WO2025083294A2 - Protéine de fusion aat-fc - Google Patents

Protéine de fusion aat-fc Download PDF

Info

Publication number
WO2025083294A2
WO2025083294A2 PCT/EP2025/055585 EP2025055585W WO2025083294A2 WO 2025083294 A2 WO2025083294 A2 WO 2025083294A2 EP 2025055585 W EP2025055585 W EP 2025055585W WO 2025083294 A2 WO2025083294 A2 WO 2025083294A2
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
less
seq
aat
domain
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/EP2025/055585
Other languages
English (en)
Other versions
WO2025083294A3 (fr
Inventor
Nikolay ZHUKOVSKY
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.)
Ageronix Sa
Original Assignee
Ageronix Sa
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 Ageronix Sa filed Critical Ageronix Sa
Publication of WO2025083294A2 publication Critical patent/WO2025083294A2/fr
Publication of WO2025083294A3 publication Critical patent/WO2025083294A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • 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
    • A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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

  • AAT-Fc fusion protein FIELD OF THE INVENTION discloses a fusion protein comprising an immunoglobulin IgG Fc domain and an alpha1-antitrypsin (AAT) polypeptide.
  • the present invention also discloses a method of treating or preventing a disorder or disease comprising administering a fusion protein comprising an immunoglobulin IgG Fc domain and an alpha1-antitrypsin (AAT) polypeptide to a subject.
  • AAT Alpha-1 antitrypsin
  • AAT is a protease inhibitor with diverse roles throughout the body.
  • AAT protects the lung by inhibiting neutrophil proteinases, but AAT has many other non-proteolytic functions that are anti-inflammatory, antiviral, and homeostatic.
  • AAT has many other non-proteolytic functions that are anti-inflammatory, antiviral, and homeostatic.
  • AATD is classically a disease of neutrophilic inflammation with an aggressive and damaging innate immune response contributing to emphysema and other pathologies.
  • AATD is one of the most common genetic disorders but considerably under-recognised (Crossley et al., Alpha-1 Antitrypsin Deficiency and Accelerated Aging: A New Model for an Old Disease?, June 2019, Drugs & Aging 36:823- 840).
  • the most clinically advanced therapy for the treatment of AATD is a fusion protein comprising an AAT polypeptide and an IgG4 Fc domain, as described in WO2013/003641.
  • AAT constructs are also useful in the treatment of diseases or disorders of the nervous system and acquired inflammatory conditions. For example, treatment with plasma-derived AAT has been shown to be effective in the treatment of Charcot-Marie-Tooth disease (CMT) in a mouse model (WO2023247736).
  • CMT Charcot-Marie-Tooth disease
  • PNS peripheral nervous system
  • CNS central nervous system
  • GNS Guillain-Barré Syndrome
  • PNs peripheral neuropathies
  • AD Alzheimer’s diseases
  • MS Multiple Sclerosis
  • ALS Amyotrophic Lateral Sclerosis
  • ischemia ischemia and traumatic brain injuries
  • depression and autism spectrum disorder have been all linked to mechanisms driven by activated microglia (Skaper et al. 2018).
  • CMT1A Charcot-Marie-Tooth disease
  • the major pathogenic component of peripheral neuropathies is damage to the myelin sheath, either after its abnormal development (dysmyelination) in the inherited forms (CMT1A-F and -X) or direct damage in the acquired ones (acute/chronic inflammatory demyelinating polyneuropathy; AIDP/CIDP).
  • Myelin is produced by Schwann cells (SCs) in the PNS and is crucial for proper transmission of electric impulses in the nerves.
  • SCs Schwann cells
  • TACE/ADAM-17 is a transmembrane protein that includes an extracellular zinc-dependent protease domain.
  • ADAM-17 is known for its inhibitory effect on SCs mediated myelination by cleaving NRG1-III in the epidermal growth factor domain in a ligand independent manner (La Marca, R., 2011, Nat Neurosci 14(7): 857-865.).
  • Conflicting evidence has been reported in the literature with respect to the role of the human protease AAT. Specifically, in 2013, AAT was shown not to interact with TACE/ADAM-17 (van't Wout E. F.
  • the present invention provides a fusion protein comprising an immunoglobulin IgG Fc domain and an alpha1-antitrypsin (AAT) polypeptide, wherein the AAT polypeptide is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain.
  • the fusion protein further comprises one or more features which reduce the immunogenicity of the fusion protein and/or increase the serum half-life of the fusion protein.
  • the fusion protein may comprise any of the features (a) to (c), or any combination thereof: (a) the Fc domain comprises mutations corresponding to L234A or F234A, L235A, and P329G (“LALAPG mutation”); (b) the Fc domain comprises mutations corresponding to M252Y, S254T, and T256E (“YTE mutation”); and/or (c) the fusion protein comprises a linker between the Fc domain and the AAT polypeptide, wherein the linker comprises at least 5 amino acids, optionally wherein the linker is a glycine-serine linker and/or comprises the amino acid sequence of SEQ ID NO: 24.
  • fusion proteins comprising features (a), (b) and/or (c) have reduced immunogenicity compared with fusion proteins not comprising these features.
  • fusion proteins of the invention may have reduced immunogenicity compared with the fusion protein of SEQ ID NO: 20 or 34 (as described in WO2013/003641), which is currently the most clinically advanced AAT-Fc construct, without any significant loss of ADAM-17 inhibitory activity.
  • Fusion proteins of the invention which comprise the YTE mutation, or a combination of the LALAPG and YTE mutations, have reduced immunogenicity compared to equivalent fusion proteins not comprising these mutations, or fusion proteins comprising the LALAPG mutation alone.
  • fusion proteins comprising the combination of the LALAPG and YTE mutations have particularly low immunogenicity. Reduced immunogenicity is advantageous as it reduces the risk of side effects in patients and allows higher doses of the fusion protein to be used safely, thereby increasing the efficacy of the treatment.
  • This result is particularly surprising in the context of fusion proteins of the invention comprising an IgG1 Fc domain, as the literature suggests that the IgG1 Fc domain is more immunogenic than the IgG4 Fc domain (such as the IgG4 Fc domain present in SEQ ID NO: 20 or 34) (see e.g. Wang W, Maliecrud JCL, Damelang T, Vidarsson G, Heck AJR, Reiding KR.
  • the increased immunogenicity described for the IgG1 Fc domain compared to the IgG4 Fc domain may be due to one or more of the following factors: - The Fc region of IgG1 binds more strongly to activating Fc receptors (such as Fc ⁇ RIIIa) compared to IgG4; - IgG4 binds more effectively to the Fc ⁇ RIIB inhibitory receptor, which can suppress immune activation. This reduced activation makes IgG4 less immunogenic and less likely to induce inflammatory responses; - IgG4 is unique in that it has a propensity to undergo "Fab-arm exchange", where the heavy chains of different IgG4 molecules swap, resulting in antibodies that have different antigen-binding arms.
  • Fc receptors such as Fc ⁇ RIIIa
  • IgG1 has a more stable structure and does not undergo this Fab-arm exchange, making it more recognisable, which can trigger an up-regulation in cytokine response.
  • the present inventors have surprisingly found that fusion proteins comprising a combination of the LALAPG and YTE mutations have an increased half-life compared to equivalent fusion proteins not comprising this combination of mutations, or compared to fusion proteins comprising only the YTE mutation, and compared to a fusion protein of SEQ ID NO: 20 or 34.
  • fusion proteins comprising a combination of the LALAPG and YTE mutations, show an increased yield following purification than equivalent proteins not comprising a combination of these mutations.
