WO2025249934A1 - Composition pharmaceutique pour injection sous-cutanée comprenant un antagoniste du récepteur de l'hormone de croissance et un support à action prolongée - Google Patents

Composition pharmaceutique pour injection sous-cutanée comprenant un antagoniste du récepteur de l'hormone de croissance et un support à action prolongée

Info

Publication number
WO2025249934A1
WO2025249934A1 PCT/KR2025/007364 KR2025007364W WO2025249934A1 WO 2025249934 A1 WO2025249934 A1 WO 2025249934A1 KR 2025007364 W KR2025007364 W KR 2025007364W WO 2025249934 A1 WO2025249934 A1 WO 2025249934A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
terminus
acid residue
seq
growth hormone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2025/007364
Other languages
English (en)
Korean (ko)
Inventor
박순재
김규완
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alteogen Inc
Original Assignee
Alteogen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alteogen Inc filed Critical Alteogen Inc
Publication of WO2025249934A1 publication Critical patent/WO2025249934A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH

Definitions

  • the present invention relates to a fusion protein comprising 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 other amino acids, and a long-acting carrier, and a pharmaceutical composition comprising the fusion protein, preferably a pharmaceutical composition for subcutaneous injection.
  • Growth hormone is an endocrine hormone that promotes growth.
  • Human growth hormone hereinafter referred to as "hGH"
  • hGH Human growth hormone
  • hGHR human growth hormone receptor
  • IGF-1 insulin-like growth factor I
  • hGH receptor antagonists are an alternative treatment option because they prevent hGH from binding to the hGH receptor by occupying the hGH receptor in place of hGH (J.J.
  • hGHRA which requires long-term administration for treatment, can improve patients' quality of life by reducing the frequency of administration.
  • Pegvisomant is a commercially available pegylated version of the hGH antagonist (M.O. Thorner, C.J. Strasburger, Z. Wu, M. Straume, M. Bidlingmaier, S.S. Pezzoli, K. Zib, J.C. Scarlett, W.F. 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.; V. Goffin, P.
  • pegvisomant The long-term mechanism of action of pegvisomant is to attach a polyethylene glycol polymer to the therapeutic protein, thereby increasing its molecular size and allowing it to bypass the kidneys and remain in the bloodstream (R.J. Ross, K.C. Leung, M. Maamra, W. Bennett, N. Doyle, M.J. Waters, K.K. Ho, Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimer, J. Clin.
  • pegylation Another advantage of pegylation is that it protects the protein from proteases (S. Jevsevar, M. Kunststoffelj, V.G. Porekar, PEGylation of therapeutic proteins, Biotechnol. J. 5 (2010) 113-128.).
  • proteases S. Jevsevar, M. Kunststoffelj, V.G. Porekar, PEGylation of therapeutic proteins, Biotechnol. J. 5 (2010) 113-128.
  • PEGylation of therapeutic proteins may have limited utility. The PEGylation process requires a series of chemical reactions, which incurs additional production costs, and the difficulty in obtaining a homogeneous reaction product necessitates additional purification steps.
  • PEGylated proteins appear safe when using small polyethylene glycols (e.g., 5 kDa), safety concerns remain, as evidenced by reports of renal vacuolation and antibody development in response to PEGylation in animal studies. Furthermore, PEGylated proteins tend to have reduced binding affinity to protein receptors compared to native proteins (R.P. Garay, R. El-Gewely, J.K. Armstrong, G. Garratty, P. Richette, Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents, Expert Opin. Drug Deliv. 9 (2012) 1319-1323. ; A. Bendele, J. Seely, C. Richey, G. Sennello, G.
  • Human growth hormone is typically administered via subcutaneous injection once to seven times a week. However, repeated subcutaneous injections into the same area can cause side effects such as adipose tissue atrophy. However, subcutaneous administration (or subcutaneous injection) of hyaluronidase together with therapeutic drugs causes the hyaluronidase to hydrolyze hyaluronic acid distributed in the extracellular matrix, reducing the viscosity of the subcutaneous tissue and increasing substance permeability. This reduces side effects and facilitates the delivery of growth hormone into the body.
  • Hyal1 and Hyal2 are expressed in most tissues, and PH20/SPAM1 (hereinafter referred to as PH20) is expressed in the plasma membrane and acrosome membrane of sperm.
  • HyalPS1 is a pseudogene and is not expressed.
  • PH20 is an enzyme (EC 3.2.1.35) that cleaves the ⁇ -1,4 bond between N-acetylglucosamine and glucuronic acid, which are constituent sugars of hyaluronic acid.
  • Human hyaluronidase PH20 has an optimal pH of 5.5 but shows some activity at pH 7-8, whereas other human hyaluronidases, including Hyal1, have an optimal pH of 3-4 and are very weakly active at pH 7-8. Since the human subcutaneous area is approximately neutral at pH 7.4, among the various types of hyaluronidase, PH20 is widely used in clinical practice. Examples of clinical uses of PH20 include subcutaneous injection of antibody therapeutics, as an ocular relaxant and anesthetic injection additive during ophthalmic surgery, hydrolyzing hyaluronic acid in the extracellular matrix of tumor cells to increase the accessibility of anticancer therapeutics to tumor cells, and promoting the reabsorption of excessive body fluids and blood within tissues.
  • the PH20 currently widely used commercially is extracted from the testes of cows or sheep.
  • Examples include Amphadase (bovine hyaluronidase) and Vitrase (sheep hyaluronidase).
  • Amphadase bovine hyaluronidase
  • Vitrase sheep hyaluronidase
  • Repeated administration of high doses of animal-derived hyaluronidase to humans may produce neutralizing antibodies, and other animal-derived biological substances included as impurities in addition to PH20 may cause allergies.
  • the use of PH20 extracted from cows is restricted due to concerns about mad cow disease. To improve these problems, research on recombinant human PH20 proteins has been conducted.
  • Recombinant human PH20 proteins have been reported to be expressed in yeast (P. pastoris), DS-2 insect cells, and animal cells (Chen et al., 2016; Hofinger et al., 2007). Recombinant PH20 proteins produced in insect cells and yeast differ from human PH20 in the pattern of N-glycosylation during posttranslational protein modification.
  • a structure-function relationship study on human PH20 reported that the C-terminal region of PH20 is important for protein expression and enzyme activity, and in particular, the C-terminal region ending at amino acids 477-483 is reported to be important for enzyme expression and activity (Frost, 2007).
  • the activity of full-length PH20 (amino acids 1-509) or a PH20 mutant with a C-terminal truncation after amino acid 467 was less than 10% of the enzyme activity of a PH20 mutant with a C-terminal truncation at either amino acid 477 or 483 (Frost, 2007).
  • Halozyme Therapeutics developed rHuPH20 (amino acids 36-482), a recombinant protein in which the C-terminus of mature PH20 was truncated at Y482 (Bookbinder et al., 2006; Frost, 2007).
  • the PH20 variant according to the applicant's prior invention can be applied to a pharmaceutical composition or formulation containing various different drugs, and thus, the pharmaceutical composition and formulation containing the PH20 variant together with various drugs can be used for subcutaneous administration, and the activity of the drugs and the PH20 variant is very stable and can be maintained for a long period of time (WO 2020-197230 A1, WO 2021-150079 A1).
  • the present invention aims to provide a subcutaneous pharmaceutical composition of a growth hormone receptor antagonist fusion protein that fuses a more effective growth hormone receptor antagonist with a carrier as a cost-effective and active alternative candidate to hGHRA.
  • the present invention aims to provide a pharmaceutical composition for subcutaneous injection that increases the content of the injection drug and/or increases the subcutaneous absorption rate of the drug in order to eliminate the inconvenience of having to inject a conventional growth hormone receptor antagonist every day.
  • An example of a candidate growth hormone receptor antagonist is the antagonist disclosed in Korean Patent Publication No. 10-2019-0074958.
  • the present invention aims to provide a suitable carrier and hyaluronidase through research and improvement of such a candidate, and to provide a pharmaceutical composition for subcutaneous injection that is suitable for use in terms of pharmacokinetics and pharmacodynamics.
