WO2024011946A1 - Dimères polypeptidiques pour le traitement de la sclérose systémique - Google Patents

Dimères polypeptidiques pour le traitement de la sclérose systémique Download PDF

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WO2024011946A1
WO2024011946A1 PCT/CN2023/082966 CN2023082966W WO2024011946A1 WO 2024011946 A1 WO2024011946 A1 WO 2024011946A1 CN 2023082966 W CN2023082966 W CN 2023082966W WO 2024011946 A1 WO2024011946 A1 WO 2024011946A1
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Prior art keywords
polypeptide dimer
systemic sclerosis
polypeptide
skin
administered
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PCT/CN2023/082966
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English (en)
Inventor
Hejian ZOU
Yu XUE
Weiguo WAN
Wenjing YE
Qian Wang
Wenyu GUO
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I-Mab Biopharma (Hangzhou) Co., Ltd
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Publication of WO2024011946A1 publication Critical patent/WO2024011946A1/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5431—IL-11
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • SSc Systemic sclerosis
  • ILD Interstitial lung disease
  • PAH pulmonary arterial hypertension
  • a gp130 homodimer is the second subunit of the IL-6 receptor complex. Dimerization of gpl30 results in transduction of an IL-6 or IL-11 signal. Initially described as the interleukin-6 signal transducer, gpl30 is a transducer chain shared by many cytokines, such as IL-6, IL-11, leukaemia inhibitory factor (LIF) , oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) .
  • cytokines such as IL-6, IL-11, leukaemia inhibitory factor (LIF) , oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) .
  • cytokines act via a bi-or tripartite receptor complex in which signaling is triggered by homodimerization (for IL-6) or heterodimerization of gpl30 with LIF-R (for LIF, CT-I, OSM, CLC and CNTF) or OSM-R (for OSM) .
  • LIF-R for LIF, CT-I, OSM, CLC and CNTF
  • OSM-R for OSM
  • the disclosure includes a polypeptide dimer as disclosed herein or a pharmaceutically acceptable salt thereof, or a composition containing either or both entities, for the treatment of systemic sclerosis (SSc) in a subject.
  • the disclosure also includes a method of treating systemic sclerosis (SSc) in a subject by administering the polypeptide dimer or a pharmaceutically acceptable salt thereof, or a composition containing either or both entities.
  • the disclosure further includes use of such a polypeptide dimer or a pharmaceutically acceptable salt thereof, or a composition containing either or both entities in the manufacture of a medicament for treating systemic sclerosis (SSc) in a subject.
  • the treatment comprises at least a second active agent.
  • the subject is human.
  • the systemic sclerosis comprises one or more of these diseases: diffuse cutaneous systemic sclerosis (dcSSc) , limited cutaneous systemic sclerosis (lcSSc) , overlap type of Systemic Sclerosis, undifferentiated type of Systemic Sclerosis, Systemic Sclerosis sine scleroderma, localized scleroderma, scleroderma without skin sclerosis, skin fibrosis, scleroderma, nephrogenic fibrosing dermopathy (NFD) , nephrogenic systemic fibrosis (NSF) , keloid formation, systemic sclerosis-associated pulmonary hypertension, systemic sclerosis-associated kidney failure, systemic sclerosis-associated malabsorption and systemic sclerosis-associated interstitial lung disease.
  • dcSSc diffuse cutaneous systemic sclerosis
  • lcSSc limited cutaneous systemic sclerosis
  • overlap type of Systemic Sclerosis undifferentiated type of Systemic Sclerosis
  • Systemic Sclerosis sine
  • the polypeptide dimer comprises two monomers, and each monomer has an amino acid sequence that has at least 90%sequence identity to SEQ ID NO: 1, for use in the treatment of systemic sclerosis (SSc) in a subject.
  • one or both of the monomers comprise an amino acid sequence of SEQ ID NO: 1.
  • one or both of the monomers comprise a Fc domain having an amino acid sequence of Cys-Pro-Pro-Cys, and the monomer dimerizes through one or more disulfide bonds formed by cysteine residues in the sequence of Cys-Pro-Pro-Cys.
  • each molecule of the polypeptide dimer contains no more than 6 molecules of galactose-alpha-1, 3-galactose.
  • the polypeptide dimer comprises glycans, and an average of at least 52%or 54%of the glycans include one or more sialic acid residues.
  • the polypeptide dimer is administered in a dosage from 0.5 mg to 5 g. In some embodiments, the dosage is administered at 0.75 mg, 7.5 mg, 60 mg, 75 mg, 150 mg, 300 mg, 600 mg or 750 mg. In some embodiments, the dosage is administered at 300 mg or 600 mg.
  • the polypeptide dimer is administered once or twice every 1, 2, 3, 4, 5, 6, 7, 14, 30 days, two months, three months, four months, or six months.
  • the polypeptide dimer is administered parenterally. In some embodiments, the polypeptide dimer is administered intraperitoneally, intravenously or subcutaneously.
  • the present disclosure provides a pharmaceutically acceptable salt of the polypeptide dimer described herein.
  • the present disclosure provides a composition comprising the polypeptide dimer described herein and/or the pharmaceutically acceptable salt described herein.
  • the polypeptide dimer is the active pharmaceutical ingredient (API) .
  • the composition additionally comprises a second active agent or a second pharmaceutical ingredient.
  • the present disclosure also provides a vector comprising a nucleic acid encoding an amino acid sequence of the polypeptide dimer described herein.
  • FIG. 1 shows that Collagen III, IL-11 and IL-11R ⁇ are significant elevated in skin of SSc.
  • C The relative expression of COL3, IL-11 and IL-11 R ⁇ .
  • FIG. 2 shows expressed IL-11 cells in skin tissue of SSc.
  • Duct cells (upper left) , hair root sheath cells (upper right) , endothelial cells (middle left) , keratinocytes (middle right) , apocrine cells (lower left) , fibroblasts (lower right) .
  • Black arrow IL-11 positive cells.
  • FIG. 3 shows western blot (left) and qPCR (right) of IL-11 overexpressed cells and controls. Cells were stable cultured for 48 hours before harvested. In FIG. 3, 4, 12 and 17, Mock: controls (Mock cells without any additional treatment) ; IL-11-His: IL-11 overexpressed cells.
  • FIG. 4 shows transwell assay for IL-11 overexpressed cells and controls.
  • FIG. 5 shows differentially expressed genes (DEGs) analysis for IL-11 overexpressed cells compared to controls using volcano plot for cells steady cultured for 24 hours.
  • DEGs differentially expressed genes
  • FIG. 6 shows DEGs analysis for IL-11 overexpressed cells compared to controls using volcano plot for cells steady cultured for 72 hours.
  • FIG. 7 shows DEGs associated with fibrosis in 24 hours (padj ⁇ 0.05 &fold-change >1.5 or ⁇ 0.67) .