  • the present invention provides a fusion protein comprising: (i) an immunoglobulin IgG Fc domain, and (ii) an alpha1-antitrypsin (AAT) polypeptide, wherein the AAT polypeptide is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain, and wherein: (a) the Fc domain comprises mutations corresponding to L234A or F234A, L235A, and P329G; (b) the Fc domain comprises mutations corresponding to M252Y, S254T, and T256E; and/or (c) the fusion protein comprises a linker between the Fc domain and the AAT polypeptide, wherein the linker comprises at least 5 amino acids, optionally wherein the linker is a glycine-serine linker and/or comprises the amino acid sequence of SEQ ID NO: 24.
  • AAT alpha1-antitrypsin
  • the present invention provides a dimer comprising a fusion protein of the invention and a second protein.
  • the present invention provides a vector comprising a nucleic acid sequence encoding a fusion protein or a dimer of the invention.
  • the present invention provides a host cell comprising, or expressing, a fusion protein, a dimer or a vector of the invention.
  • the present invention provides a pharmaceutical composition comprising a fusion protein, a dimer, a vector, or a host cell of the invention.
  • the present invention provides a fusion protein, a dimer, a vector, a host cell, or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder.
  • the present invention provides a method of treating or preventing a disease or disorder comprising administering a fusion protein, a dimer, a vector, a host cell, or a pharmaceutical composition of the invention to a subject.
  • the present invention provides the use of a fusion protein, a dimer, a vector, a host cell, or a pharmaceutical composition of the invention in the manufacture of a medicament for use in a method of treating or preventing a disease or disorder.
  • the present invention provides a method of inhibiting ADAM-17 in a cell, the method comprising contacting the cell with a fusion protein, a dimer, a vector, a host cell, or a pharmaceutical composition of the invention.
  • the present invention provides a method of promoting remyelination and/ or arresting demyelination of the axons of the PNS in a subject, the method comprising administering to the subject a fusion protein, a dimer, a vector, a host cell, or a pharmaceutical composition of the invention.
  • the present invention provides a method of producing a fusion protein or a dimer of the invention.
  • the present invention provides a method of purifying a fusion protein or a dimer of the invention.
  • the present invention provides a fusion protein for use in the prevention and/or treatment of a disease or disorder of the nervous system, wherein said fusion protein comprises (i) an immunoglobulin IgG Fc domain, a variant or a fragment thereof, and (ii) an alpha1-antitrypsin (AAT) polypeptide, a variant, an isoform and/or a fragment thereof, wherein the AAT polypeptide, variant, isoform and/or fragment thereof, is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain, variant or fragment thereof.
  • AAT alpha1-antitrypsin
  • the present invention also provides a nucleic acid sequence encoding a fusion protein of the invention.
  • the present invention provides a vector comprising a nucleic acid sequence encoding a fusion protein for use of anyone of the invention.
  • the present invention also provides a host cell comprising, or expressing, a fusion protein for use of the invention or a vector of the invention.
  • the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of i) a fusion protein for use of the invention, ii) a vector of the invention or iii) a host cell of the invention, and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • the present invention further provides a method of treatment and/or prevention of a disease or disorder of the nervous system, comprising administering a fusion protein of the invention or a pharmaceutical composition of the invention, to a subject in need thereof.
  • FIGURES Figure 1. SDS-PAGE coomassie-stained with AAT from multiple sources Figure 2. Inhibitory effects of AAT-Fc and of AAT from different sources on the neutrophil elastase activity expressed as percent of control (untreated) enzymatic activity Figure 3. The graph shows ADAM-17 activity. Plasma-derived AAT (Sigma Aldrich). Figure 4. Scheme of the experiment. Test plasma-derived AAT and AAT-derived peptides on TNF ⁇ stimulated HSC. Figure 5. NF-kB immunostaining. Blue nuclei (staining with DAPI) and green NF-kB protein. As a result of TNF ⁇ stimulation, NF-kB translocates from the cytoplasm to the nucleus.
  • the IC50 value is 0.25 ⁇ M.
  • Figure 7. MHCII reporter expression in microglia derived from iPSCs. The graph represents the MHCII luciferase expression normalized to cell viability for the indicated treatments. Interferon ⁇ (IFN ⁇ ) was used to stimulate MHCII response. The experiment was conducted in technical triplicates.
  • Figure 8. MHCII reporter expression in microglia derived from iPSCs co-cultured with normal and Parkinson’s neurospheres. Interferon ⁇ (IFN ⁇ ) was used to stimulate MHCII response. The AAT treatment (50 ⁇ M, Sigma Aldrich) decreased MHCII reporter activity both in microglia co-cultured with normal neurospheres (Fig.
  • ADAM-17 activity was measured using a fluorescence-based enzymatic inhibition assay at different concentrations (8, 6, 3, 2.5, 2, 1, 0.5, 0.1 ⁇ M). The experiment was conducted in technical triplicates. IC50 values were determined using GraphPad Prism based on percentage of ADAM-17 activity inhibition. Figure 13. Neutrophil elastase activity inhibition by AAT-Fc derivatives and IC50 values were measured using a neutrophil elastase inhibition assay.
  • MeOSuc-AAPV- pNA l00 ⁇ M Substrate MeOSuc-AAPV- pNA l00 ⁇ M, purified human neutrophil elastase (ELA2) 2.2 ⁇ U/ ⁇ L and several concentrations of AAT-Fc (100, 30, 10, 5, 3, 2 nM) were incubated together for 30 min. Absorbance was measured every minute in a spectrophotometer reader (A405 nm). Vmax value was determined with the trendline slope during 10 min. The experiment was repeated twice. Error bars indicate standard deviation. Figure 14. The mRNA levels of pro-inflammatory cytokines (IL-1 ⁇ , TNF ⁇ , IL-6, and IL-4) upon AAT treatment in Schwann cells (SCs).
  • IL-1 ⁇ pro-inflammatory cytokines
  • mRNA levels of pro-inflammatory cytokines IL-6, TNF ⁇ , and NF ⁇ BIA
  • SCs Schwann cells
  • Human SCs were plated in a 24-well plate at a density of 70000 cells per well. Two days after seeding, AAT-Fc pre-treatment was initiated for 1 day at 3 ⁇ M with or without LPS 1 ⁇ g/mL or TNF ⁇ 10 ng/mL. Then, the medium was replaced with fresh medium containing 3 ⁇ M AAT-Fc for 2 days with or without LPS 1 ⁇ g/mL or TNF ⁇ 10 ng/mL. Subsequently, the cells were harvested for RNA extraction. Gene expression was analysed by qPCR, with relative expression normalized to a housekeeping gene and visualized using GraphPad Prism. Figure 16D represent the 2(DCt) raw data.
  • the present invention provides a fusion protein comprising: (i) an immunoglobulin IgG Fc domain, and (ii) an alpha1-antitrypsin (AAT) polypeptide, wherein the AAT polypeptide is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain, and wherein: (a) the Fc domain comprises mutations corresponding to L234A or F234A, L235A, and P329G; (b) the Fc domain comprises mutations corresponding to M252Y, S254T, and T256E; and/or (c) the fusion protein comprises a linker between the Fc domain and the AAT polypeptide, wherein the linker comprises at least 5 amino acids, optionally wherein the linker is a glycine-serine linker and/or comprises the amino acid sequence of SEQ ID NO: 24.