  • the present invention aims to provide a method for increasing the amount of drug absorbed into the body without a rapid increase in drug concentration by using hyaluronidase as a method for increasing the amount of drug absorbed into the body.
  • One object of the present invention is to provide a pharmaceutical composition for subcutaneous injection comprising a fusion protein of a growth hormone receptor antagonist and a carrier, and hyaluronidase, which comprises a growth hormone variant in which one or more amino acids in the amino acid sequence of natural human growth hormone are substituted with other amino acids.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by human growth hormone, comprising a growth hormone receptor antagonist fusion protein and hyaluronidase.
  • Another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a carrier capable of increasing the biological activity of the growth hormone receptor antagonist and improving the pharmacodynamic activity and/or pharmacodynamic activity.
  • the present invention provides an antagonist-carrier fusion protein suitable for increasing the biological activity of a growth hormone receptor antagonist, which comprises a growth hormone variant in which one or more amino acids in the amino acid sequence of a natural human growth hormone are substituted with other amino acids, and provides a subcutaneous injection formulation capable of improving pharmacokinetic/pharmacodynamic activity.
  • growth hormone mutant refers to a growth hormone in which one or more amino acids in the amino acid sequence of the growth hormone are substituted with other amino acids. In other words, it refers to a growth hormone having one or more amino acid substitutions.
  • the substitution may include a substitution of the 120th amino acid in the amino acid sequence of the growth hormone (more specifically, a substitution with lysine or arginine). It may also include a substitution of the 46th amino acid (more specifically, a substitution with lysine).
  • the substitution may include a substitution of amino acid position 174 (more specifically, a substitution with serine) in the amino acid sequence of the growth hormone, and may include a substitution of amino acid position 21 (more specifically, a substitution with asparagine).
  • the substitution may include a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 46th amino acid, the 54th amino acid, the 64th amino acid, the 120th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid in the amino acid sequence.
  • the substitution may include one or more substitutions selected from H18D, H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K172R, E174S, F176Y, and I179T in the amino acid sequence of the growth hormone.
  • the above variants may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, or amidation.
  • the above variant may include those described in Republic of Korea Patent Publication No. 10-2019-0074958.
  • the growth hormone variant may be a protein having an amino acid sequence of any one of SEQ ID NOs: 1 to 9 disclosed in Table 1 below.
  • SEQ ID NO: 10 represents a human growth hormone sequence.
  • GHR Crowth Hormone Receptor
  • growth hormone receptor antagonist refers to an agent that antagonizes the binding of growth hormone to the growth hormone receptor, thereby suppressing the side effects that arise from excessive binding of growth hormone to the growth hormone receptor.
  • 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 effects of growth hormone.
  • the growth hormone receptor antagonist may be a fusion protein fused with a long-acting carrier.
  • the term "long-acting carrier” refers to a substance capable of increasing the in vivo half-life.
  • a long-acting agent can be used that antagonizes the growth hormone receptor while also increasing the in vivo half-life.
  • various carriers capable of reducing renal clearance specifically, at least one 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, repeating unit polymers 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 high molecular weight polymers, may be used. More specifically, alpha-1 antitrypsin (hereinafter, interchangeably referred to as “A1AT” in the specification and drawings) or variants thereof may be used as the persistent carrier.
  • A1AT alpha-1 antitrypsin
  • Alpha-1 antitrypsin or its variants are disclosed in Korean Patent Publication Nos. 10-2013-0136883 and 10-2013-0029713.
  • A1AT is one of the most abundant proteins in human plasma, with a concentration of 1.5-3.5 grams per liter, and is primarily synthesized in hepatocytes and secreted into the blood.
  • the alpha-1 antitrypsin variants are engineered to have additional mutations in A1AT to increase glycosylation and eliminate intrinsic activity.
  • the alpha-1 antitrypsin variants can be fused to target proteins to extend their half-life.
  • alpha-1 antitrypsin variant technology is its non-immunogenicity.
  • human plasma A1AT has already been used to treat patients with A1AT deficiency due to emphysema and other lung diseases, at a very high weekly dose of 60 mg per kg of body weight. No serious side effects have been reported, demonstrating the safety of A1AT as a therapeutic agent.
  • a long-lasting hGHRA fusion protein is provided by fusing an alpha-1 antitrypsin variant to a growth hormone variant.
  • the alpha-1 antitrypsin variant may comprise at least one substitution among amino acids at positions 1 to 25 in the sequence of alpha-1 antitrypsin, and the growth hormone variant may comprise any one or more substitutions selected from H18D, H21N, Q46K, F54P, R64K, G120K, R167N, K168A, D171S, K172R, E174S, F176Y, and I179T in the amino acid sequence of growth hormone.
  • the alpha-1 antitrypsin variant may have the amino acid sequence of SEQ ID NO: 11 disclosed in Table 2 below.
  • SEQ ID NO: 11 is used interchangeably with “NexP.”
  • a growth hormone receptor antagonist fusion protein in which an alpha-1 antitrypsin variant is fused to a growth hormone variant as a carrier not only has a higher binding affinity to the growth hormone receptor compared to a pegylated growth hormone variant, but can also more potently antagonize the effect of growth hormone.
  • alpha-1 antitrypsin variant As a persistent carrier, it is also predicted that natural alpha-1 antitrypsin exhibits an effect similar to that of the alpha-1 antitrypsin variant, and furthermore, some fragments of alpha-1 antitrypsin exhibit the same effect. Therefore, natural alpha-1 antitrypsin, alpha-1 antitrypsin variants, fragments of natural alpha-1 antitrypsin, and fragments of alpha-1 antitrypsin variants can also be used as persistent carriers.
  • Immunoglobulin Fc exists as a homodimer or heterodimer. While heterodimers of immunoglobulin Fc are expected to be more advantageous for acting as antagonists against growth hormone receptor dimers, the scope of the present invention is not limited thereto.
  • the heterodimer of the immunoglobulin Fc comprises a variant CH3 domain containing amino acid mutations that promote heterodimer formation. These mutations include mutations at positions 366, 394, 405, and 407 of the amino acid sequence, and may further include mutations at positions 390 and 400. Examples of heterodimers that may include, but are not limited to, a Knop-in-Hole structure, as described in U.S. Patent No. 7,695,936.
  • heterodimers of immunoglobulin Fc include the amino acid sequences disclosed in Table 3 below.
  • the persistent carrier may be fused to at least one of the N-terminus and the C-terminus of the growth hormone mutant.
  • the growth hormone receptor antagonist can bind to the N-terminus and C-terminus of both chains or one chain of the immunoglobulin Fc heterodimer, respectively.
  • the growth hormone receptor antagonist can include, but is not limited to, a dimer in which the N-terminus of SEQ ID NO: 12 and SEQ ID NO: 13 are bound to and expressed simultaneously, a dimer in which the N-terminus of SEQ ID NO: 12 is bound only to and expressed simultaneously with SEQ ID NO: 13, a dimer in which the N-terminus of SEQ ID NO: 13 is bound only to and expressed simultaneously with SEQ ID NO: 12, a dimer in which the C-terminus of SEQ ID NO: 12 and SEQ ID NO: 13 are bound only to and expressed simultaneously with SEQ ID NO: 13, and a dimer in which the C-terminus of SEQ ID NO: 13 is bound only to and expressed simultaneously with SEQ ID NO: 12.
  • growth hormone receptor antagonist fusion proteins fused to the N-terminus of the mutant have significantly higher binding affinity to the growth hormone receptor or can potently antagonize the effects of growth hormone compared to those fused to the C-terminus.