  • FIG. 9 shows relative expression of ADAM10 in skin of SSc patients.
  • HC healthy controls.
  • Saline controls;
  • BLM BLM-induced-SSc mice.
  • FIG. 11 shows western blot assessing protein levels of supernatant sIL-11R ⁇ (left) or cell membrane IL-11R ⁇ (right) .
  • FIG. 12 shows expression of COL3, STAT3 and pSTAT3 in IL-11 overexpressed fibroblasts and control cells by Western blot. Iono 1 ⁇ M stimulated for 48h.
  • FIG. 13 shows western blot assessing protein levels of COL3, STAT3 and pSTAT3 in fibroblast.
  • Iono 1 ⁇ M and/or rhIL-11 150 ng/ml stimulated for 48h.
  • Iono Ionomycin
  • FIG. 16 shows skin thickness of three groups.
  • saline control group
  • BLM BLM group (BLM-induced-SSc mice)
  • TJ301 TJ301 intervention group (BLM-induced-SSc mice using TJ301 as intervention reagent) .
  • FIG. 17 shows western blot assessing protein levels of COL3, pSTAT3 and STAT3 in IL-11 overexpressedfibroblasts.
  • IL-11 overexpressedfibroblasts stimulated with Iono 1 ⁇ M and/or TJ301 1 ⁇ g/ml for 48h.
  • FIG. 18 shows western blot assessing protein levels of COL3, pSTAT3 and STAT3 for different stimulate in fibroblasts.
  • Cells stimulated with Iono 1 ⁇ M and/or rhIL-11 150 ng/ml and/or WP1066 4 ⁇ M for 48h. **** p ⁇ 0.0001.
  • FIG. 20 shows body weight curve of mice in 4 treatment groups.
  • FIG. 21 shows the blood IL-6 level in each group as determined by the enzyme-linked immunosorbent assay.
  • FIG. 22 shows the blood sIL-6R level in each group as quantified by the enzyme-linked immunosorbent assay.
  • FIG. 23 shows the immunohistochemical staining of ⁇ -SMA in the skin via Masson's trichrome, H&E, and Masson's trichrome staining.
  • FIG. 24 shows the skin thickness in the 4 cohorts.
  • the error bar represents the mean ⁇ standard deviation.
  • FIG. 25 shows the percentage of collagen in the skin, as determined by the Masson's trichrome.
  • the error bar represents the mean ⁇ standard deviation.
  • FIG. 26 shows the staining score of the fibroblasts in the skin that are ⁇ -SMA-positive.
  • the error bar represents the mean ⁇ standard deviation
  • FIG. 27 shows the collagen/wet weight ratio of collagen in the skin as determined by hydroxyproline quantification.
  • the error bar represents the mean ⁇ standard deviation
  • FIG. 28 shows the mRNA ratio of CD31, ⁇ -SMA, fibronectin, and collagen 1A2 in the 4 cohorts, respectively.
  • the error bar represents the mean ⁇ standard deviation.
  • FIG. 29 shows the western blot result of ⁇ -SMA, CD31, and type I collagen in the 4 cohorts.
  • FIG. 30 shows the bar chart of relative density of the ⁇ -SMA, CD31, and type I collagen as compared to GAPDH in the skin tissue transformed from the western blot result of FIG. 29.
  • the error bars represent the mean ⁇ standard deviation.
  • FIG. 31 shows immunohistochemical staining of SMA in lungs via H&E, Masson's trichrome and immunohistochemical (IHC) staining.
  • FIG. 32 shows the Ashcroft score of the lungs, indicating that BLM+saline group has developed fibrosis.
  • the error bars reflect the mean ⁇ standard deviation of three different tests.
  • FIG. 33 shows the percentage of collagen in the lungs by Masson's trichrome staining.
  • the error bars reflect the mean ⁇ standard deviation of three different tests.
  • FIG. 34 shows the staining score of ⁇ -SMA positive fibroblasts.
  • the error bars reflect the mean ⁇ standard deviation of three different tests.
  • FIG. 35 shows the collagen/wet weight ratio of lung collagen determined by hydroxyproline measurement.
  • the error bars reflect the mean ⁇ standard deviation of three different tests.
  • FIG. 36 shows the mRNA ratio encoding the Sma, Fibronectin, Collagen 1A2 (Col1A2) , and CD31, respectively.
  • the error bars reflect the mean ⁇ standard deviation of three different tests.
  • FIG. 37 shows the western blot of CD31, type I collagen, and ⁇ -SMA expressed in the lungs, respectively.
  • FIG. 38 shows the relative density of CD31, type I collagen, and ⁇ -SMA as compared to GAPDH in the lungs, respectively.
  • the error bars reflect the mean ⁇ standard deviation of three different tests.
  • FIG. 39 shows the phosphorylation level of Stat3 (Tyr931) , Akt (Ser473) and Smad3 (S423/S425) in the mice’s skin. Values are represented by mean ⁇ SD, pooled from three independent experiments.
  • FIG. 40 shows phosphorylation level of Stat3 (Tyr931) , Akt (Ser473) and Smad3 (S423/S425) in mice’s lung tissues. Values are represented by mean ⁇ SD, pooled from three independent experiments.
  • FIG. 41 shows the level of IL-6 and IL-6R in the supernatant of cells cultured in DMEC with 15%FBS for 6 hours after treatment by the enzyme-linked immunosorbent assay.
  • FIG. 42 shows the cell proliferation viability of HFF-1 cells within 48h.
  • FIG. 43 shows the mRNA ratio for collagen 1A2, fibronectin, and ⁇ -SMA under various treatment settings.
  • FIG. 44 shows the western blot result of type I collagen and ⁇ -SMA proteins.
  • HFF-1s Human foreskin fibroblasts
  • 15%SSc patients serum, 5 ng/ml of human TGF- ⁇ 1, or 50 ng/ml of IL-6 and 200 ng/ml of IL-6R complex, respectively, whereas the control HFF-1 group was treated with 15%FBS.
  • TGF- ⁇ group HFF-1s were given 5 ng/ml of human TGF- ⁇ 1 and 15%FBS; TGF- ⁇ +TJ301 group: HFF-1s were given 5 ng/ml of human TGF- ⁇ 1, 15%FBS and 200ng/mL of TJ301.
  • Control group HFF-1s were treated with 15%FBS. The average standard deviation was from three different tests represented each outcome.
  • FIG. 45 shows the expression level of IL-6 and IL-6R in the supernatant of DMEC cultured cells containing 15%FBS for 6h after therapy, as measured by ELISA.
  • FIG. 46 shows the migration rate of HDMECs examined at 12h, 24h and 48h in different cultures.