  • AAT alpha1-antitrypsin
  • the fusion protein may have the following features: (a) the Fc domain is an IgG1 Fc domain and comprises mutations corresponding to L234A, L235A, and P329G; (b) the Fc domain is an IgG1 Fc domain and comprises mutations corresponding to M252Y, S254T, and T256E; (c) the Fc domain is an IgG1 Fc domain and comprises mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (d) the Fc domain is an IgG4 Fc domain and the fusion protein comprises a linker between the Fc domain and the AAT polypeptide, wherein the linker comprises at least 5 amino acids, optionally wherein the linker is a glycine-serine linker and/or comprises the amino acid sequence of SEQ ID NO: 24.
  • the fusion proteins and the dimers of the invention have lower immunogenicity than a fusion protein consisting of the amino acid sequence of SEQ ID NO: 20 or 34, or a dimer consisting of two fusion proteins consisting of the amino acid sequence of SEQ ID NO: 20 or 34.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, and P329G have lower immunogenicity than equivalent fusion proteins not comprising mutations corresponding to L234A, L235A, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E have lower immunogenicity than equivalent fusion proteins not comprising mutations corresponding to M252Y, S254T, and T256E.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G have lower immunogenicity than equivalent fusion proteins not comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E and comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25 have lower immunogenicity than equivalent fusion proteins not comprising mutations corresponding to M252Y, S254T, and T256E and not comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • immunogenicity may refer to the inflammation and/or cytokine release triggered by treatment with a fusion protein or a dimer of the invention. Immunogenicity may also refer to expression of pro-inflammatory transcription factors such as NF ⁇ BIA.
  • the fusion proteins and the dimers of the invention trigger a reduced inflammatory response in a cell and/or in a subject compared to a fusion protein consisting of the amino acid sequence of SEQ ID NO: 20 or 34, or a dimer consisting of two fusion proteins consisting of the amino acid sequence of SEQ ID NO: 20 or 34.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, and P329G trigger a reduced inflammatory response in a cell and/or in a subject compared to an equivalent fusion proteins not comprising mutations corresponding to L234A, L235A, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E trigger a reduced inflammatory response in a cell and/or in a subject compared to an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G trigger a reduced inflammatory response in a cell and/or in a subject compared to an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E and comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25 trigger a reduced inflammatory response in a cell and/or in a subject compared to an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E and not comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • the immunogenicity is measured by contacting human Schwann cells with the fusion protein or the dimer.
  • immunogenicity may be measured by contacting the cells with the fusion protein or the dimer, harvesting the cells, extracting RNA, and analysing the expression of at least one cytokine and/or pro-inflammatory transcription factor.
  • the method may further comprise treating the Schwann cells with pro-inflammatory factors, such as LPS and/or TNF ⁇ .
  • the at least one cytokine is selected from IL-1 ⁇ , TNF ⁇ , IL-6, and/or IL-4.
  • the at least one cytokine comprises IL-1 ⁇ .
  • the at least one cytokine comprises TNF ⁇ .
  • the at least one cytokine comprises IL-6.
  • the at least one cytokine comprises IL-4. In some embodiments, the at least one cytokine comprises IL-1 ⁇ , TNF ⁇ , IL-4, or IL-6, or any combination thereof. In some embodiments, the pro-inflammatory transcription factor is NF ⁇ BIA.
  • the fusion protein promotes less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cytokine and/or pro- inflammatory transcription factor expression promoted by a fusion protein consisting of the amino acid sequence of SEQ ID NO: 20 or 34, or a dimer consisting of two fusion proteins consisting of the amino acid sequence of SEQ ID NO: 20 or 34.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, and P329G promote less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cytokine and/or pro-inflammatory transcription factor expression promoted by an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E promote less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cytokine and/or pro-inflammatory transcription factor expression promoted by an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G promote less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cytokine and/or pro- inflammatory transcription factor expression promoted by an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E and comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25 promote less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cytokine and/or pro-inflammatory transcription factor expression promoted by an equivalent fusion proteins not comprising mutations corresponding to M252Y, S254T, and T256E and not comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • the fusion protein or the dimer can inhibit and/or impair the activity of ADAM metallopeptidase domain 17 (ADAM-17).
  • ADAM-17 ADAM metallopeptidase domain 17
  • inhibition of ADAM-17 is measured by incubating ADAM-17 with increasing concentrations of the fusion protein or the dimer and a fluorogenic ADAM-17 substrate, measuring the fluorescence, and calculating an IC50 value for the fusion protein or the dimer.
  • the IC50 of the fusion protein or the dimer is less than 10 ⁇ M, less than 9 ⁇ M, less than 8 ⁇ M, less than 7 ⁇ M, less than 6 ⁇ M, less than 5 ⁇ M, less than 4 ⁇ M, less than 3 ⁇ M, less than 2 ⁇ M, or less than 1 ⁇ M.
  • the IC50 of the fusion protein or the dimer is between 0.5 ⁇ M and 10 ⁇ M. In some embodiments, the IC50 of the fusion protein or the dimer is between 0.5 ⁇ M and 5 ⁇ M. In some embodiments, the IC50 of the fusion protein or the dimer is between 0.5 ⁇ M and 4 ⁇ M. In some embodiments, the IC50 of the fusion protein or the dimer is between 0.5 ⁇ M and 2 ⁇ M. In some embodiments, the IC50 of the fusion protein or the dimer is between 0.5 ⁇ M and 1 ⁇ M. In some embodiments, the fusion protein or the dimer can inhibit and/or impair the activity of neutrophil elastase (NE).
  • NE neutrophil elastase
  • inhibition of NE is measured by incubating NE with the fusion protein or the dimer and a fluorogenic NE substrate, and measuring the fluorescence, and calculating an IC50 value for the fusion protein or the dimer.
  • the IC50 of the fusion protein or the dimer is less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, or less than 10nM. In some embodiments, the IC50 of the fusion protein or the dimer is between 1nM and 100nM.
  • the IC50 of the fusion protein or the dimer is between 1nM and 50nM. In some embodiments, the IC50 of the fusion protein or the dimer is between 1nM and 40nM. In some embodiments, the IC50 of the fusion protein or the dimer is between 1nM and 20nM. In some embodiments, the IC50 of the fusion protein or the dimer is between 1nM and 10nM. In some embodiments, the fusion protein or the dimer has a lower IC50 for NE compared to a fusion protein consisting of the amino acid sequence of SEQ ID NO: 20 or 34, or a dimer consisting of two fusion proteins consisting of the amino acid sequence of SEQ ID NO: 20 or 34.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, and P329G have a lower IC50 for NE compared to an equivalent fusion proteins not comprising mutations corresponding to L234A, L235A, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E have a lower IC50 for NE compared to an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G have a lower IC50 for NE compared to an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E and comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25 have a lower IC50 for NE compared to an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E and not comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • the fusion protein or the dimer has increased serum half-life compared to a fusion protein consisting of the amino acid sequence of SEQ ID NO: 20 or 34, or a dimer consisting of two fusion proteins consisting of the amino acid sequence of SEQ ID NO: 20 or 34.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, and P329G have increased serum half-life compared to an equivalent fusion proteins not comprising mutations corresponding to L234A, L235A, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E have increased serum half-life compared to an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E.
  • fusion proteins of the invention comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G have increased serum half-life compared to an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G.
  • fusion proteins of the invention comprising mutations corresponding to M252Y, S254T, and T256E and comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25 have increased serum half-life compared to an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E and not comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • the serum half-life is measured by administering the fusion protein or the dimer to a mouse, taking blood samples from the mouse at various time points (for example at day 1, day 2, day 3, day 4, day 6, day 8, day 10, day 14, day 17, and day 21), and measuring the concentration of the fusion protein or the dimer in the samples using an ELISA.