  • the persistent carrier may be directly fused with the growth hormone receptor antagonist, or may be fused via a linker.
  • substitution of amino acid residues at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363 is more preferably, but not limited to, one or more selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G.
  • the novel PH20 variant or fragment thereof according to the present invention is characterized by comprising substitutions of amino acid residues M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T, and may further be characterized by comprising substitutions of one or more amino acid residues selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N and N363G, but is not limited thereto.
  • the PH20 variant or fragment thereof according to the present invention may be any one selected from the group consisting of, but is not limited thereto.
  • an expression in which a one-letter amino acid residue name and a number are written together refers to an amino acid residue at each position in the amino acid sequence according to SEQ ID NO: 222.
  • S347 means that the amino acid residue at position 347 in the amino acid sequence of SEQ ID NO: 222 is serine.
  • S347T means that the 347th serine in SEQ ID NO: 222 is substituted with threonine.
  • the PH20 variant or fragment thereof according to the present invention is interpreted to mean also a variant or fragment thereof in which an amino acid residue is conservatively substituted at a specific amino acid residue position.
  • “conservative substitution” means a modification of a PH20 variant that includes replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the PH20 variant.
  • a “conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain.
  • Classes of amino acid residues with similar side chains are well-defined and known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine
  • PH20 variants of the present invention may still retain activity even if they have conservative amino acid substitutions.
  • the PH20 variant or fragment thereof according to the present invention is interpreted to mean also including PH20 variants or fragments thereof that have substantially the same function and/or effect as the PH20 variant or fragment thereof according to the present invention and have an amino acid sequence homology of 80% or 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.
  • the PH20 variants or fragments thereof according to the present invention exhibit increased expression in animal cells compared to mature wild-type PH20, increased protein folding, and thus increased thermal stability. Furthermore, despite the increased thermal stability, their enzymatic activity is increased or similar to that of mature wild-type PH20.
  • the PH20 variant or fragment thereof according to the present invention is characterized by, but is not limited to, substitution of some amino acid residues in the alpha helix 8 region (S347 to C381) and/or the connecting region between alpha helices 7 and 8 (A333 to R346) of wild-type PH20, and additional deletion of some amino acid residues in the C-terminus and/or N-terminus.
  • the PH20 variant or fragment thereof according to the present invention comprises a truncation before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminus of the amino acid sequence of SEQ ID NO: 222, preferably before an amino acid residue of L36, N37, F38, R39, A40, P41 or P42, so that some amino acid residues at the N-terminus are deleted, and/or after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus, preferably after an amino acid residue selected from the group consisting of V455 to S490, most preferably V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, It may be characterized by deletion of some amino acid residues at the C-terminus due to truncation at amino acid residues P486, T
  • cleavage occurred in front of an amino acid residue selected from the group consisting of M1 to P42 of the N-terminus means that the amino acid residue immediately preceding the amino acid residue selected from M1 to P42 of the N-terminus was cleaved and deleted.
  • the expression that cleavage occurred before amino acid residues L36, N37, F38, R39, A40, P41 or P42 means that in the sequence of SEQ ID NO: 222, the amino acid residues M1 to T35 immediately preceding L36, M1 to L36 immediately preceding N37, M1 to N37 immediately preceding F38, M1 to F38 immediately preceding R39, M1 to R39 immediately preceding A40, M1 to A40 immediately preceding P41, and M1 to P41 immediately preceding P42 were cleaved and removed.
  • cleavage occurred after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminus means that cleavage occurred and deletion occurred starting from the amino acid residue immediately following the amino acid residue selected from V455 to L509 at the C-terminus.
  • the expression that cleavage occurred at an amino acid residue of V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 of the C-terminus means that cleavage occurred at an amino acid residue of V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 in the sequence of SEQ ID NO: 222, respectively. This means that the next amino acid residue was cut and removed.
  • the PH20 variant or fragment thereof according to the present invention may be selected from the group consisting of amino acid sequences of SEQ ID NO: 25 to SEQ ID NO: 70, but is not limited thereto.
  • the PH20 variant or fragment thereof according to the present invention is characterized by having an amino acid sequence of SEQ ID NO: 64.
  • the PH20 variant having the amino acid sequence of SEQ ID NO: 64 is characterized by having 15 amino acid residues substituted as T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and being cleaved before the F38 residue at the N-terminus and cleaved after the F468 residue at the C-terminus.
  • the sequences of amino acids substituted or truncated in the PH20 mutants produced by the applicant are as described in Table 5.
  • the applicant produced PH20 mutants having higher stability than wild-type PH20 by replacing a specific alpha helix forming the secondary structure of PH20 with an alpha helix of another human hyaluronidase, and these mutants are characterized in that they are mutants having constant enzymatic activity regardless of the C-terminal truncation position, since the interaction of the substituted alpha helix domain with other parts forming the secondary structure of PH20 is different from that of wild-type PH20.
  • the novel PH20 variant or fragment thereof according to the present invention which has increased enzyme activity and thermal stability compared to mature wild-type PH20, comprises a substitution of one or more amino acid residues selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T in the amino acid sequence of wild-type PH20, and is characterized in that some amino acids located in the alpha helix 8 region (S347 to C381) and/or the junction region of alpha helix 7 and alpha helix 8 (A333 to R346) are substituted with other amino acids.
  • amino acid substitutions at the junctions of alpha helix 8 and alpha helix 7 and alpha helix 8 include substitutions of some amino acids in the T341 ⁇ N363, T341 ⁇ I361, L342 ⁇ I361, S343 ⁇ I361, I344 ⁇ I361, M345 ⁇ I361, or M345 ⁇ N363 sections.
  • HM6 is a mutant in which the amino acids in the M345 to N363 region are substituted with the amino acid sequence of Hyal1 (M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T are substituted in SEQ ID NO: 222), and is a mutant with the fewest substitutions in the alpha helix 8 region and the junction region of alpha helix 7 and alpha helix 8 among the PH20 mutants according to the present invention that do not contain additional C-terminal truncation (i.e., in the form in which the C-terminal amino acid residue is S490, like the mature wild-type PH20).
  • Hyal1 M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D,
  • HM10 is a mutant in which the amino acids in the L342 ⁇ I361 section are substituted with the amino acid sequence of Hyal1 (L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T are substituted in SEQ ID NO: 222). It also does not include additional C-terminal truncation and is the mutant with the highest thermal stability among the PH20 mutants according to the present invention while having an enzymatic activity similar to that of the mature wild-type PH20.