  • FIG. 47 shows the mRNA ratio of ⁇ -SMA and CD31 respectively in the 3 groups.
  • FIG. 48 shows the immunofluorescence of ⁇ -SMA and CD31 respectively in the 3 groups.
  • FIG. 49 shows the western blot result of phosphorylated Stat3 (Tyr931) and Akt (Ser473) in HFF-1 with TGF- ⁇ 1 alone or in combination with TJ301.
  • FIG. 50 shows the western blot result of phosphorylated Stat3 (Tyr931) and Akt (Ser473) in HDMEC with TGF- ⁇ 1 alone or in combination with TJ301.
  • the term "about” when modifying the quantity (e.g., mM, or M) of a substance or composition, the percentage (v/v or w/v) of a formulation component, the pH of a solution/formulation, or the value of a parameter characterizing a step in a method, or the like refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like.
  • "about” can mean a variation of ⁇ 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%or 10%.
  • disulfide bond includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors) . Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • pharmaceutically effective amount or “effective amount” means an amount whereby sufficient therapeutic composition or formulation is introduced to a patient to treat a diseased or condition.
  • mis level may vary according the patient's characteristics such as age, weight, etc.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as "to a patient in need of treatment” or "a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
  • “Pharmaceutically acceptable” refers to excipients (vehicles, additives) and compositions that can reasonably be administered to a subject to provide an effective dose of the active ingredient employed and that are "generally regarded as safe” e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human.
  • this term refers to molecular entities and compositions approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • Constantly modified variants or “conservative substitution” refers to substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule, even in essential regions of the polypeptide. Such exemplary substitutions are preferably made in accordance with those set forth in Table 1 as follows:
  • Sequence identity can be determined using a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.
  • the following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al, (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al, (1993) Nature Genet. 3: 266-272; Madden, T.L., et al, (1996) Meth. Enzymol. 266: 131-141; Altschul, S.F., et al, (1997) Nucleic Acids Res.
  • a "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation.
  • the reconstituted formulation is suitable for administration, (e.g. parenteral administration) , and may optionally be suitable for subcutaneous administration.
  • Reconstitution time is the time that is required to rehydrate a lyophilized formulation with a solution to a particle-free clarified solution.
  • a “stable formulation” is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage.
  • Various analytical techniques for measuring protein stability' are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N. Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993) .
  • Stability can be measured at a selected temperature for a selected time period.
  • a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2-8°C) for at least 12 months.
  • a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2-8°C) for at least 18 months.
  • stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 3 months.
  • stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 6 months.
  • stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 12 months.
  • stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 18 months.
  • the criteria for stability for a polypeptide dimer formulation are as follows.
  • the formulation is colorless, or clear to slightly opalescent by visual analysis.
  • the concentration, pH and osmolality of the formulation have no more than +/-10%change.
  • the disclosure includes a polypeptide dimer as disclosed herein which is a dimer comprising gp130 (also called “fusion protein comprising gp130” , or called “fusion protein” for short) or its pharmaceutically acceptable salt.
  • a polypeptide dimer as disclosed herein which is a dimer comprising gp130 (also called “fusion protein comprising gp130” , or called “fusion protein” for short) or its pharmaceutically acceptable salt.
  • the processes for preparing the dimer comprising gp130 are described in PCT/CN2021/143870 or PCT/EP2007/005812.
  • CHO-K1 cells were bought from German Collection of Microorganisms and Cell Cultures (Braunschweig, German) .
  • the disclosure includes a polypeptide dimer as disclosed herein which is a dimer of gp130-Fc fusion monomers or its pharmaceutically acceptable salt.
  • the dimer of gp130-Fc fusion monomers is soluble gp130-Fc (sgp130Fc) .
  • a molecule or ion with charge opposite to a drug when combining with the drug to form a salt, can improve certain undesirable physicochemical or biopharmaceutical properties of the drug, such as changing solubility or dissolution, reducing hygroscopicity, improving stability, or changing melting point for the drug.
  • the final determination of an ideal salt form requires proper balance between physicochemical properties and biopharmaceutical properties. Requirements, e.g. solubility, hygroscopicity, and stability to environmental factors in different states, should be given priority in selecting a pharmaceutically acceptable salt form of a drug.
  • the pharmaceutically acceptable salts of the polypeptide dimer of the present application may be in any suitable pharmaceutically acceptable salt form.
  • the pharmaceutically acceptable salt of the polypeptide dimer is a trifluoroacetate.
  • the pharmaceutically acceptable salt of the polypeptide dimer is an acetate.
  • the pharmaceutically acceptable salt of the polypeptide dimer is a hydrochloride.
  • the pharmaceutically acceptable salt of the polypeptide dimer is a phosphate.
  • the pharmaceutically acceptable salt of the polypeptide dimer is an acetate or a hydrochloride.
  • the polypeptide dimers may be produced, for example, by expressing the monomers, e.g. monomers comprising SEQ ID NO: 1, in cells.
  • Preferred polypeptide dimers of the disclosure include a dimer of gp130-Fc fusion monomers (e.g., two monomers of SEQ ID NO: 1) .
  • the polypeptide of SEQ ID NO: 1 exists as a dimer linked by two disulfide linkages at Cys601 (amino acid positions 601 of SEQ ID NO: 1) and Cys604 (amino acid positions 604 of SEQ ID NO: 1) .
  • Polypeptide dimers described herein preferably comprise gpl30-Fc monomers having the sequence corresponding to SEQ ID NO: l.
  • polypeptide dimers described herein comprise polypeptides having at least 90%, 95%, 97%, 98%, 99%or 99.5%sequence identity to SEQ ID NO: 1.
  • the polypeptide comprises the gpl30 D6 domain (in particular amino acids TFTTPKFAQGE: amino acid positions 585-595 of SEQ ID NO: 1) , AEGA in the Fc domain hinge region (amino acid positions 609-612 of SEQ ID NO: 1) and does not comprise a linker between the gpl30 portion and the Fc domain.
  • the disclosure provides a polypeptide dimer comprising two monomers having an amino acid sequence at least 90%sequence identify to SEQ ID NO: 1, wherein the amino acid sequence comprises the gpl30 D6 domain, AEGA in the Fc domain hinge region, and there is no linker present between the gpl30 portion and the Fc domain.
  • the monomer comprises a Fc domain having an amino acid sequence of Cys-Pro-Pro-Cys, and the monomer dimerizes through one or more disulfide bonds formed by cysteine residues in the sequence of Cys-Pro-Pro-Cys.
  • polypeptides it is desirable for polypeptides to be substantially free of galactose-alpha-l, 3-galactose moieties, as these are associated with an immunogenic response.
  • the polypeptide dimers of the disclosure have low levels of such moieties.
  • each molecule of the polypeptide dimer contains no more than 6 molecules of galactose-alpha-1, 3-galactose.