  • the fusion proteins and the dimers of the invention have a serum half-life of at least 100 hours, at least 110 hours, at least 120 hours, at least 130 hours, at least 140 hours, at least 150 hours, at least 160 hours, at least 170 hours, at least 180 hours, at least 190 hours, at least 200 hours, at least 210 hours, at least 220 hours, or at least 230 hours.
  • the fusion proteins of the invention have: (a) an IC50 of between 0.5 ⁇ M and 1 ⁇ M for ADAM-17; (b) an IC50 of between 1nM and 10nM for NE; and (c) a serum half-life of at least 210 hours.
  • the present invention also contemplates an amino acid sequence as set forth in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO. 3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO.
  • the fusion protein can form a homodimer or a heterodimer.
  • one AAT-Fc dimerizes with a second AAT-Fc domain thereby forming a homodimer wherein the sequence of the second AAT-Fc is similar to the sequence of the first AAT-Fc (i.e. symmetric homodimer).
  • a non-limiting example of a homodimeric fusion protein is represented in Figure 9 that can be formed by 2X SEQ ID NO.9, 2X SEQ ID NO. 10, or 2X SEQ ID NO.16.
  • the two AAT-Fcs can be linked through one or more disulfide bond(s).
  • the present invention provides a dimer comprising a fusion protein of the invention and a second protein.
  • the second protein is identical to the fusion protein, i.e. the fusion protein and the second protein comprise or consist of the same amino acid sequence.
  • one AAT-Fc dimerizes with an other domain thereby forming a heterodimer (i.e. asymmetric heterodimer) wherein the other domain is an Fc domain or an Fc comprising a signal domain.
  • a non-limiting example of a heterodimeric fusion protein is represented in Figure 10 that can be formed by SEQ ID NO. 15 and SEQ ID NO.12 or SEQ ID NO.11 and SEQ ID NO.12.
  • the fusion protein and second protein may comprise so called “knob in hole” mutations in their respective Fc domains.
  • the Fc domain of the fusion protein may comprise a mutation corresponding to T366W (according to Kabat numbering of the Fc domain).
  • the Fc domain of the second protein may comprise one or more mutations corresponding to T366S, L368A, Y407V, H435R, and/or Y436F (according to Kabat numbering of the Fc domain).
  • the second protein does not comprise an AAT polypeptide.
  • the second protein comprises or consists of an amino acid sequence of SEQ ID NO: 23.
  • Alpha1-Antitrypsin is a protein that naturally occurs in the human body. It is derived from blood plasma (pd) by pharmaceutical companies worldwide for the treatment of AAT-deficiency, a hereditary disorder.
  • the AAT of the invention is a human sequence, most preferably as set forth in SEQ ID No. 1 (see Table 1) or SEQ ID NO: 28, 35, or 36.
  • AAT protein can be obtained by isolation from blood (e.g. human blood) or can be produced recombinantly.
  • the AAT polypeptide comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of any one of SEQ ID NO: 28, 35, or 36.
  • the AAT polypeptide comprises or consists of an amino acid sequence that is identical to the amino acid sequence of any one of SEQ ID NO: 28, 35, or 36.
  • a "fragment" of an AAT protein or Fc protein, peptide or polypeptide of the invention refers to a sequence containing less amino acids in length than the AAT protein, peptide or polypeptide of the invention. This sequence can be used as long as it exhibits the same properties, i.e. is biologically active, as the native AAT sequence from which it derives.
  • the fragment shares about 5 consecutive amino-acids, at least about 7 consecutive amino-acids, at least about 15 consecutive amino-acids, at least about 20 consecutive amino- acids, at least about 25 consecutive amino-acids, at least about 30 consecutive amino-acids, at least about 35 consecutive amino-acids, at least about 40 consecutive amino-acids, at least about 45 consecutive amino-acids, at least about 50 consecutive amino-acids, at least about 55 consecutive amino-acids, at least about 60 consecutive amino-acids, at least about 100 consecutive amino-acids, at least about 150 consecutive amino-acids, at least about 200 consecutive amino-acids, at least about 300 consecutive amino-acids, etc... or more of the native human AAT amino acid sequence.
  • the AAT fragment is a sequence containing less amino acids in length than the C-terminal AAT sequence 374-418 (SEQ ID NO.2).
  • the AAT fragment comprises, or consists of, SEQ ID NO. 2.
  • AAT or Fc that is an amino acid sequence that vary from the native sequence by amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles.
  • the amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence, e.g. at the N- or C-terminal sequence or within the amino acid sequence.
  • Substitutions can also be conservative, in this case, the conservative amino acid substitutions are herein defined as exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly II. Polar, positively charged residues: His, Arg, Lys III.
  • Non-limiting examples of a variant e.g. of a variant of an Fc sequence, is shown in SEQ ID NO. 11 (substitution T366W), SEQ ID NO. 12 (substitutions T366S, L368A, Y407V, H435R, and Y436F), SEQ ID NO. 11 / SEQ ID NO. 15 (substitution T366W).
  • “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% or at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified sequence.
  • homology can be determined by readily available computer programs or by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
  • the peptidic variants may be linear peptides or cyclic peptides and may be selected from the group comprising short cyclic peptides derived from the C-terminal sequence as set forth in SEQ ID No. 2.
  • the short cyclic peptides derived from the C-terminal sequence of Alpha1-Antitrypsin will be selected from the non-limiting group comprising Cyclo- (CPFVFLM)-SH, Cyclo-(CPFVFLE)-SH, Cyclo-(CPFVFLR)-SH, and Cyclo-(CPEVFLM)- SH, or any combination thereof.
  • an "isoform" of an AAT protein, peptide or polypeptide of the invention refers to a splice variant resulting from alternative splicing of the AAT mRNA. Isoforms of AAT are known in the art (see e.g.
  • amino acid sequence of AAT, the variant, isoform or fragment thereof, as described herein is at least 80% identical to the corresponding amino acid sequence in SEQ ID NO: 1.
  • the amino acid sequence of AAT, the variant, isoform or fragment thereof is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a corresponding amino acid sequence in SEQ ID NO: 1.
  • the molecule that increases or potentiates the capacity of AAT to inhibit a human protease polypeptide, fragment or variant thereof is preferably an Fc domain of an immunoglobulin G (IgG), a fragment or variant of thereof.
  • IgG immunoglobulin G
  • the Fc domain of an IgG is preferably a Fc domain of a mouse or a human IgG, most preferably of a human IgG1, IgG2, IgG3 or IgG4, a fragment or a variant thereof.
  • the Fc domain of a human IgG is selected from the group comprising, or consisting of, an IgG1 Fc (SEQ ID No. 3), an IgG2 Fc (SEQ ID No. 6), an IgG3 Fc (SEQ ID No. 7), and an IgG4 Fc (SEQ ID No.8), a fragment, a variant, or a combination of one or more of these sequences.
  • the Fc domain comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of any one of SEQ ID NOs: 29 to 33. In some embodiments, the Fc domain comprises or consists of an amino acid sequence that is identical to the amino acid sequence of any one of SEQ ID NOs: 29 to 33. In some embodiments, the Fc domain comprises a wild-type hinge region. The hinge region is described in, for example, Rispens, T., Huijbers, M.G. The unique properties of IgG4 and its roles in health and disease. Nat Rev Immunol 23, 763–778 (2023). In some embodiments, the Fc domain comprises a truncated hinge region.
  • the Fc domain is an IgG1 Fc domain, and the IgG1 Fc domain comprises a hinge region, optionally wherein the hinge region comprises or consists of the amino acid sequence of SEQ ID NO: 25; or (b) the Fc domain is an IgG4 Fc domain, and the IgG4 Fc domain comprises a hinge region, optionally wherein the hinge region comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27.