  • HM21 is a mutant in which the amino acids in the T341 ⁇ I361 section are substituted with the amino acid sequence of Hyal1 (T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T are substituted in SEQ ID NO: 222). It also does not contain additional C-terminal truncation, and is a mutant with an enzyme activity approximately twice as high as that of the wild-type PH20 at pH 7.0.
  • the HM6-based PH20 variant produced in the applicant's prior invention has the N-terminus truncated at L36 and the C-terminus truncated at I465, F468, or P471 as shown in Table 5.
  • the PH20 mutants according to the present invention exhibited enzymatic activity similar to that of mature wild-type PH20, regardless of the truncation position at the C-terminus.
  • HM21-based PH20 mutants (HP34: 38-470, HP46: 38-468) had in common that the N-terminus was truncated before the F38 residue and the C-terminus was truncated after the F468 or K470 residue. They contained amino acid substitutions in helix 8 and the T341-I361 region at the junction of helices 7 and 8, using HM21 as a template, which has approximately twice the enzymatic activity of mature wild-type PH20.
  • mutants which were truncated before the F38 residue at the N-terminus and truncated after the F468 or K470 residue at the C-terminus, retained the high enzymatic activity of HM21 regardless of the C-terminal truncation position.
  • mutants HM40, HM13, HM41, HM24, HM42, and HM25 in which the N-terminus was truncated before residues N37, F38, R39, A40, P41, or P42 of the amino acid sequence of SEQ ID NO: 222 were constructed using HM6 as a template.
  • Sequence Number Substitution Sequence HM1 25 12 amino acids are substituted with M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G from SEQ ID NO: 222.
  • a PH20 variant or fragment thereof comprising a substitution, deletion and/or insertion of one or more amino acid residues in the hyaluronidase variant having the amino acid sequence of SEQ ID NO: 64, and optionally a deletion of a portion of the amino acid residues at the N-terminus and/or C-terminus.
  • the PH20 variants or fragments thereof usable in the present invention exemplified in Table 6, showed increased protein expression levels when expressed in ExpiCHO cells compared to mature wild-type PH20, and the protein aggregation temperature increased by about 4 to 11.5 °C, thereby having high thermal stability and being able to be produced efficiently.
  • the PH20 variants or fragments thereof exemplified in Table 6 that can be used in the present invention have the effect of improving protein refolding and renaturing faster than the mature wild-type PH20 in the substrate-gel assay, which is one of the experiments measuring the activity of hyaluronidase, and have the effect of maintaining the original enzyme activity regardless of the truncation position of the C-terminus.
  • PH20 variants or fragments thereof according to the examples in Table 6 that can be used in the present invention have low immunogenicity, and thus can be repeatedly administered to the human body.
  • SEQ ID NO Substitution Sequence HM63 71 One amino acid residue R346M is substituted from SEQ ID NO: 64, cleavage occurs before F38 amino acid residue at N-terminus of PH20, and cleavage occurs after F468 amino acid residue at C-terminus of PH20.
  • FRAPPVIPNVPFLWAWNAPSEFCLGKFDEP A DMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRS IELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQ QSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGSWENTRTKESCQAIKEYMDTTLNPYIIN VTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAF
  • amino acid sequence in Table 7 above is the amino acid sequence of wild-type human PH20 consisting of 509 amino acids (SEQ ID NO: 222).
  • the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by human growth hormone, comprising a growth hormone receptor antagonist.
  • a pharmaceutical composition for the prevention or treatment of diseases caused by human growth hormone comprising a growth hormone receptor antagonist.
  • the terms used herein are as described above.
  • human growth hormone-induced disease refers to a disease caused by causes such as the pituitary gland not being able to normally regulate the secretion of growth hormone and secreting excessively. Examples may include acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, lung inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • GDD growth hormone deficiency
  • CLI chronic renal insufficiency
  • diseases induced by human growth hormone include diseases that occur due to increased secretion of IGF (insulin-like growth factor)-1 caused by excessive growth hormone action.
  • the present invention provides a method for producing a growth hormone receptor antagonist, comprising the step of culturing a cell containing a polynucleotide encoding a growth hormone variant in which one or more amino acids in the amino acid sequence of a native human growth hormone having the amino acid sequence represented by SEQ ID NO: 10 are substituted with other amino acids.
  • a method for producing a growth hormone receptor antagonist comprising the step of culturing a cell containing a polynucleotide encoding a growth hormone variant in which one or more amino acids in the amino acid sequence of a native human growth hormone having the amino acid sequence represented by SEQ ID NO: 10 are substituted with other amino acids.
  • the above cells may be cells into which an expression vector of a growth hormone variant has been transferred, and non-limiting examples thereof include CHO (Chinese hamster ovary)-K1.
  • the present invention provides a method for preventing or treating a disease caused by human growth hormone, comprising administering to a subject a pharmaceutical composition comprising the growth hormone receptor antagonist described above.
  • a pharmaceutical composition comprising the growth hormone receptor antagonist described above.
  • administration refers to introducing the pharmaceutical composition of the present invention into a patient by any suitable method.
  • the administration route of the composition of the present invention may be administered through various oral or parenteral routes as long as it can reach the target tissue, and preferably refers to subcutaneous administration.
  • the formulation according to the present invention can be prepared in various dosage forms depending on the intended administration method.
  • administration may be performed prophylactically or therapeutically.
  • the frequency of administration of the formulation of the present invention is not particularly limited, but may be administered once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days, or may be administered in divided doses several times a day.
  • the subject of administration of the formulation according to the present invention may refer to any animal, including humans.
  • the animal may include, but is not limited to, mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, as well as humans.
  • a pharmaceutical composition containing a growth hormone receptor antagonist the subject of the present invention
  • administration of a pharmaceutical composition containing a growth hormone receptor antagonist is carried out periodically over a long period of time. Therefore, for the convenience of the patient, it is necessary to administer it by subcutaneous injection in a sustained-release formulation that can limit the number of administrations.
  • a sustained-release formulation that can limit the number of administrations.
  • methods include using a pharmaceutical composition containing a high concentration of the antagonist or using hyaluronidase to contain a high content of the antagonist. The present invention proposes this through specific experimental examples.
  • the present invention provides a pharmaceutical composition comprising the growth hormone receptor antagonist for the prevention or treatment of diseases caused by human growth hormone.
  • the terms used herein are as described above.
  • a fusion protein comprising a growth hormone receptor inhibitor and a long-acting carrier according to the present invention has strong binding affinity to the growth hormone receptor and can exhibit a sustained antagonistic effect.
  • a pharmaceutical composition for subcutaneous injection comprising a growth hormone receptor inhibitor according to the present invention, a carrier, and hyaluronidase has strong binding affinity to the growth hormone receptor and can exhibit a sustained antagonistic effect.
  • the pharmaceutical composition for subcutaneous injection comprising a growth hormone receptor inhibitor, a carrier, and hyaluronidase according to the present invention can increase the content of the drug absorbed into the human body without a rapid increase in the concentration of the growth hormone receptor inhibitor by increasing the content of the subcutaneous injection drug.
