapeutically.
- the administration frequency of the agent of the present invention is not particularly limited, but it may be administered once a day or divided into several doses.
- the subject to which the preparation according to the present invention is administered may mean all animals including humans.
- the animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,
- the present invention provides a pharmaceutical composition comprising the above-described growth hormone receptor antagonist for use in the prevention or treatment of diseases caused by human growth hormone.
- the terms used herein are as described above.
- the growth hormone receptor inhibitor according to the present invention has strong binding force to the growth hormone receptor and can exhibit antagonistic action continuously.
- FIG. 1 shows the structure of a complex in which growth hormone and growth hormone receptor are bound (PDB id: 3HHR).
- Figure 2 shows hGH efficacy against hGH, hGH-A1 and hGH-A2.
- FIG. 3 is a chart showing purification by chromatography of hGHR-A3 as a growth hormone receptor antagonist of the present invention.
- FIG. 3A shows the chromatogram of the second ion exchange column chromatography for the purification of hGHR-A3, and
- FIG. 3B shows the results of 10% SDS-polyacrylamide gel electrophoresis of each purified fraction.
- Figure 3C shows the purity of purified hGHR-A3 as analyzed by size exclusion HPLC.
- Figure 4 shows the results of hGH receptor binding assay and hGH competitive inhibition assay for hGH-A7, hGH-A9, hGH-A10, and hGH-A11 and pegvisomant.
- FIG. 5 is a plot of the relative binding capacity of hGH-A1 to hGH-A11 and the comparative example pegvisomant and the results on the competitive potency of hGH.
- FIG. 6 is a diagram illustrating an example of a process for producing the growth hormone receptor of the present invention.
- cDNA clones of hGH-NexP were prepared according to known methods.
- the hGH receptor antagonist (hGHRA-NexP) was produced by site-directed mutagenesis of the gene of hGH-NexP. Mutations were subsequently confirmed by DNA sequencing.
- CHO (Chinese hamster ovary) -K1 cells were transiently transfected with a plasmid containing the nucleotide sequence of each hGHRA-NexP clone. Transformed cells were grown in IMDM medium (Isocove's Modified Dulbecco's Medium) supplemented with 10% FBS for 7 days in a 5% CO 2 humidified incubator.
- IMDM medium Isocove's Modified Dulbecco's Medium
- the mutant of hGHRA-NexP was purified through a series of column chromatography from transiently transformed CHO-K1 cells.
- the culture supernatant was diluted with the same volume of buffer A (20 mM sodium phosphate, pH 8.0) and applied to an ion-exchange column equilibrated with buffer A. After washing with Buffer A, protein was eluted with a linear gradient of NaCl in Buffer A.
- the fraction containing hGHRA-NexP was then loaded directly onto the affinity column equilibrated with buffer B (20 mM Tris-HCl, 100 mM NaCl, pH 7.5). In buffer B the fraction eluted with MgCl 2 gradient.
- SE-HPLC size exclusion high performance liquid chromatography
- the purpose of this application is to produce inhibitors of growth hormone receptor antagonists using the NexP technology to induce persistence by using site specific mutagenesis for the hGH sequence, (hGH-A1: G120K) and Example 2 (hGH-A2: G120R) were prepared. Further, for the above purpose, Examples 3 to 7 were prepared by introducing an additional mutation to site 1 of hGH-A1 in the hGH sequence. Specifically, substituted amino acids in the examples are shown in Table 3 below. Example 9 introduced the Q46K mutation to induce cation-pi interactions through the introduction of lysine.
- 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 and 10 hGH-A7, hGH-A10 H21N / F54P / R64K / G120K / R167N / D171S / E174S / F176Y
- Example 8 hGH-A8 H18D / H21N / G120K / R167N / K168A / D171S / K172R / E174S
- hGH-A1 to hGH-A9 are obtained by fusion of NexP with the C-terminus of the mutant, and hGH-A10 and hGH-A11 are fused with N-terminal of the mutant.