  • a "fragment" of an IgG polypeptide of the invention refers to a sequence containing less amino acids in length than the IgG polypeptide of the invention. This sequence can be used as long as it exhibits the same properties, i.e.
  • the fragment shares about 5 consecutive amino-acids, at least about 7 consecutive amino-acids, at least about 15 consecutive amino-acids, at least about 20 consecutive amino- acids, at least about 25 consecutive amino-acids, at least about 30 consecutive amino-acids, at least about 35 consecutive amino-acids, at least about 40 consecutive amino-acids, at least about 45 consecutive amino-acids, at least about 50 consecutive amino-acids, at least about 55 consecutive amino-acids, at least about 60 consecutive amino-acids, at least about 100 consecutive amino-acids, at least about 150 consecutive amino-acids, at least about 200 consecutive amino-acids, or more of the native human IgG amino acid sequence.
  • variant refers to an IgG Fc domain polypeptide having an amino acid sequence that differs to some extent from the Fc domain native sequence peptide, that is an amino acid sequence that varies from the Fc native sequence by amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles.
  • the amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence, e.g. at the N- or C-terminal sequence or within the amino acid sequence. Substitutions can also be conservative, in this case, the conservative amino acid substitutions are herein defined as exchanges within one of the following five groups: I.
  • the reference sequence is the human IgG Fc fragment amino acid sequence as set forth in any one of sequences IgG1 Fc (SEQ ID No. 3), an IgG2 Fc (SEQ ID No. 6), an IgG3 Fc (SEQ ID No. 7), and an IgG4 Fc (SEQ ID No. 8), a fragment or a combination of one or more of these sequences.
  • IgG Fc domain variants are known in the art and described, e.g.
  • AAT, fragment, or variant thereof is covalently fused to the N-terminus or the C-terminus of the Fc domain by, or via a linker, e.g.
  • the polypeptide linker consists primarily of stretches of Gly and Ser residues (“GS” linker) or Gly- Gly and Ser residues (“GGS” linker) or Gly-Gly-Gly-Gly and Ser residues (“GGGGS” linker) followed or not by one or more Arg residue ("R" residue).
  • the linker comprises at least 5 amino acids.
  • the linker is between 5 and 15 amino acids in length.
  • the linker comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in length.
  • the linkers comprise 10-30 amino-acids, optionally, 10- 25 amino-acids, and further optionally 15-25 amino-acids.
  • GGS and GGGGS linkers are disclosed herein. Examples of linkers comprise: SEQ ID NO: 4 – GGS linker GGSGGSGGSGGSGGSGGSGGS SEQ ID NO: 5 – GGGGS linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 24 – GGGGS linker GGGGSGGGGS.
  • one or more signal peptide is fused to the to the N-terminus and/or the C-terminus of the AAT-Fc fusion protein.
  • the signal peptide comprises 10-30 amino-acids, preferably, 10-25 amino-acids, and more preferably 15-25 amino-acids.
  • the first amino acid of the signal peptide is methionine ("M").
  • M methionine
  • a non-limiting example of a signal peptide is disclosed herein (e.g. SEQ ID NO. 14).
  • AAT, a fragment, or a variant thereof is not fused to the Fc domain, fragment, or variant thereof, but both are present in the same composition of the invention and administered simultaneously or staggered in time.
  • the fusion protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of any one of SEQ ID NOs: 17 to 19, 21, or 22. In some embodiments, the fusion protein comprises or consists of an amino acid sequence that is identical to the amino acid sequence of any one of SEQ ID NOs: 17 to 19, 21, or 22.
  • General definitions The term “comprise/comprising” is generally used in the sense of include/including, that is to say permitting the presence of one or more features or components. The terms “comprise(s)” and “comprising” also encompass the more restricted ones “consist(s)", “consisting” as well as “consist/consisting essentially of", respectively.
  • nucleic acid molecule polynucleotide
  • nucleotide sequence are intended to refer to a polymeric chain of any length of nucleotides, including deoxyribonucleotides, ribonucleotides, or analogues thereof.
  • the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides).
  • the nucleic acid molecule, polynucleotide or nucleotide sequence may consist of DNA.
  • the nucleic acid molecule, polynucleotide or nucleotide sequence may be mRNA. Since the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).
  • the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence).
  • the nucleotide or amino acid residues at each position are then compared.
  • a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the amino acids are identical at that position.
  • the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence has at least 90% identity to SEQ ID NO: 19, SEQ ID NO: 19 would be the reference sequence. To assess whether a sequence has at least 90% identity to SEQ ID NO: 19 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 19, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 19. If at least 90% of the positions are identical, the test sequence is at least 90% identical to SEQ ID NO: 19. If the sequence is shorter than SEQ ID NO: 19, the gaps or missing positions should be considered to be non-identical positions.
  • An alignment between two sequences can be accomplished using a mathematical algorithm. For example, an alignment may be performed using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J Mol Biol.;48(3):443- 53) which aligns the sequences optimally over the entire length). Sequences of substantially different lengths may alternatively be aligned using a local alignment algorithm (e.g.15 Smith and Waterman algorithm (Smith and Waterman, 1981, J Theor Biol.
  • Fusion proteins of the invention may comprise mutations in the Fc domain “corresponding to” mutations at specified positions. In these embodiments, the residues are typically numbered according to the EU index of Kabat.
  • an Fc domain comprising a mutation corresponding to L234A refers to an Fc domain with an A at the position that best aligns to L234 in a wild-type Fc domain wherein the residues are numbered according to the EU index of Kabat. It is within the capabilities of the person skilled in the art to determine which amino acids in an alternative amino acid sequence “correspond to” a given residue in a wild-type Fc domain numbered according to the EU index of Kabat.
  • the person skilled in the art merely needs to perform a sequence alignment of the alternative amino acid sequence with a wild-type Fc domain numbered according to the EU index of Kabat using a suitable alignment algorithm such as that of Needleman and Wunsch described above, and determine which region of the alternative amino acid sequence best aligns to the specified residues in the wild-type Fc domain numbered according to the EU index of Kabat.
  • a suitable alignment algorithm such as that of Needleman and Wunsch described above
  • the skilled person is able to align the alternative amino acid sequence with SEQ ID NO: 3 and determine which amino acid best aligns, and therefore corresponds to, e.g. residue L234 of SEQ ID NO: 3 numbered according to the EU index of Kabat.
  • the invention provides a fusion protein for use in the prevention and/or treatment of a disease or disorder of the nervous system, wherein said fusion protein comprises (i) an immunoglobulin IgG Fc domain, a variant or a fragment thereof, and (ii) an alpha1-antitrypsin (AAT) polypeptide, a variant, an isoform and/or a fragment thereof, wherein the AAT polypeptide, variant, isoform and/or fragment thereof, is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain, variant or fragment thereof.
  • AAT alpha1-antitrypsin
  • treatment means any administration of a composition, pharmaceutical composition, therapeutic agent, compound, etc... of the disclosure to a subject for the purpose of: (i) inhibiting the disease, that is, arresting the development of clinical symptoms described herein; and/or (ii) relieving the disease, that is, causing the regression of clinical symptoms described herein.
  • prevention means any administration of a composition, pharmaceutical composition, therapeutic agent, compound, etc.
  • a disease or disorder for example a disease or disorder of the peripheral nervous system (PNS), more preferably a demyelinating and/or dysmyelinating PNS disease or associated disorder, that is, causing the clinical symptoms of the disease not to develop.