  • Figure 1 is a drawing comparing the human growth hormone receptor binding properties of Pegvisomant and growth hormone receptor antagonist - carrier candidate substance 3, where Figure 1A shows the result of confirming binding to the water-soluble portion of the human growth hormone receptor, and Figure 1B shows the result of confirming the biological activity of inhibiting growth using HEK293F cells in which the human growth hormone receptor is overexpressed.
  • Figure 3 shows the results of measuring the IGF-1 level in serum after a single subcutaneous injection of growth hormone receptor antagonist - carrier candidate substance 3 in combination with hyaluronidase “ALT-B4” (SEQ ID No: 99 of WO 2020-022791 A1) at different doses in NZW rabbits.
  • Figure 4 shows the results of pharmacokinetic and pharmacodynamic analysis for the results of Figure 3.
  • Figure 5 is a photograph showing the swelling phenomenon that appears at the injection site during the subcutaneous injection of Figure 3.
  • the present invention is a.
  • a pharmaceutical composition comprising hyaluronidase, preferably a pharmaceutical composition for subcutaneous injection.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist comprises a substitution of lysine at the 46th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10, a substitution of lysine or arginine at the 120th amino acid, or a combination of these substitutions.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist further comprises a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 54th amino acid, the 64th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist further comprises a substitution of the 174th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with serine, a substitution of the 21st amino acid with asparagine, or a combination of these substitutions.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist comprises at least one substitution selected from the group consisting of a substitution of the 18th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with aspartic acid, a substitution of the 21st amino acid with asparagine, a substitution of the 46th amino acid with lysine, a substitution of the 54th amino acid with proline, a substitution of the 64th amino acid with lysine, a substitution of the 120th amino acid with lysine or arginine, a substitution of the 167th amino acid with asparagine, a substitution of the 168th amino acid with alanine, a substitution of the 171st amino acid with serine, a substitution of the 172nd amino acid with arginine, a substitution of the 174th amino acid with serine, a substitution of the 176th amino acid with tyrosine, and
  • the present invention also relates to a pharmaceutical composition for subcutaneous injection, wherein the fusion protein has an amino acid sequence of one of SEQ ID NOs: 18 to 24, but it is apparent to a person skilled in the art that the present invention is not limited to this sequence.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the persistent carrier is fused to at least one of the N-terminus or C-terminus of the growth hormone receptor antagonist.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the persistent carrier is selected from the group consisting of, but not limited to, polyethylene glycol, fatty acid, albumin or a fragment thereof, an albumin-binding substance, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or a fragment thereof, a repeating unit polymer of a specific amino acid sequence, an antibody or a fragment thereof, an FcRn binding substance, an in vivo connective tissue or a derivative thereof, a nucleotide, fibronectin, transferrin, a saccharide, and a high molecular weight polymer, although preferred examples thereof are not limited thereto.
  • the persistent carrier is selected from the group consisting of, but not limited to, polyethylene glycol, fatty acid, albumin or a fragment thereof, an albumin-binding substance, alpha-1 antitrypsin or a variant thereof, immunoglobulin Fc or a fragment thereof, a repeat
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, comprising immunoglobulin Fc or a fragment thereof as the persistent carrier.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the immunoglobulin Fc or a fragment thereof is a heterodimer.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the immunoglobulin Fc comprises an amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 17.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the growth hormone receptor antagonist is fused with the long-acting carrier directly or through a linker.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the linker is a peptidic linker or a non-peptidic linker.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the non-peptide linker is not limited to the examples below, but is preferably 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, chitin, hyaluronic acid, or a combination thereof.
  • the non-peptide linker is not limited to the examples below, but is preferably polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA),
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the peptide linker is a pharmaceutical composition in which two or more amino acids are linked.
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, for the prevention or treatment of a disease selected from the group consisting of diseases induced by human growth hormone, such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • a disease selected from the group consisting of diseases induced by human growth hormone, such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • a disease selected from the group consisting of diseases induced by human growth hormone, such as acromegaly, gig
  • the present invention relates to a pharmaceutical composition, preferably a pharmaceutical composition for subcutaneous injection, wherein the hyaluronidase is a human or mammalian hyaluronidase or a variant thereof, having hyaluronidase activity.
  • the hyaluronidase refers to the naturally occurring PH20, Hyal1, Hyal2, Hyal3, Hyal4 of mammals such as humans, sheep, cows, and apes, and their mature forms.
  • mutants refer to those in which one or more amino acids in the amino acid sequence of the above-described natural and/or mature hyaluronidase are substituted, deleted or added, and specifically, mutants in which amino acids 1-35, 1-36, 1-37, 1-38, 1-39, 1-40 or 1-41 of human PH20 are truncated and/or all amino acids after 480 or after any one of amino acids 468 to 490 are truncated, and the hyaluronidase may be one selected from SEQ ID NOs: 25 to 222, and in addition, WO 2004-078140 A2, WO 2006-091871 A1, WO 2010-077297 A1, WO 2013-102144 A2, WO 2015-003167 A1, WO Examples include, but are not limited to, various hyaluronidase variants disclosed in WO 2020-22791 A1, WO 2021-150079 A1, etc.
  • the present invention relates to a fusion protein comprising a growth hormone receptor antagonist in which one or more amino acids in the amino acid sequence of growth hormone are substituted with other amino acids and an immunoglobulin Fc or a fragment thereof as a long-acting carrier.
  • the present invention relates to a fusion protein wherein the immunoglobulin Fc or a fragment thereof is a heterodimer.
  • the present invention relates to a fusion protein, wherein the immunoglobulin Fc comprises an amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 17.
  • the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist comprises a substitution of lysine at the 46th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10, a substitution of lysine or arginine at the 120th amino acid, or a combination of these substitutions.
  • the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist further comprises a substitution at any one or more positions selected from the group consisting of the 18th amino acid, the 21st amino acid, the 54th amino acid, the 64th amino acid, the 167th amino acid, the 168th amino acid, the 171st amino acid, the 172nd amino acid, the 174th amino acid, the 176th amino acid, and the 179th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10.
  • the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist further comprises a substitution of the 174th amino acid from the N-terminus of the human growth hormone amino acid sequence represented by SEQ ID NO: 10 with serine, a substitution of the 21st amino acid with asparagine, or a combination of these substitutions.