- the hGHRA-NexP protein was prepared by the transformation of CHO-K1 cells and a series of column chromatographies according to the preparation example described above. Typically, the chromatogram of the second ion exchange column chromatography is shown in Figure 3A. Pure protein was obtained from chromatography, and the purified protein migrated to a position between 100 kDa and 70 kDa (Fig. 3B). After collecting fractions of fractions 7-14, the solution was dialyzed against phosphate buffered saline (PBS). The purity of the protein was found to show a high purity of 95% as seen in the main peak of the SE-HPLC chromatogram (Fig. 3C).
- PBS phosphate buffered saline
- the hGH receptor gene was introduced into the chromosome of HEK293F cells containing the luciferase gene, which could be induced by the hGH receptor signal.
- the resulting cell line was named hGHR / Luc / HEK293F.
- a series of dilutions of hGH and hGHRA-NexP were added to each of the 96-well white plates containing hGHR / Luc / HEK293F. Plates were incubated in a 5% CO 2 incubator at 37 ° C for 24 hours.
- luciferase assay reagent 100 ⁇ L (Steady-Glo ® luciferase assay system, Promega) was added to the each well, the plate was immersed was protected from light. After 5 minutes at room temperature, the luminescence from the wells was analyzed using a multimode microplate reader (SpectraMax M5, Molecular Devices).
- the binding affinity of hGRA-NexP to the hGH receptor was assessed by binding assays using the recombinant hGH receptor Fc chimera.
- TMB 5,5'-tetramethylbenzidine
- the binding profiles of hGH-A7 (), hGH-A9 () and Pegvisomant () were measured and are shown in FIG.
- the results of measurement of hGH-A10 (), hGH-A11 (DELTA) and Pegvisomant () are shown in FIG.
- hGH-A7 and hGH-A9 had higher hGH receptor binding capacity than pegvisomant.
- hGH-A10 and hGH-A11 were found to have significantly higher hGH receptor binding potency than pegvisomant.
- hGH competition assay To each well of a 96-well plate containing hGHR / Luc / HEK293F a series of dilutions of Examples 1-11 and Pegvisomant were added. Plates were incubated in a 5% CO 2 incubator at 37 ° C for 24 hours. After incubation, luciferase assay reagent of 100 ⁇ L (Steady-Glo ® luciferase assay system, Promega) was added to the each well, the plate was immersed was protected from light. After 5 minutes at room temperature, the luminescence from the wells was analyzed using a multimode microplate reader (SpectraMax M5, Molecular Devices).
- the binding profiles were measured for hGH-A7 ( ⁇ ), hGH-A9 ( ⁇ ) and Pegvisomant ( ⁇ ) and are shown in Figure 4 (B). The measurement was also performed for hGH-A10 (), hGH-A11 (), and Pegvisomant (), and is shown in FIG. 4 (D).
- hGH-A7 and hGH-A9 had higher hGH competitive inhibitory activity than pegvisomant.
- hGH-A10 and hGH-A11 had significantly higher hGH competitive inhibitory activity than pegvisomant.
- hGH-A4, hGH-A6, hGH-A7, hGH-A8, hGH-A9, hGH-A10 and hGH-A11 are highly competitive with hGH receptor binding ability as compared with conventionally known Pegvisomant
- hGH-A10 and hGH-A11, in which NexP was fused with the N-terminus of the mutant exhibited remarkably high competitive inhibitory activity.