  • PNS peripheral nervous system
  • the disease or disorder is selected from the group consisting of alpha-1 antitrypsin deficiency (AATD), chronic obstructive pulmonary disease (COPD), Charcot- Marie-Tooth disease (CMT), Guillain-Barré Syndrome (GBS), Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), neuropathic pain, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), amyloid polyneuropathy (Transthyretin-Related Familial Amyloid Polyneuropathy), panniculitis, vasculitis, anti-proteinase-3 (PR3)-related vasculitis, inflammatory bowel disease (IBD), ulcerative colitis, hypothyroidism, Graft-versus-Host disease (GVHD), Type 1 diabetes mellitus, Type 2 diabetes mellitus, diabetic neuropathies, cardiovascular disease, arterial stiffness, hypertension, osteoporosis, sarcopenia, Alzheimer’s disease, neurodegenerative conditions, viral
  • the disease may be a disease or disorder of the peripheral nervous system (PNS), more preferably a demyelinating and/or dysmyelinating PNS disease or associated disorder.
  • PNS peripheral nervous system
  • the terms "subject”/"patient”, are well-recognized in the art, and are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human.
  • the subject is a subject in need of treatment or a subject with a disease or disorder.
  • the subject can be a normal subject. The term does not denote a particular age or sex.
  • the subject is a human, most preferably a human patient having a disease or disorder of the nervous system, preferably a disease or disorder of the peripheral nervous system (PNS), more preferably a demyelinating and/or dysmyelinating PNS disease or associated disorder.
  • a disease or disorder of the nervous system preferably a disease or disorder of the peripheral nervous system (PNS), more preferably a demyelinating and/or dysmyelinating PNS disease or associated disorder.
  • PNS peripheral nervous system
  • the disease or disorder of the nervous system described herein is a demyelinating and/or dysmyelinating disease or disorder of the peripheral nervous system (PNS).
  • Demyelinating diseases are common neurological disorders that affect the central nervous system (CNS) and peripheral nervous system (PNS). They cause substantial disability, and some are associated with a high mortality rate if not treated promptly.
  • Peripheral nerve demyelinating diseases discussed herein are Guillain–Barré syndrome, chronic inflammatory demyelinating neuropathy, anti-myelin-associated glycoprotein neuropathy, and POEMS syndrome.
  • ADAM-17 activity modulation is involved in myelin regulation and as an inflammation hallmark of chronic inflammatory demyelinating polyneuropathy. Inhibiting and/or impairing the activity of ADAM-17 by a compound, a molecule, a method or a composition of the invention will be beneficial for a subject in need thereof, i.e.
  • the demyelinating and/or dysmyelinating disease or disorder of the PNS is selected from the group comprising Charcot–Marie–Tooth (CMT), Guillain-Barré Syndrome (GBS), neuropathic pain and chronic inflammatory demyelinating polyneuropathy (CIDP).
  • CMT Charcot–Marie–Tooth
  • GRS Guillain-Barré Syndrome
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • CMT Charcot-Marie-Tooth
  • PMP22, GJB1, MPZ, and MFN2 genes are the most prevalent category of demyelinating and/or dysmyelinating disease or disorder of the PNS. It is an inherited neuropathy with autosomal dominant inheritance pattern being the most common, though there also are X-linked and autosomal recessive subtypes. In addition to a variety of inheritance patterns, there are a myriad of genes associated with CMT, reflecting the heterogeneity of this disorder. Currently, more than 90 distinct genetic variations have been implicated in causing or contributing to the clinical picture of these neuropathies. The most encountered genetic mutations affect the PMP22, GJB1, MPZ, and MFN2 genes.
  • CMT1 as demyelinating subtype (autosomal dominant); CMT2 as axonal subtype (autosomal dominant or recessive CMTA2A); CMTX with intermediate conduction velocities (most classically X-linked, though there are autosomal dominant and recessive intermediate variants); CMT3 also known as Dejerine-Sottas disease and CMT4.
  • CMT1 can be further subcategorized as: CMT1A, caused by a 1.4 Mb duplication of the PMP22 gene; CMTX1, a common form of hereditary motor and sensory neuropathy that has an X- linked dominant inheritance pattern; CMT1B, caused by mutations in the MPZ gene (1q22).
  • GBS Guillain-Barré Syndrom
  • MFS Miller Fisher syndrome
  • GBS is the most common form of neuromuscular paralysis. It is an acute inflammatory demyelinating polyradiculoneuropathy which is characterized by rapidly progressive proximal and distal symmetric weakness, sensory loss, and depressed reflexes. GBS mostly affects young people and can cause long-term residual disability.
  • Chronic inflammatory demyelinating polyneuropathy also known as chronic inflammatory demyelinating polyradiculoneuropathy
  • CIDP chronic inflammatory demyelinating polyradiculoneuropathy
  • the inventors have discovered that, in contrast to what has been described in previous articles (see e.g. Lee S, Lee Y, Hong K, Hong J, Bae S, Choi J, Jhun H, Kwak A, Kim E, Jo S, Dinarello CA, Kim S.
  • AAT-Fc ⁇ 1-antitrypsin Fc-fused
  • NE Neutrophil Elastase
  • ADAM-17 Metalloprotease 17
  • ADAM-17 also called TACE (tumor necrosis factor- ⁇ - converting enzyme) refers to a 70 kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases.
  • the disease or disorder is AAT deficiency (AATD) or Chronic Obstructive Pulmonary Disease (COPD).
  • AATD is a genetic disease characterized by insufficient levels of AAT which may cause lung disease such as emphysema, loss of lung function, and decreased life expectancy.
  • COPD is a chronic lung condition characterized by inflammation in the airways of the lungs, resulting in reduced airflow and breathing difficulties.
  • Nucleic acids Further provided is a nucleic acid sequence encoding one or more recombinant constructs of the invention, such as e.g., the fusion proteins of the invention.
  • Non-limiting examples of an nucleic acid sequence encoding one or more recombinant constructs of the invention comprise a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, or any variant, fragment of combination thereof.
  • nucleic acid refers to any kind of deoxyribonucleotide (e.g. DNA, cDNA, ...) or ribonucleotide (e.g. RNA, mRNA, ...) polymer or a combination of deoxyribonucleotide and ribonucleotide (e.g. DNA/RNA) polymer, in linear or circular conformation, and in either single or double stranded form.
  • analogue of a particular nucleotide has the same base-pairing specificity, i.e. an analogue of A will base-pair with T.
  • Vectors, host cells and compositions Further contemplated is a plasmid or a vector comprising a nucleic acid sequence of the invention.
  • vector refers to a viral vector or to a nucleic acid (DNA or RNA) molecule such as e.g. a plasmid or other vehicle, which contains one or more heterologous nucleic acid sequence(s) of the invention and, preferably, is designed for transfer between different host cells.
  • expression vector refers to any vector that is effective to incorporate and express one or more nucleic acid(s) of the invention, in a cell, preferably under the regulation of a promoter.
  • a cloning or expression vector may comprise additional elements, for example, regulatory and/or post- transcriptional regulatory elements in addition to a promoter.
  • the vector may be introduced into a host cell (autologous, allogeneic or heterologous) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
  • the cloning vectors of the invention may contain, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • the origin of replication may be selected to promote autonomous replication of the vector in the host cell.
  • the present disclosure provides isolated host cells, or population of cells, comprising, or expressing, the fusion protein, the vector or plasmid provided herein.
  • the host cells, or population of cells, containing the vector or plasmid may be useful in expression or cloning of the polynucleotide contained in the vector.
  • Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
  • Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
  • the vector or plasmid can be introduced to the host cell, or population of cells, using any suitable methods known in the art, including, without limitation, DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector or plasmid of interest are well known in the art.