  • each molecule of the polypeptide dimer contains no more than 5, 4, 3, 2, or 1 molecules of galactose-alpha-1, 3-galactose or even an undetectable level of galactose-alpha-1, 3 -galactose (e.g., as measured by WAX-HPLC, NP-HPLC or WAX, preferably as determined by WAX-HPLC) .
  • the polypeptide dimer contains less than 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or even 0.1%of galactose-alpha-1, 3-galactose, relative to the total amount of glycans, either by mass or on a molar basis.
  • polypeptide of the disclosure is sialylated. This has the advantage of increasing the half-life of polypeptides of the disclosure.
  • Each chain of the polypeptide dimer contains 10 N-glycosylation sites; nine N-glycosylation sites are located in the gpl30 portion and one N-glycosylation site is located in the Fc portion.
  • the polypeptide therefore contains a total of 20 glycosylation sites. In certain embodiments, an average of at least 52% (e.g. at least 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%) or at least 54% (e.g.
  • the polypeptide of the disclosure has an approximate molecular weight of 220 kDa; each 93 kDa having an additional ⁇ 20 kDa molecular weight derived from 10 N-glycosylation chains.
  • the polypeptide dimers provided herein is TJ301 (having two monomers of SEQ ID NO: 1) as described in the EXEMPLIFICATION.
  • TJ301 exists as a dimer linked by two disulfide linkages at Cys601 (amino acid positions 601 of SEQ ID NO: 1) and Cys604 (amino acid positions 604 of SEQ ID NO: 1) .
  • Each molecule of TJ301 contains no more than 6, 5, 4, 3, 2, or 1 molecule of galactose-alpha-1, 3-galactose or even an undetectable level of galactose-alpha-1, 3 -galactose.
  • An average of at least 52%or at least 54%of glycans on TJ301 include a sialic acid residue, such as an average from 52-65%.
  • Processes for the preparation of TJ301 can be found in PCT/CN2021/143870 as the preparation of IM001.
  • a vector (also called “carrier” and “vehicle” ) comprising a nucleic acid encoding an amino acid sequence of the polypeptide dimer (e.g. SEQ ID NO: 1) is transfected into cells.
  • the design of the expression vector including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and so forth.
  • Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer) , Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer) , adenovirus, (e.g., the adenovirus major late promoter (AdMLP) ) , polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the host cell may be a mammalian, insect, plant, bacterial, or yeast cell, preferably the cell is a mammalian cell such as a CHO cell.
  • microvascular injury is thought to cause increased endothelial cell activation.
  • Activated endothelial cells are believed to express adhesion molecules resulting in altered capillary permeability allowing migration of inflammatory cells through the endothelium and entrapment in the vessel wall.
  • the immune activation is thought to contribute to sustained endothelial activation, which results in the breakdown of endothelial cells. This process is believed to contribute to the loss of elasticity and narrowing of the vessels commonly observed in scleroderma patients.
  • microvascular injury contributes to perivascular infiltrates of mononuclear cells in the dermis which is thought to contribute to the activation of fibroblasts and may of the associated hallmark symptoms of scleroderma.
  • Fibroblasts provide a structural framework (stroma) for many tissues, play an important role in wound healing and are the most common cells of connective tissue in animals. Fibroblasts are morphologically heterogeneous with diverse appearances depending on their location and activity.
  • scleroderma There are two major forms of scleroderma: limited systemic sclerosis/scleroderma and diffuse systemic sclerosis/scleroderma.
  • limited cutaneous scleroderma the fibrosis of the skin is generally confined to the area proximal to the elbow.
  • Patients with limited cutaneous scleroderma generally experience vascular impairment. Cutaneous and organ fibrosis generally progresses slowly in patients with limited scleroderma.
  • Patients with diffuse scleroderma generally experience fibrosis of skin and organs that progresses more rapidly than in limited scleroderma and/or widespread inflammation and/or more severe internal organ involvement than is seen in limited scleroderma.
  • the systemic sclerosis comprises diffuse cutaneous systemic sclerosis (dcSSc, or diffuse systemic sclerosis (dSSc) ) , limited cutaneous systemic sclerosis (lcSSc, or limited systemic sclerosis, (lSSc) ) , overlap type of Systemic Sclerosis, undifferentiated type of Systemic Sclerosis, Systemic Sclerosis sine scleroderma, localized scleroderma, scleroderma without skin sclerosis, skin fibrosis, scleroderma, nephrogenic fibrosing dermopathy (NFD) , nephrogenic systemic fibrosis (NSF) , keloid formation systemic sclerosis-associated pulmonary hypertension, systemic sclerosis-associated kidney failure, systemic sclerosis-associated malabsorption and systemic sclerosis-associated interstitial lung disease.
  • dcSSc diffuse cutaneous systemic sclerosis
  • lcSSc limited cutaneous systemic sclerosis
  • lSSc
  • Scleroderma is most commonly diagnosed by inspection of skin symptoms.
  • Tests to diagnosis include but are not limited to visual and/or manual inspection of the skin, blood pressure testing, chest x-ray, lung CT, echocardiogram, urinalysis, skin biopsy, and blood tests including antinuclear antibody testing, antitopoisomerase antibody testing, anticentromere antibody testing, anti-U3 antibody testing, anti-RNA antibody testing, other types of antibody testing, erythrocyte sedimentation rate, and rheumatoid factor.
  • the patient with systemic sclerosis has been classified according to the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for the classification of systemic scleroderma based on major criterion: proximal diffuse (truncal) sclerosis (skin tightness, thickening, and non-pitting induration) ; and minor criteria: (1) sclerodactyly (only fingers and/or toes) , (2) digital pitting scars or loss of substance of the digital finger pads (pulp loss) , and (3) bilateral basilar pulmonary fibrosis, wherein a patient with systemic sclerosis should fulfill the major criterion or two of the three minor criteria.
  • major criterion proximal diffuse (truncal) sclerosis (skin tightness, thickening, and non-pitting induration)
  • minor criteria (1) sclerodactyly (only fingers and/or toes) , (2) digital pitting scars or loss of substance of the digital finger pads (pul
  • the SSc is mediated via interleukin-11 (IL-11) and/or interleukin-6 (IL-6) pathway. In some embodiments, the SSc is mediated by upregulation of IL-11 and/or IL-6 pathway. In some embodiments, the polypeptide dimer provided herein down-regulates or blocks the IL-11 and/or IL-6 pathway.
  • IL-11 interleukin-11
  • IL-6 interleukin-6
  • no other medicament than the polypeptide dimer of the disclosure is administered to the subject to treat the SSc.
  • the polypeptide dimer is an active pharmaceutical ingredient (API) or active agent.
  • the polypeptide dimer is administered with a second active agent.