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Abstract
Description
서열번호 1 | FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF |
서열번호 2 | FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF |
서열번호 3 | FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTFLRIVQCRSVEGSCGF |
서열번호 4 | FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTFLRTVQCRSVEGSCGF |
서열번호 5 | FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTYLRIVQCRSVEGSCGF |
서열번호 6 | FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTYLRIVQCRSVEGSCGF |
서열번호 7 | FPTIPLSRLFDNAMLRAHRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCPSESIPTPSNKEETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNKDMSKVSTYLRIVQCRSVEGSCGF |
서열번호 8 | FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF |
서열번호 9 | FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLKNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEKIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVSTFLRTVQCRSVEGSCGF |
서열번호 10 | EDPQGDAANKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLHTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAINMSIPPEVKFNKPFVFLMIDQNTKSPLFMGKVVNPTQK |
Name | Mutations | |
실시예 1 | hGH-A1 | G120K |
실시예 2 | hGH-A2 | G120R |
실시예 3 | hGH-A3 | H21N/G120K/E174S |
실시예 4 | hGH-A4 | H18D/H21N/G120K/E174S/I179T |
실시예 5 | hGH-A5 | H21N/G120K/E174S/F176Y |
실시예 6 | hGH-A6 | H21N/F54P/R64K/G120K/E174S/F176Y |
실시예 7 및 10 | hGH-A7, hGH-A10 | H21N/F54P/R64K/G120K/R167N/D171S/E174S/F176Y |
실시예 8 | hGH-A8 | H18D/H21N/G120K/R167N/K168A/D171S/K172R/E174S/I179T |
실시예 9 및 11 | hGH-A9, hGH-A11 | H18D/H21N/Q46K/G120K/E174S/I179T |
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 |
Claims (18)
- 성장 호르몬(growth hormone)의 아미노산 서열에서 하나 이상의 아미노산이 다른 아미노산으로 치환된 성장 호르몬 변이체(growth hormone variant)를 포함하는, 성장 호르몬 수용체 길항제(growth hormone receptor antagonist).
- 제1항에 있어서, 상기 다른 아미노산으로의 치환은 N-말단으로부터 46번째 아미노산의 라이신으로 치환, 120번째 아미노산의 라이신 또는 아르기닌으로 치환, 또는 이들 치환의 조합을 포함하는 것인, 성장 호르몬 수용체 길항제.
- 제2항에 있어서, 상기 다른 아미노산으로의 치환은 N-말단으로부터 18번째 아미노산, 21번째 아미노산, 54번째 아미노산, 64번째 아미노산, 167번째 아미노산, 168번째 아미노산, 171번째 아미노산, 172번째 아미노산, 174번째 아미노산, 176번째 아미노산, 및 179번째 아미노산으로 이루어진 군에서 선택되는 어느 하나 이상의 위치의 치환을 추가로 포함하는 것인, 성장 호르몬 수용체 길항제.
- 제2항에 있어서, 상기 다른 아미노산으로의 치환은 N-말단으로부터 174번째 아미노산의 세린으로 치환, 21번째 아미노산의 아스파라긴으로 치환, 또는 이들 치환의 조합을 추가로 포함하는 것인, 성장 호르몬 수용체 길항제.
- 제1항에 있어서, 상기 다른 아미노산으로의 치환은 N-말단으로부터 18번째 아미노산의 아스파트산으로 치환, 21번째 아미노산의 아스파라긴으로 치환, 46번째 아미노산의 라이신으로 치환, 54번째 아미노산의 프롤린으로 치환, 64번째 아미노산의 라이신으로 치환, 120번째 아미노산의 라이신 또는 아르기닌으로 치환, 167번째 아미노산의 아스파라긴으로 치환, 168번째 아미노산의 알라닌으로 치환, 171번째 아미노산의 세린으로 치환, 172번째 아미노산의 아르기닌으로 치환, 174번째 아미노산의 세린으로 치환, 176번째 아미노산의 타이로신으로 치환, 및 179번째 아미노산의 트레오닌으로 치환으로 이루어진 군으로부터 선택되는 어느 하나 이상의 치환을 포함하는 것인, 성장 호르몬 수용체 길항제.
- 제1항 내지 제5항 중 어느 한 항에 있어서, 상기 성장 호르몬 변이체는 지속성 담체(long-acting carrier)가 연결된 형태인 것인, 성장 호르몬 수용체 길항제.
- 제6항에 있어서, 상기 지속성 담체는 상기 성장 호르몬 변이체의 N-말단 또는 C-말단에 융합된 것인, 성장 호르몬 수용체 길항제.