  • the host cell (autologous, allogeneic or heterologous) will preferably be selected from the group comprising a glial cell, a neuronal cell, a fibroblast cell, a cell line, a stem cell or a progenitor of any one of these cells.
  • the present invention also contemplates compositions as well as pharmaceutical compositions.
  • the pharmaceutical composition of the invention comprises a therapeutically effective amount of i) a fusion protein for use of the invention, ii) a vector of the invention or iii) a host cell of the invention, and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • the pharmaceutical composition of the invention comprises a therapeutically effective amount of a fusion protein described herein, and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • a pharmaceutical composition can include a dimer of AAT-Fc (symmetric or asymmetric) or a monomer of AAT-Fc or AAT cleaved from the Fc or combinations thereof.
  • the pharmaceutical composition of the invention comprises a therapeutically effective amount of a plasmid or a vector described herein, and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • the pharmaceutical composition of the invention comprises a therapeutically effective amount of an isolated host cell, or population of cells described herein, and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • the pharmaceutical composition described above can further comprise, or be administered in combination with, another therapy.
  • a "therapeutically effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • the effective amount may depend on the individual patient’s history, age, weight, family history, genetic makeup, stage of the thyroid-related autoimmune disease, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.
  • a therapeutically effective amount of the pharmaceutical composition of the invention can be any amount that reduces the severity, or occurrence, of symptoms of the disease, disorder and/or condition to be treated without producing significant toxicity to the subject.
  • an effective amount of the pharmaceutical composition of the invention can be any amount that reduces the number of diseased cells (e.g. dysregulated immune cells), autoantibodies, and/or other disease markers (e.g. cytokines) without producing significant toxicity to the subject.
  • the therapeutically effective amount of the pharmaceutical composition of the invention (and any additional therapeutic agent) can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject ⁇ s response to treatment.
  • the frequency of administration can be any frequency that reduces the severity, or occurrence, of symptoms of the disease, disorder and/or condition to be treated without producing significant toxicity to the subject.
  • Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the disease, disorder and/or condition may require an increase or decrease in the actual effective amount administered.
  • compositions of the present invention may be administered to a subject by different routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, via buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular or combinations thereof.
  • the composition may be administered as a suitably acceptable formulation in accordance with normal human practice. The skilled artisan will readily determine the dosing regimen and route of administration that is most appropriate for a particular patient.
  • compositions of the invention may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gone guns”, or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound.
  • EP electroporation
  • compositions of the invention may be administered alone or in combination with other therapies.
  • compositions of the present invention may also be delivered to the patient, by several technologies including DNA injection of nucleic acid encoding the fusion protein, a variant, an isoform and/or a fragment thereof of the invention (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, recombinant vectors such as recombinant lentivirus, recombinant adenovirus, and recombinant adeno-associated virus (AAV) as described herein.
  • Methods of producing and purifying fusion proteins In some embodiments, the present invention provides a method of producing a fusion protein or a dimer according to the invention.
  • the method comprises expressing the fusion protein or the dimer in cells, for example CHO HK1 cells.
  • expressing the fusion protein or the dimer comprises transfecting a cell with a nucleic acid or vector encoding the fusion protein or the dimer.
  • the present invention provides a method of purifying a fusion protein according to the invention.
  • the method comprises a step of affinity chromatography.
  • the step of affinity chromatography uses a column comprising protein A.
  • the protein A comprises a Z domain.
  • fusion proteins comprising an immunoglobulin IgG Fc domain comprising: (i) mutations corresponding to M252Y, S254T, and T256E; (ii) mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (iii) mutations corresponding to M252Y, S254T, and T256E and a wild-type hinge region, e.g. a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25, and a second polypeptide domain have an increased yield compared to equivalent fusion proteins not comprising features (i) to (iii).
  • the Fc domain is an IgG1 Fc domain.
  • Fc affinity columns for example the MabSelect SuRe TM column from Cytiva, comprise modified protein A which comprises a modified Fc-binding domain known as the Z domain (see Yu F, Järver P, Nygren P ⁇ (2013) Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG1. PLOS ONE 8(2)).
  • the Z domain is known to contact the Fc domain of IgG1 at positions 252-254.
  • the present invention provides a fusion protein comprising an immunoglobulin IgG Fc domain comprising: (i) mutations corresponding to M252Y, S254T, and T256E; (ii) mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (iii) mutations corresponding to M252Y, S254T, and T256E and a wild-type hinge region, e.g.
  • the Fc domain is an IgG1 Fc domain.
  • the fusion protein has increased affinity for protein A compared to an equivalent fusion protein not comprising features (i) to (iii).
  • the fusion protein has an increased affinity for the Z domain of protein A.
  • the fusion protein has increased yield during a step of affinity chromatography using a column comprising protein A comprising a Z domain compared to an equivalent fusion protein not comprising features (i) to (iii).
  • the present invention provides a method of purifying a fusion protein comprising an immunoglobulin IgG Fc domain comprising: (i) mutations corresponding to M252Y, S254T, and T256E; (ii) mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (iii) mutations corresponding to M252Y, S254T, and T256E and a wild-type hinge region, e.g. a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25, and a second polypeptide domain.
  • the method comprises a step of affinity chromatography.
  • the step of affinity chromatography uses a column comprising protein A.
  • the protein A comprises a Z domain.
  • the method has a higher yield than an equivalent method carried out on a fusion protein not comprising features (i) to (iii).
  • the present invention provides a method of increasing the yield of a fusion protein comprising an immunoglobulin IgG Fc domain, the method comprising introducing into the Fc domain: (i) mutations corresponding to M252Y, S254T, and T256E; (ii) mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (iii) mutations corresponding to M252Y, S254T, and T256E wherein the Fc domain comprises a wild-type hinge region, e.g. a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • a wild-type hinge region e.g. a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25.
  • the method comprises a step of affinity chromatography using a column comprising protein A comprising a Z domain.
  • the present invention provides the use of a fusion protein comprising an immunoglobulin IgG Fc domain comprising: (i) mutations corresponding to M252Y, S254T, and T256E; (ii) mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (iii) mutations corresponding to M252Y, S254T, and T256E and a wild-type hinge region, e.g.
  • the yield of the fusion protein is the yield of a purification method.
  • the purification method comprises a step of affinity chromatography using a column comprising protein A comprising a Z domain.
  • the yield is improved compared to an equivalent purification method performed on a fusion protein not comprising features (i) to (iii).
  • Reduced samples were prepared for analysis by mixing with NuPage 4x LDS sample buffer (Life Technologies) and NuPage 10x sample reducing agent (Life Technologies), and incubated at 70°C, 10 min. For non-reduced samples, the reducing agent and heat incubation were omitted. 5 ⁇ g of protein was loaded per lane. Samples were electrophoresed on 4-20% Mini- PROTEAN® Precast Gels (BioRad) with TGS buffer. Then gels were fixed for 30 minutes at room temperature in fixation buffer (50% Methanol + 10% acetic acid + 40% H2O). Coomassie blue (Sigma Aldrich) was added at final concentration of 0.25% and incubated for additional 15 minutes.
  • ADAM-17 protein Human recombinant ADAM-17 protein (930-ADB-010, R&D system) and fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (ES003 R&D system) were used for the assay.
  • Hr-ADAM-17 protein was reconstituted in sterile water at 100 ⁇ g/ml and the peptide substrate was diluted to 2mM in DMSO. 500 ⁇ g of AAT-Fc (YbdYbiotech, SEQ ID NO. 9) were resuspended in sterile water at 60 ⁇ M.