  • the second active agent can be one or more immunosuppressive agents, non-steroidal anti-inflammatory drugs (NSAIDs) , disease modifying anti-rheumatic drugs (DMARDs) , methotrexate (MTX) , anti-B-cell surface marker antibodies, anti-CD20 antibodies, rituximab, TNF-inhibitors, corticosteroids, and co-stimulatory modifiers, or any combination thereof.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs disease modifying anti-rheumatic drugs
  • MTX methotrexate
  • anti-B-cell surface marker antibodies anti-CD20 antibodies
  • rituximab TNF-inhibitors
  • corticosteroids corticosteroids
  • co-stimulatory modifiers or any combination thereof.
  • the second active agent as set forth herein are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99%of the heretofore-employed dosages. If such additional drugs are used at all, preferably, they are used in lower amounts than if the polypeptide dimer were not present, especially in subsequent dosings beyond the initial dosing with the polypeptide dimer, so as to eliminate or reduce side effects caused thereby.
  • the combined administration of a second active agent includes co-administration (concurrent administration) , using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents (medicaments) simultaneously exert their biological activities.
  • the polypeptide dimer or the second agent is administered in a dosage from 0.5 mg to 5 g (e.g. from 0.75 mg to 4g, from 0.75 mg to 3g, from 0.75 mg to 2g, from 0.75 mg to 1g, from 0.75 mg to 950 mg, from 0.75 mg to 900 mg, from 0.75 mg to 850 mg, from 0.75 mg to 800 mg, from 0.75 mg to 750 mg, from 0.75 mg to 700 mg, from 0.75 mg to 650 mg, from 0.75 mg to 600 mg, from 0.75 mg to 550 mg, from 0.75 mg to 500 mg, from 0.75 mg to 450 mg, from 0.75 mg to 400 mg, from 0.75 mg to 350 mg, from 0.75 mg to 300 mg, from 0.75 mg to 250 mg, from 0.75 mg to 200 mg, from 0.75 mg to 150 mg, from 0.75 mg to 100 mg, from 0.75 mg to 95 mg, from 0.75 mg to 90 mg, from 0.75 mg to 85 mg, from 0.75 mg to 80 mg, from 0.75 mg to 75
  • the dosage is administered at 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg or 1 g.
  • the dosage is administered at 0.75 mg, 7.5 mg, 60mg, 75 mg, 150 mg, 300 mg, 600 mg or 750 mg.
  • the polypeptide dimer and/or the second active agent is administered once every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, two months, three months, four months, or six months. In some embodiments, the polypeptide dimer and/or the second active agent is administered twice every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, two months, three months, four months, or six months. In a preferred embodiment, the polypeptide dimer and/or the second active agent is administered once every 14 days in human.
  • the polypeptide dimer and/or the second active agent is administered parenterally.
  • the polypeptide dimer and/or the second active agent is administered intradermally, inhalational, transdermaly (topically) , transmucosally, intraperitoneally, intravenously and/or subcutaneously. In a preferred embodiment, the polypeptide dimer and/or the second active agent is administered intravenously and/or subcutaneously.
  • Eligible study subjects were patients who fulfilled the 2013 ACR/EULAR classification criteria for SSc from Rheumatology department of Huashan Hosipital, Fudan University between November 1, 2020 and February 28, 2022. Skin tissue and whole blood were obtained from SSc and healthy (control) donors. The study was approved by ethics committee of Huashan Hospital, Fudan University (2021-470) , and written informed consent was obtained from all participants. The study complies with the declaration of Helsinki. The demographic information of the participants was summarized in Table 2 and 3.
  • DcSSc diffuse cutaneous SSc
  • lcSSc limited cutaneous SSc
  • mRSS modified Rodnan skin score
  • ILD interstitial lung disease
  • PAH pulmonary arterial hypertension
  • DU digital ulcer
  • NVC nailfold videocapillaroscopy.
  • IHC staining Human skin punch biopsies sample or mouse skin/pulmonary sample were fixed in formalin, embedded in paraffin and sectioned for haematoxylin and eosin (H&E) , masson or immunohistochemistry (IHC) staining.
  • Primary antibodies for IHC staining were anti-COL3 (1: 1000 dilution in 5%Bovine Serum Albumin [BSA] ) (Proteintech) , anti-IL-11 (1: 200 dilution in 5%BSA) (ThermoFisher) , anti-IL-11R ⁇ (1: 500 dilution in 5%BSA) (Abcam) and ADAM10 (1: 200 dilution in 5%BSA) (Affinity) .
  • Semi-quantitative analysis of IHC was performed by blinded investigators using image-pro plus (version 6.0) .
  • mice were housed under specific pathogen-free (SPF) conditions in our animal facility.
  • SPF pathogen-free
  • a total of 30 male C57/BL6 mice (6-8 weeks) were purchased from Shanghai SLAC Laboratory and separated to three group randomly.
  • Bleomycin (BLM) model group was injected with BLM (2U/kg) (Hanhui) subcutaneously into a single location on shaved backs of the mice daily for 28 days.
  • the intervention group was administrated with BLM daily and intraperitoneal injected with TJ301 (30 mg/kg) twice a week during the experiment for 28 days, i.e., two injections of TJ301 in week 1 (day 1 to day 7 of BLM injection) , two injections of TJ301 in week 2, two injections of TJ301 in week 3, and two injections of TJ301 in week 4 (day 22 to day 28 of BLM injection) .
  • Two control groups either subcutaneously injected with BLM as mentioned above or saline instead
  • mice After 28 days, mice were sacrificed and the skin and pulmonary were collected. Cells and RNA-seq analysis
  • IL-11 overexpressed cell line was established from human dermal fibroblast (HFF-1) (ATCC) .
  • HFF-1 human dermal fibroblast
  • IL-11 NCBI Accession No. NM_000641.4
  • IL-11R ⁇ NCBI Accession No. NM_001142784.3
  • HFF-1 overexpressed cells and control cells HFF-1 were steady for 24 or 72 hours, washed with pre-cold D-PBS (Gibico) , then added with trizol regent for extract RNA.
  • RNA-seq was performed by Illumina and analyzed by linux system and R v4.1.2.
  • IL-11R ⁇ overexpressed fibroblasts were incubated with serum-free medium and stimulated with 1 ⁇ M of ionomycin (Iono) or DMSO as negative control for 1 hour at 37°C.
  • Supernatant were harvested and centrifugated at 1, 2000g for 10 min using Amicon ultra-0.5 centrifugal filters (Millipore) , and cells were also harvested. Concentrated supernatant and harvested cells were prepared for western blot analysis as described below.