  • the present invention relates to a fusion protein, wherein the growth hormone receptor antagonist comprises at least one substitution selected from the group consisting of a substitution of the 18th amino acid from the N-terminus with aspartic acid, a substitution of the 21st amino acid with asparagine, a substitution of the 46th amino acid with lysine, a substitution of the 54th amino acid with proline, a substitution of the 64th amino acid with lysine, a substitution of the 120th amino acid with lysine or arginine, a substitution of the 167th amino acid with asparagine, a substitution of the 168th amino acid with alanine, a substitution of the 171st amino acid with serine, a substitution of the 172nd amino acid with arginine, a substitution of the 174th amino acid with serine, a substitution of the 176th amino acid with tyrosine, and a substitution of the 179th amino acid with threonine in the human growth hormone amino acid sequence represented by
  • the present invention relates to a fusion protein in which the carrier is fused to at least one of the N-terminus or C-terminus of the growth hormone receptor antagonist.
  • the present invention relates to a fusion protein in which the growth hormone receptor antagonist is fused directly or through a linker to the long-acting carrier.
  • the present invention relates to a fusion protein, wherein the linker is a peptidic linker or a non-peptidic linker.
  • the present invention relates to a fusion protein in which the peptidic linker is composed of two or more amino acids linked together.
  • the present invention relates to a fusion protein, characterized in that the fusion protein is for the prevention or treatment of a disease caused by human growth hormone.
  • the present invention relates to a fusion protein having an amino acid sequence of one of SEQ ID NOs: 18 to 24, but it is obvious to a person skilled in the art to which the present invention pertains that the present invention is not limited to this sequence.
  • the present invention relates to a fusion protein characterized in that the disease induced by the human growth hormone is selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • the disease induced by the human growth hormone is selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • the present invention relates to a pharmaceutical composition for preventing or treating a disease caused by human growth hormone, comprising the fusion protein.
  • the present invention relates to a nucleic acid encoding the fusion protein, a vector for expressing the fusion protein comprising the nucleic acid, a host cell comprising the vector, and a pharmaceutical composition for preventing or treating a disease caused by human growth hormone comprising the nucleic acid, vector, or host cell. Furthermore, the present invention relates to a method for producing a fusion protein using the nucleic acid, vector, or host cell, and a method for producing a pharmaceutical composition for preventing or treating a disease caused by human growth hormone using the nucleic acid, vector, or host cell.
  • the hGH receptor gene was introduced into the chromosomes of HEK293F cells containing the luciferase gene, which can be induced by hGH receptor signaling.
  • the resulting cell line was designated hGHR/Luc/HEK293F.
  • Serial dilutions of hGH and hGHRA-NexP were added to each 96-well white plate containing hGHR/Luc/HEK293F. The plates were incubated for 24 h in a 5% CO 2 incubator at 37°C.
  • luciferase assay reagent (Steady-Glo ® luciferase assay system, Promega) was added to each well, and the plates were sealed and protected from light. After 5 minutes at room temperature, the luminescence of each well was analyzed using a multi-mode microplate reader (SpectraMax M5, Molecular Devices).
  • Example 1-2 hGH receptor binding assay
  • the binding affinity of hGRA-NexP for the hGH receptor was evaluated in a binding assay using a recombinant hGH receptor Fc chimera.
  • Microplates were coated with the hGH receptor chimera overnight at 25°C. After washing with TPBS buffer (PBS buffer containing 0.05% Tween-20), samples (candidates and Pegvisomant) were loaded into each well. The samples were washed three times and then conjugated with anti-hGH polyclonal antibody-biotin. After an additional washing step, 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well to perform the TMB reaction. The absorbance signal from the reaction was recorded at 450-650 nm.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Example 1-3 hGH competitive inhibitory activity assay
  • the inhibitory activity of the candidate compounds on downstream signal transduction was analyzed as follows using an hGH competition assay. Serial dilutions of the growth hormone receptor antagonists of Examples 1 to 11 and Pegvisomant were added to each well of a 96-well plate containing hGHR/Luc/HEK293F. The plates were incubated for 24 h in a 5% CO 2 incubator at 37°C. After incubation, 100 ⁇ L of luciferase assay reagent (Steady-Glo ® luciferase assay system, Promega) was added to each well, and the plates were sealed and protected from light. After 5 minutes at room temperature, the luminescence of each well was analyzed using a multi-mode microplate reader (SpectraMax M5, Molecular Devices).
  • Example 2 Analysis of biological activity of growth hormone receptor antagonist-carrier according to the type and location of the carrier.
  • polyethylene glycol, NexP (SEQ ID NO: 11) presented in the present invention, and immunoglobulin Fc were used to analyze growth hormone receptor antagonist-carriers according to the type of carrier and the binding position of the fusion protein and carrier, using the method of Example 1. The results were compared with those of Pegvisomant (Table 8).
  • Candidate 1 is a fusion protein in which a growth hormone receptor antagonist having a sequence number of 7 is linked to the C-terminus of NexP (SEQ ID NO: 11)
  • Candidate 2 is a fusion protein in which a linker (GGGGS) and SEQ ID NO: 12 are sequentially linked to the C-terminus of the growth hormone receptor antagonist having a sequence number of 7, and expressed together with SEQ ID NO: 13
  • Candidate 3 is a fusion protein in which a linker (GGGGS) and a growth hormone receptor antagonist having a sequence number of 7 are sequentially linked to the C-terminus of SEQ ID NO: 12, and expressed together with SEQ ID NO: 13.
  • Table 8 shows that Candidate 3 appears to have excellent binding affinity and efficacy.
  • Example 3-2 Comparative test of candidate substance 3 by dose and comparative test of formulation containing hyaluronidase
  • pharmacokinetic analysis was performed by measuring the serum IGF-1 level after administering candidate substance 3 once at each dose to NZW rabbits and collecting blood samples.
  • the experimental results are presented in Fig. 3.
  • Candidate substance 3 was administered once at 10 mg/kg, 15 mg/kg, and 20 mg/kg, respectively, and each sample was administered with hyaluronidase ALT-B4 and compared.
  • Serum IGF-1 levels were measured 24 and 12 hours before administration, immediately before administration, and 12, 24, 48, 72, 96, 120, 144, 168, 192, and 240 hours after administration.
  • the obtained pharmacokinetic and pharmacodynamic results are presented in Fig. 4, and photographs of some test animals immediately after administration are presented in Fig. 5.
  • Example 4 Analysis of hGH competitive inhibitory activity of additional growth hormone receptor antagonist-carrier variants.
  • the function of the growth hormone receptor fusion protein according to the binding position of the fusion protein and the carrier was analyzed using immunoglobulin Fc as a carrier by the method of Example 1, using Candidate 3 of Example 2 as a control (Table 9).
  • Candidate 4 is a fusion protein produced by expressing sequence number 21 together with sequence number 14
  • Candidate 5 is a fusion protein produced by expressing sequence number 22 together with sequence number 15
  • Candidate 6 is a fusion protein produced by expressing sequence number 23 together with sequence number 12
  • Candidate 7 is a homodimer fusion protein produced by expressing sequence number 24.
  • the present invention can be used for the prevention or treatment of diseases selected from the group consisting of diseases induced by human growth hormone, such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • diseases induced by human growth hormone such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner syndrome, Prader-Willi syndrome, small for gestational age, and chronic renal insufficiency (CRI).
  • diseases induced by human growth hormone such as acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, growth hormone deficiency (GHD), idiopathic short stature, Turner