- 제6항 또는 제7항에 있어서, 상기 지속성 담체는 폴리에틸렌 글리콜, 지방산, 알부민 또는 이의 절편, 알부민-결합 물질, 알파-1 안티트립신 또는 이의 변이체, 면역글로불린 Fc 또는 이의 절편, 특정 아미노산 서열의 반복단위 중합체, 항체 또는 이의 단편, FcRn 결합물질, 생체 내 결합 조직 또는 이의 유도체, 뉴클레오타이드, 파이브로넥틴, 트랜스페린, 사카라이드, 및 고분자 중합체로 이루어진 군에서 선택되는 것인, 성장 호르몬 수용체 길항제.
- 제7항에 있어서, 상기 지속성 담체는 알파-1 안티트립신 또는 이의 변이체인 것인, 성장 호르몬 수용체 길항제.
- 제9항에 있어서, 상기 알파-1 안티트립신 변이체는 하나 이상의 아미노산이 다른 아미노산으로 치환되어, 상기 치환은 N-말단으로부터 1번째 아미노산 내지 25번째 아미노산 중 적어도 하나 이상의 위치의 치환을 포함하는 것인, 성장 호르몬 수용체 길항제.
- 제9항 또는 제10항에 있어서, 상기 알파-1 안티트립신 변이체는 N-말단으로부터 9번째 아미노산의 아스파라긴으로 치환, 232번째 아미노산의 세린으로 치환, 37번째 아미노산의 아스파라긴으로 치환, 및 359번째 아미노산의 트레오닌으로 치환으로 이루어지는 군으로부터 선택되는 어느 하나 이상의 치환을 포함하는 것인, 성장 호르몬 수용체 길항제.
- 제6항 내지 제11항 중 어느 한 항에 있어서, 상기 성장 호르몬 변이체는 상기 지속성 담체와 직접적으로 또는 링커를 통하여 융합되는 것인, 성장 호르몬 수용체 길항제.
- 제12항에 있어서, 상기 링커는 펩타이드성 링커 또는 비펩타이드성 링커인 것인, 성장 호르몬 수용체 길항제.
- 제13항에 있어서, 상기 비펩타이드성 링커는 폴리에틸렌 글리콜, 폴리프로필렌 글리콜, 에틸렌 글리콜 및 프로필렌 글리콜의 공중합체, 폴리옥시에틸레이티드 폴리올, 폴리비닐 알코올, 폴리사카라이드, 덱스트란, 폴리비닐 에틸 에테르, 폴리락트산(PLA), 폴리락트-글리콜산(PLGA), 지질 폴리머, 키틴류, 히알루론산, 및 이들의 조합인 것인, 성장 호르몬 수용체 길항제.
- 제13항에 있어서, 상기 펩타이드성 링커는 2개 이상의 아미노산이 연결된 인, 성장 호르몬 수용체 길항제.
- 제1항 내지 제15항 중 어느 한 항에 따른 성장 호르몬 수용체 길항제를 포함하는, 인간 성장 호르몬에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물.
- 제16항에 있어서, 상기 질환은 말단 비대증, 거인증, 암, 당뇨병성 신 병증, 관절염, 폐 염증, 성장 호르몬 결핍증(GHD), 특발성 저신장, 터너 증후군, 프라더-윌리(Prader-Willi) 증후군, 저체중아 (small for gestational age), 및 만성 신부전증(CRI)로 이루어지는 군으로부터 선택되는 것인, 약학적 조성물.
- 성장 호르몬(growth hormone)의 아미노산 서열에서 하나 이상의 아미노산이 다른 아미노산으로 치환된 성장 호르몬 변이체를 코딩하는 폴리뉴클레오티드를 포함하는 세포를 배양하는 단계를 포함하는, 성장 호르몬 수용체 길항제의 제조방법.
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JP2020534611A JP7175983B2 (ja) | 2017-12-20 | 2018-12-20 | 新規な成長ホルモン受容体拮抗剤及びその融合タンパク質 |
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