  • Cells were lysed by radioimmunoprecipitation assay (RIPA) lysis buffer and collected after centrifugation at 3000g for 15 min. The protein concentration was quantified and approximately 30 ⁇ g of total protein was loaded and run on 10%SDS-PAGE. The protein in the SDS-PAGE was transferred onto PVDF membranes (Millipore) . Then, the membranes were blocked with 5%non-fat milk and incubated with primary antibodies (Table 4) at 4 °C overnight, followed by incubation with the appropriate HRP-conjugated secondary antibody at room temperature for 1 h. The blots were scanned using imaging system (Biorad) .
  • imaging system Biorad
  • IHC immunohistochemistry
  • WB western blot.
  • a total of 2 ⁇ 10 3 cells without FBS DMEM was added to the upper chamber of the 24 well transwell plate (Corning) , and 10%FBS DMEM was added to the lower chamber. After culturing for 24 hours, cells were removed gently from inner of the upper chamber, and cells outside the upper chamber was stained and captured for analysis.
  • Plasma IL-11 levels in patients with SSc showed a tendency of elevation compared with controls
  • the unexpected low expression of plasma IL-11 is speculated to be associated with glucocorticoid usage.
  • Glucocorticoid may downregulate inflammation response, such as downregulate plasma IL-11 expression level.
  • dcSSc diffuse cutaneous SSc
  • mRSS modified Rodnan skin score
  • ESR erythrocyte sedimentation rate
  • ILD interstitial lung disease
  • PAH pulmonary arterial hypertension
  • DU digital ulcer
  • NVC nailfold videocapillaroscopy.
  • IL-11 and IL-11R ⁇ were significantly elevated in skin tissue of SSc patients
  • IL-11 and IL-11R ⁇ were significantly increased (2.5 folds and 2 folds, respectively) in skin of SSc patients than that of healthy controls (FIG. 1B and 1C) .
  • Several type of skin cells expressed IL-11 such as duct cells (upper left) , hair root sheath cells (upper right) , endothelial cells (middle left) , keratinocytes (middle right) , apocrine cells (lower left) , fibroblasts (lower right) (FIG. 2) .
  • Fibroblasts with high IL-11 expression showed a pro-fibrotic phenotype
  • IL-11 overexpressed skin fibroblasts was established and studied.
  • COL3 level was increased in the IL-11 overexpressed cells, and the ratio of pSTAT3/STAT3 was elevated as compared to controls (Mock cells without any additional treatment) in western blot (FIG. 3) .
  • Transwell assay showed that IL-11 overexpressed cells also exhibited higher ability of migration (FIG. 4) .
  • RNA-Seq analysis was performed in two time points. Volcano plot showed that the number of up-and down-regulation gene in 24 hours were much more than 72 hours (FIG. 5 and 6) , and differentially expressed genes (DEGs) associated with fibrosis such as FBN1, TGFB2, FGF2, EGF were significant up-regulated, and TIMP1, VEGFB were significant down-regulated in 24 hours samples (FIG. 7, p. adj ⁇ 0.05 &fold-change >1.5 or ⁇ 0.67) .
  • DEGs differentially expressed genes
  • DEGs were enriched in several pro-fibrosis biological process, including regulation of supramolecular fiber organization, response to transforming growth factor beta, tissue remodeling, collagen metabolic process for 24h (Table 6) and extracellular matrix organization, extracellular structure organization, wound healing, regulation of angiogenesis for 72h (data not shown) .
  • dysregulation of fibrosis-associated pathways was not found in Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (data not shown) .
  • IL-11 performed pro-fibrosis effect through trans-signal transduction pathway
  • IL-11 seems to play at least a role in promoting fibrosis through classic pathway via binding to its membrane receptor IL-11R ⁇ . Thus, whether the trans-signaling pathway of IL-11 participants in fibrosis process was investigated. Since ADAM10 could cut cell membrane IL-11R ⁇ into soluble IL-11R ⁇ (sIL-11R ⁇ ) , ADAM10 may reflect the relative level of sIL-11R ⁇ . Thus, IHC was performed, and ADAM10 expression level in the skin of SSc patients or skin and pulmonary of BLM induced-SSc mice was significantly elevated than their controls (FIG. 8-10) . Ionomycin (Iono) could cut cell membrane IL-11R ⁇ into soluble form by activating ADAM10 (FIG. 11) .
  • Ionomycin Iono
  • Stimulating IL-11 overexpressed cells with Iono could dramatically increase COL3 protein expression and increased STAT3 phosphorylation as compared to control group (using GAPDH as control group, FIG. 12 and 13) .
  • Intervention of IL-11 trans-signal transduction pathway by TJ301 can ameliorate the pro-fibrotic response by IL-11
  • TJ301 (sgp130Fc) , an inhibitor of IL-11 trans-signaling, was used as intervention reagent.
  • TJ301 ameliorates skin and lung fibrosis in BLM induced-SSc mice by reducing skin thickness, collagen deposition and expression level of COL3 (FIG. 14-16) and inhibits COL3 expression induced by IL-11 and Iono co-stimulation in fibroblasts (FIG. 17) .
  • the three groups are BLM group, TJ301 intervention group and control group.
  • BLM group refers to BLM-induced-SSc mice
  • TJ301 intervention group refers to BLM-induced-SSc mice using TJ301 as intervention reagent
  • control group refers to mice without SSc but injected saline instead of BLM.
  • TJ301 which ameliorates skin and lung fibrosis in BLM induced-SSc mice.
  • mice From the SLRC Laboratory in Shanghai, China, wild-type C57BL/6 mice were purchased. In a pathogen-free environment, they were maintained at 24°C with a 12 hour light/12 hour dark cycle. In order to begin the treatment, male mice between the ages of 6 and 8 weeks were utilized. All experiments were carried out according to the Animal Care and Use Committee at Fudan University specified the guidelines.
  • bleomycin was filtered after being diluted in phosphate buffer saline at a concentration of 300 g/ml.
  • 0.5U bleomycin (BLM) was subcutaneously administered (daily) to the mice's shaved backs using a 27-gauge needle for four weeks. On day 29, mice were sacrificed and samples were collected.
  • mice were treated with saline (BLM+saline) , MR16-1 (BLM+MR16-1) or TJ301 (BLM+TJ301) , respectively as described below.
  • Anti-mouse IL-6R antibody (herein after “MR16-1” ) was purchased from Chugai Pharmaceutical and administered to mice after dilution with saline to 0.26 mg/ml (100 ⁇ g per mouse, i. p. weekly) from day 0.
  • Sgp130-Fc (TJ301) was manufactured by Ferring Pharmaceuticals and was administered to mice after dilution with saline to 1.5mg/ml (30 mg/kg, i. p., twice a week) from day 0.
  • saline was administered i. p. weekly with the same volume as that of the MR16-1.
  • mice were injected with 200 ⁇ L saline subcutaneously on mice’s back (daily) , while the saline was also i. p. injected once per week with the same volume as that of MR16-1.