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne : une protéine de fusion comprenant un antagoniste du récepteur de l'hormone de croissance et un support à action prolongée, l'antagoniste du récepteur de l'hormone de croissance comprenant un variant d'hormone de croissance dans lequel un ou plusieurs acides aminés dans la séquence d'acides aminés de l'hormone de croissance sont substitués par un autre acide aminé ; et une composition pharmaceutique, de préférence une composition pharmaceutique pour injection sous-cutanée, comprenant la protéine de fusion. Une composition pharmaceutique comprenant un inhibiteur du récepteur de l'hormone de croissance, un support et de l'hyaluronidase, selon la présente invention, peut avoir une forte affinité de liaison au récepteur de l'hormone de croissance et peut présenter un antagonisme prolongé. Une composition pharmaceutique pour injection sous-cutanée comprenant un inhibiteur du récepteur de l'hormone de croissance, un support et de l'hyaluronidase, selon la présente invention, peut augmenter la quantité d'un médicament à injecter par voie sous-cutanée, ce qui permet d'augmenter la quantité du médicament absorbé dans le corps humain sans augmentation rapide de la concentration de l'inhibiteur du récepteur de l'hormone de croissance.
PCT/KR2025/007364 2024-05-29 2025-05-29 Composition pharmaceutique pour injection sous-cutanée comprenant un antagoniste du récepteur de l'hormone de croissance et un support à action prolongée Pending WO2025249934A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2024-0069837 2024-05-29
KR20240069837 2024-05-29