  • mice were treated as below:
  • Control cohort injected subcutaneously with 200 ⁇ L saline daily, and saline ip.qw for 4 weeks.
  • BLM+saline cohort injected with BLM ih. qd. and saline ip. qw for 4 weeks.
  • BLM+MR16-1 cohort injected with BLM ih. qd. and MR16-1 ip. qw for 4 weeks.
  • BLM+TJ301 cohort injected with BLM ih. qd. and TJ301 ip. biw for 4 weeks.
  • IL-6 and sIL-6R ⁇ immunoassay kits (R&D Infrastructure, Minneapolis, US) in accordance with the instructions of the manufacturer to measure the concentrations of IL-6 and sIL-6R in mouse serum were used.
  • cytokine/receptor level was determined by human IL-6 and IL-6R ⁇ immunoassay kit (Neobio Science, China) following the instructions.
  • Skin and lung tissues were fixed in 10%formalin, deposited in paraffin and cut into 4mm thick segments. A hematoxylin and eosin stain or Masson's trichrome stain was applied to each segment to determine its fibrosis level (Beyotime, China) .
  • Skin and lung fibrosis were assessed histologically and in addition by quantification of collagen via a hydroxyproline kit (Quick Zyme, Netherlands) to detect the collagen in tissue. The average distance between the points where the epidermis met the dermis and the dermis met the subcutaneous fat was used to calculate the dermal thickness. Image J was used to do this on 5 skin slices from every mouse. On stained histology specimens, the interstitial lung alterations were also quantified via the Ashcroft scale. These analyses were conducted by three independent observers. The collagen volume fraction of skin and lung tissues were assessed via Image J, basing on Masson’s trichrome staining.
  • the endogenous peroxidase activity was stopped by immersing the slides in a 3 percent H 2 O 2 solution after they had been cleansed of wax and rehydrated. To recover the antigens, the slides were boiled in an antigen removal solution. Slides were incubated for 30 minutes with UK-based Vector Laboratories' 5 percent goat serum. The tissue slices were incubated with a primary antibody against ⁇ -SMA as per usual protocols (Abcam, US) . Images were captured using standard light microscopes (a DMI4000B from Leica) magnified 100x, 200x, or 400x. Staining intensity was semi-quantitated as negative, +, ++, or +++, where +++ was defined as the concentration of intensity in the positive control slide.
  • HFF-1s and Human Dermal Microvascular Endothelial Cells were starved and differently treated with transformation growth factor-1 (Cellular Signaling Technology, US) at 5 ng/ml, recombinant human IL-6 (50ng/ml; Med Chem Express, US) and recombinant human IL-6R (200ng/ml; Med Chem Express, US) for 48h, comparing with SSc serum (15%) treated cells from the very start. Then cells received the therapy with TGF- ⁇ 1 (purchased from Cellular Signaling Technology, US) at 5 ng/ml, with or without TJ301 at 200ng/ml for 48h.
  • TGF- ⁇ 1 purchased from Cellular Signaling Technology, US
  • HFF-1s were washed with PBS, after that, each well received 10 ⁇ L of the working solution, which was then kept at 37°C for one hour. The absorbance at 450 nm was measured using a microplate reader.
  • Each well of a 6-well plate was seeded with 4 ⁇ 10 5 cells, and the cells were allowed to develop until confluence.
  • the monolayer was scratched with a point and cleaned with PBS.
  • the cells were cultured in full media with or without TGF- ⁇ 1 (5ng/ml) and TJ301 (200ng/ml) .
  • HDMECs were photographed at 0h, 12h, 24h and 48h.
  • the initial wound size was denoted by X0, while subsequent wound sizes were denoted by Xn.
  • RNA from the mouse clinical specimens as well as cells Trizol (Invitrogen, US) was used in accordance with the manufacturer's recommendations.
  • the PrimeScript RT reagent kit (Takara Bio, Japan) was utilized to reverse-copy RNA. Life Technologies' QuantStudio TM 6 Flex Real-Time PCR system or Applied Biosystems' ABI 7500 real-time PCR system were used for real-time RT-PCR, and Takara Bio's SYBR Green PCR Master Mix was used.
  • the fold inductions of PCR products were calculated.
  • mice ⁇ -Sma 5’-CCCAGACATCAGGGAGTAATGG -3’ (forward, SEQ ID NO: 2) and 5’-TCTATCGGATACTTCAGCGTCA-3’ (reverse, SEQ ID NO: 3) ; mouse Col1a2: 5’-TCGTGCCTAGCAACATGCC-3’ (forward, SEQ ID NO: 4) and 5’-TTTGTCAGAATACTGAGCAGCAA-3’ (reverse, SEQ ID NO: 5) ; mouse Col3a1: 5’-CTGTAACATGGAAACTGGGGAAA-3’ (forward, SEQ ID NO: 6) and 5’-CCATAGCTGAACTGAAAACCACC-3’ (reverse, SEQ ID NO: 7) ; mouse Fibronectin: 5’-GAGTACAGCTCGGATGACACG-3’ (forward, SEQ ID NO: 8) and 5’-GGCCCCTGGTGGCTATTTG-3’ (
  • Anti-Stat3 antibody Cellular Signal Technology, USA
  • Anti-phospho-Stat3 (Tyr931) antibody Cellular Signal Technology, USA
  • anti-Akt antibody Cellular Signal Technology, USA
  • anti-phosphor-Akt Ser473 antibody
  • anti- ⁇ -SMAd3 antibody Cellular Signal Technology, USA
  • a GAPDH-blocking antibody (Abcam, USA) were obtained.
  • ECL (Millipore, US) was used to visualize the immunoblots, and the Las-3000 Imaging Densitometer (Fujifilm, Japan) was utilized to measure the band densities for every phenotype. Protein concentrations were calculated by dividing the protein concentration by the GAPDH concentration.
  • HDMECs from various treatments were placed on coverslips and incubated for 20 minutes with 4 percent paraformaldehyde. Then, they received treatments for 15 minutes with 0.2 percent Triton X-100 to make them more permeable.
  • Cells were treated overnight at 4°C using mouse anti- ⁇ -SMA (1: 200; Affinity, China) and rabbit anti-CD31 antibodies after being blocked for 30 minutes with 3 percent BSA (1: 200; Affinity, China) .
  • Fluor647-conjugated anti-mouse IgG (H+L) (Affinity, China) were used as secondary antibodies.
  • the cells were counterstained with DAPI (Beyotime, China) . Images were captured by Leica DMI4000B microscope.