Publications (1)

Publication Number Publication Date
WO2025249934A1 true WO2025249934A1 (fr) 2025-12-04

Family

ID=97871054

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2025/007364 Pending WO2025249934A1 (fr) 2024-05-29 2025-05-29 Composition pharmaceutique pour injection sous-cutanée comprenant un antagoniste du récepteur de l'hormone de croissance et un support à action prolongée

Country Status (1)

Country Link
WO (1) WO2025249934A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721348A (en) * 1990-12-14 1998-02-24 University Of Connecticut DNA encoding PH-20 proteins
KR101769652B1 (ko) * 2009-07-31 2017-08-18 에프. 호프만-라 로슈 아게 피하용 항-에이치이알2 항체 제형
KR102167827B1 (ko) * 2017-12-20 2020-10-20 주식회사 알테오젠 신규한 성장 호르몬 수용체 길항제 및 이의 융합 단백질
KR20220069610A (ko) * 2020-11-20 2022-05-27 (주)알테오젠 Fgf19 변이체를 포함하는 신규 융합 단백질 및 이의 용도
KR20240038901A (ko) * 2022-09-16 2024-03-26 (주)피앤피바이오팜 신규한 히알루로니다제 ph-20 변이체 및 그 용도
KR102650991B1 (ko) * 2019-03-25 2024-03-27 (주)알테오젠 인간 히알루로니다제 ph20의 변이체와 약물을 포함하는 피하투여용 약학 조성물

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721348A (en) * 1990-12-14 1998-02-24 University Of Connecticut DNA encoding PH-20 proteins
KR101769652B1 (ko) * 2009-07-31 2017-08-18 에프. 호프만-라 로슈 아게 피하용 항-에이치이알2 항체 제형
KR102167827B1 (ko) * 2017-12-20 2020-10-20 주식회사 알테오젠 신규한 성장 호르몬 수용체 길항제 및 이의 융합 단백질
KR102650991B1 (ko) * 2019-03-25 2024-03-27 (주)알테오젠 인간 히알루로니다제 ph20의 변이체와 약물을 포함하는 피하투여용 약학 조성물
KR20220069610A (ko) * 2020-11-20 2022-05-27 (주)알테오젠 Fgf19 변이체를 포함하는 신규 융합 단백질 및 이의 용도
KR20240038901A (ko) * 2022-09-16 2024-03-26 (주)피앤피바이오팜 신규한 히알루로니다제 ph-20 변이체 및 그 용도

Similar Documents

Publication Publication Date Title
WO2014017849A1 (fr) Formulation liquide à base d'insuline à action prolongée et d'un peptide insulinotrope
WO2014017847A1 (fr) Formulation liquide à base d'un conjugué d'insuline à action prolongée
WO2012011752A2 (fr) Nouveau conjugué de glucagon à action prolongée et composition pharmaceutique le comportant pour prévention et traitement de l'obésité
WO2010011096A2 (fr) Complexe de polypeptide comprenant un polymère non-peptidylique ayant trois extrémités fonctionnelles
WO2015183054A1 (fr) Composition utilisable en vue du traitement du diabète sucré et contenant de l'insuline et un double agoniste du glp-1/glucagon
WO2012165915A2 (fr) Composition pour le traitement du diabète comprenant un conjugué d'insuline à action prolongée et un conjugué de peptide insulinotropique à action prolongée
WO2015183038A1 (fr) Composition pour le traitement du diabète comprenant un conjugué d'analogue d'insuline à longue durée d'action et un conjugué de peptide insulinotropique à longue durée d'action
WO2013133667A1 (fr) Composition pharmaceutique destinée à la prévention ou au traitement de la stéatose hépatique non alcoolique
KR102167827B1 (ko) 신규한 성장 호르몬 수용체 길항제 및 이의 융합 단백질
WO2014133324A1 (fr) Analogue novateur de l'insuline et son utilisation
WO2011122922A2 (fr) Formulation d'hormone humaine stimulant les follicules à action prolongée utilisant un fragment d'immunoglobuline
EP3152236A1 (fr) Procédé permettant de réduire l'immunogénicité d'une protéine et d'un peptide
WO2015005747A1 (fr) Conjugué fc d'immunoglobuline maintenant une force de liaison au fcrn
WO2012002745A2 (fr) Formation d'un complexe avec le facteur viia au moyen d'un fragment d'immunoglobuline
EP3240802A1 (fr) Dérivés du glucagon
WO2021182928A1 (fr) Nouvelle protéine bispécifique et son utilisation
WO2022245179A1 (fr) Composition pour polythérapie comprenant un variant de facteur de différenciation de croissance 15 et un agoniste du récepteur du peptide 1 de type glucagon
WO2021096275A1 (fr) Protéine de fusion comprenant l'interleukine -7 modifiée et le récepteur bêta ii du tgf et son utilisation
WO2024058648A1 (fr) Nouveau variant de la hyaluronidase ph-20 et son utilisation
WO2017116191A2 (fr) Nouvelle préparation en phase liquide d'un conjugué d'hormone de croissance humaine à longue durée d'action
AU2019308089A1 (en) Pharmaceutical composition comprising polypeptide
WO2018105988A1 (fr) Conjugué ayant une réponse immunitaire atténuée
WO2018004294A9 (fr) Composition pharmaceutique comprenant une protéine mutante d'hormone de croissance humaine ou une protéine de fusion de transferrine de celle-ci en tant qu'ingrédient actif
WO2019125002A1 (fr) Nouvel antagoniste du récepteur de l'hormone de croissance et sa protéine de fusion
WO2022245183A1 (fr) Composition pour la prévention ou le traitement d'une stéatose hépatique non alcoolique ou d'une stéatohépatite non alcoolique comprenant un variant de facteur de différenciation de croissance 15

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25816424

Country of ref document: EP

Kind code of ref document: A1