  • a mouse model of subcutaneous BLM-induced SSc (Liang M, Lv J, Zou L, et al. A modified murine model of systemic sclerosis: bleomycin given by pump infusion induced skin and pulmonary inflammation and fibrosis. Lab Invest. 2015; 95: 342-50) to investigate the role of IL-6 and its soluble receptor play in the onset of SSc was initially employed. The disease progressed in the model resembles symptoms in humans regarding both inflammatory and fibrotic phase was observed. All animal blood IL-6 and sIL-6R via enzyme-linked immunosorbent assay in the present or absence of MR16-1 or TJ301 (FIG. 19) was measured. Upon completion of the last injection (FIG.
  • a histological and physical examination was performed (day 29) .
  • the body weight of BLM treated mice decreased gradually during 28-day treatment period (FIG. 20) .
  • IL-6 levels in BLM-treated mice were significantly higher than in the control group (P is less than 0.01, FIG. 21) .
  • Mice of the BLM cohort showed reduced IL-6/sIL-6R expression when IL-6 signaling was disrupted (FIG. 21 and 22) .
  • the increased IL-6 and sIL-6R in BLM-treated mice group were prevented by TJ301 via inhibiting the IL-6 signaling pathway.
  • TJ301 reduced cutaneous fibrosis in SSc mice caused by BLM.
  • TJ301 a humanized soluble gp130Fc fusion protein that can cross react with mouse IL-6/sIL-6R, was then investigated if it benefits mice with BLM-induced fibrosis and skin thickening.
  • FIG. 23 to 30 demonstrated that in control mice cohort or the group treated with MR16-1 or TJ301 treatment, skin thickness and collagen deposition were significantly greater at the BLM injection site than at the saline injection location.
  • the histopathological findings clearly demonstrated that MR16-1 or TJ301 administration slowed down the thickening and fibrosis of the skin caused by BLM (FIG. 23, 24 and 25) .
  • mice that treated with BLM had a significantly larger number of myofibroblasts (P less than 0.001) .
  • BLM treated mice were administered with MR16-1 or TJ301, less myofibroblasts were observed than in the BLM+saline group (FIG. 23 and 26) .
  • Collagen in the skin was measured via testing hydroxyproline level. Both MR16-1 and TJ301 were able to drastically decrease collagen accumulation caused by BLM in the skin (FIG. 27) .
  • the mRNA and protein expression levels of skin fibrotic genes showed similar trend in the evaluation of Fibroblast-to-Myofibroblast Transition (FMT) and Endothelial to Mesenchymal Transition (EndoMT) .
  • FMT Fibroblast-to-Myofibroblast Transition
  • EndoMT Endothelial to Mesenchymal Transition
  • the protein or mRNA levels of ⁇ -SMA, type I collagen, Fibronectin, and Collagen 1A2 (mesenchymal markers) significantly increased after BLM treatment (FIG. 28, 29 and 30)
  • CD31 endothelial marker
  • FIG. 30 P less than 0.05
  • TJ301 inhibiting IL-6 signaling via IL-6 receptor antibody or sgp130-Fc (i.e. TJ301) could alleviate skin fibrosis and EndoMT in BLM-induced SSc mice.
  • TJ301 showed higher efficacy than MR16-1 in reverting BLM-induced collagen marker protein expression (FIG. 29 and 30) .
  • mice administered with MR16-1 or TJ301 had relatively normal lung structure and fewer ⁇ -SMA-positive fibroblasts, but mice given BLM had significant lung damage and aberrant collagen deposition. These alterations were consistent with the skin abnormalities (FIG. 31 and 34) .
  • BLM+MR16-1 or BLM+TJ301 group had lower levels of mRNA encoding ⁇ -SMA, Fibronectin, and Collagen 1A2 (FIG. 36) , lower levels of protein (FIG. 37 and 38) , and lower Ashcroft scores of lung fibrosis (FIG. 32) . They also had less collagen deposition as demonstrated by Masson's trichrome staining and hydroxyproline, respectively (FIG. 33 and 35) (P less than 0.05) . Mice who received MR16-1 or TJ301 revealed an increase in the endothelial cell marker CD31. Thus, these mice had lower EndoMT levels (FIG. 36, 37 and 38) .
  • TJ301 significantly decreased expression of phosphorylated Stat3, Akt and Smad3 in skin tissues of BLM-induced mice.
  • TJ301 alleviated TGF- ⁇ 1-induced FMT in fibroblasts.
  • TGF- ⁇ 1 an important fibrotic cytokine, participates in cell proliferation, differentiation and migration to regulate the healing process, thus influence the fibrosis of liver, kidney and lung.
  • TGF- ⁇ 1 substantially boosted both the protein and mRNA levels of ⁇ -SMA and type I collagen, while TJ301 reverted the increase of ⁇ -SMA, fibronectin, collagen 1A2, as well as the proliferation viability caused by TGF- ⁇ 1 (FIG. 42, 43 and 44) .
  • TJ301 alleviated TGF- ⁇ 1-induced EndoMT in endothelial cells.
  • EndoMT in conjunction to FMT, might also result in activated myofibroblasts in SSc (Manetti M, Romano E, Rosa I, et al. Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis. 2017; 76: 924-934) .
  • human dermal microvascular endothelial cells To recover the effects of TJ301 to myofibroblast transition in HDMECs treated with TGF- ⁇ 1, human dermal microvascular endothelial cells firstly received therapy with 15%serum from SSc patients, human TGF- ⁇ 1 (5ng/mls) , or IL-6 (50ng/mls) as well as IL-6R (200ng/mls) complex.
  • the assay showed that human dermal microvascular endothelial cells (HDMECs) expressed IL-6 and IL-6R, similar to that in human umbilical vein endothelial cells (FIG. 45) (Zegeye MM, Lindkvist M, K, et al.
  • TJ301 could decrease cell migration induced by TGF- ⁇ 1 significantly (P less than 0.05) (FIG. 46 and 47) .
  • TGF- ⁇ 1-treatment increased the expression of ⁇ -SMA, and reduced the expression of CD31 in HDMECs, thus induced EndoMT, while TJ301 slowed down the progression of EndoMT in endothelial cells (FIG. 48) .
  • TGF- ⁇ group HFF-1s were given 5 ng/ml of human TGF- ⁇ 1 and 15 %FBS; TGF- ⁇ +TJ301 group: HFF-1s were given 5 ng/ml of human TGF- ⁇ 1, 15 %FBS and 200ng/mL of TJ301.
  • Control group HFF-1s were treated with 15%FBS.
  • GAPDH was used to normalize the signals from total proteins and phosphorylated proteins. The ratio between phosphorylated and total proteins was then determined. All of the outcomes were the mean ⁇ standard deviation of 3 separate tests.

Abstract

L'invention concerne un dimère polypeptidique pour le traitement de la sclérose systémique.
PCT/CN2023/082966 2022-07-12 2023-03-22 Dimères polypeptidiques pour le traitement de la sclérose systémique WO2024011946A1 (fr)

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