  • Plasma-derived AAT (Sigma A9160) was resuspended in sterile water at 500 ⁇ M. The reagents were kept on ice. Just before starting the reaction, we prepared a working solution (10 ⁇ M, 4x concentrated to achieve a final concentration of 2.5 ⁇ M of AAT-Fc and plasma-derived AAT). We diluted the HR-ADAM-17 protein 1:250 and the peptide substrate 1:100 in an assay buffer. The reagents were dispensed in triplicate on a black Maxisorp plate in this order: - 25 ⁇ l/well of AAT 10 ⁇ M - 25 ⁇ l/well of HR-ADAM-17 - 50 ⁇ l/well of peptide substrate. In control wells, AAT was replaced by 25 ⁇ l of assay buffer.
  • Control negative contains 50 ⁇ l of assay buffer and 50 ⁇ l of peptide substrate. Fluorescence was measured immediately using a SpectraMax iD3 instrument using the following parameters: excitation wavelength 320 nm, emission wavelength 405 ⁇ m, kinetic mode for five minutes. The blank values were subtracted from the other values and an analysis was performed using GraphPad-Prism. Results Figure 1 shows the protein size of each AAT sources tested. Figure 2 shows that all source of AAT are not able to inhibit neutrophil elastase at 1nM.
  • recombinant AAT4 and recombinant AAT-Fc are able to inhibit completely neutrophil elastase activity in comparison to plasma-derived AAT (40% inhibition), recombinant AAT-1 (60%), recombinant AAT-2 (30%) and recombinant AAT-his 3 (0% inhibition).
  • AAT-Fc at 2.5 ⁇ M reduced ADAM-17 activity to 87%, whether there is no effect at the same concentration of plasma-derived AAT.
  • Example 2 Treatment with AAT and AAT-derived peptides on Human Schwann cells Materials and Methods Human Schwann cells (HSC) were purchased from the Innoprot company and cultured according to the manufacturer's instructions. The TNF ⁇ was used at a concentration of 10ng/ml for 1 hour to activate the downstream pathway of TNF ⁇ .
  • the AAT-derived peptides were purchased from IRIS Biotech and resuspended in DMSO. Sequence of peptide 8: Ac-LFLYVIH-NH2; sequence of peptide 9: Ac-YRAHQGE-NH2; sequence of peptide 14: 4-Hydroxy-Bzl-His-(R/S)-Phg-NH2.
  • AAT plasma-derived, SEQ ID NO. 1
  • peptides for 48 hours. Following treatment, HSC were fixed with 4% PFA and permeabilized with 0.1% Triton. An anti-NF-kB/P65 antibody (Thermofisher) was incubated for two hours at room temperature, followed by an anti-rabbit- 488 antibody (Alexa). The nuclei were counterstained with DAPI. The pictures were taken using a Leica fluorescent microscope. Results The purpose of this study was to investigate the regulation of TNF ⁇ signaling by AAT and peptides in HSC stimulated with TNF ⁇ . The cytosol-nuclear translocation of NF-kB is one of the main downstream pathways regulated by TNF ⁇ .
  • Example 3 ADAM-17 activity inhibition by AAT-Fc and IC50 values for AAT-FC Materials and Methods Human recombinant ADAM-17 protein (930-ADB-010, R&D system) and fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (ES003 R&D system) were used for the assay. Hr-ADAM-17 protein was reconstituted in sterile water at 100ug/ml. Peptide substrate was diluted to 2mM in DMSO. 50ug of AAT-Fc (Ybdybiotech, SEQ ID NO. 9) were resuspended in sterile water at 5 ⁇ M.
  • AAT-Fc was tested at the following final concentrations: 0.012, 0.03, 0.11, 0.33, and 1 ⁇ M. See Figure 4 for AAT-Fc aminoacidic sequence.
  • the concentration of 25 ⁇ M of AAT (plasma-derived, SEQ ID NO.1) was used as a positive control.
  • the reagents were kept on ice. Just before starting the reaction, we prepared a working solution.
  • the reagents were dispensed in triplicate on a black Maxisorp plate in this order: - 25 ⁇ l/well of AAT - 25 ⁇ l/well of HR-ADAM-17 - 50 ⁇ l/well of peptide substrate.
  • AAT was replaced by 25 ⁇ l of assay buffer.
  • Control negative contains 50 ⁇ l of assay buffer and 50 ⁇ l of peptide substrate. Fluorescence was measured immediately using a SpectraMax iD3 instrument using the following parameters: excitation wavelength 320 nm, emission wavelength 405 nm, kinetic mode for five minutes. The blank values were subtracted from the other values and the analysis was performed using GraphPad-Prism. Results AAT-Fc reduces ADAM-17 activity in a dose-dependent manner.
  • AAT-Fc decreases IFN ⁇ -induced expression of MHCII in microglia derived from Human Induced Pluripotent Stem Cell (hiPSCs) Materials and Methods Human microglia cells derived from hiPSCs and transduced with MHCII-FLuc reporter gene were cultured for 24 hours before treatment.
  • the IFN ⁇ was used at a concentration of 1 ⁇ g/ml for 24 hours to activate the microglia MHCII-FLuc reporter gene.
  • the AAT-derived peptides were purchased from IRIS Biotech, resuspended in DMSO and used at 50 ⁇ M for 48 hours.
  • AAT plasma-derived, SEQ ID NO. 1
  • AAT-FC Ybdybiotech, SEQ ID NO.9
  • the microglia cell viability was controlled with the Cell Counting Kit-8 (CCK-8), a sensitive colorimetric assay which determines the number of viable cells.
  • CCK-8 solution was added directly to the cells and after 1 hour the absorbance was measured at 450nm using a SpectraMax iD3 instrument.
  • the quantitation of luciferase expression was done with ONE-GloTM EX Luciferase Assay System. A volume of ONE-GloTM EX Reagent that is equal to the volume of culture medium in each well was added, and samples were incubated for at least 3 minutes to lyse cells. The luminescence was then read using a SpectraMax iD3 instrument.
  • the luciferase activity was calculated and normalized to the number of viable cells and results were displayed using GraphPad-Prism. Results Using microglia derived from hiPSCs and stable expressing the MHCII-Fluc reporter gene, we demonstrated the anti-(neuro)inflammatory properties of AAT-FC and AAT-derived peptides. We stimulated the cells with IFN ⁇ , to increase MHCII expression, thereby indicating active/inflamed microglia. We treated them with AAT and its derivatives. AAT-FC can reduce the activity of the MHCII reporter by 90% in the presence of IFN ⁇ , as shown in Figure 7. Peptide 8 slightly reduces the activity of MHCII as compared to IFN ⁇ .
  • Example 5 AAT decreases IFN ⁇ -induced expression of major histocompatibility complex II (MHCII) in co-culture of neurospheres and microglia derived from hiPSCs.
  • MHCII major histocompatibility complex II
  • Five-thousand human microglia cells derived from hiPSCs and transduced with MHCII-FLuc reporter gene were added to each control neurosphere or neurosphere mutated with the ⁇ - synuclein gene (Parkinson’s mutation) aged to 25 days of dopaminergic differentiation. Co- cultures were maintained for one week before starting the AAT (plasma-derived, SEQ ID No. 1) pre-treatment at 50 ⁇ M.
  • the IFN ⁇ was used at a concentration of 100ng/ml or 1 ⁇ g/ml for 24 hours to activate the microglia MHCII-FLuc reporter gene.
  • the quantitation of luciferase expression was done with ONE-GloTM EX Luciferase Assay System. A 1 ⁇ 2 volume of ONE-GloTM EX Reagent and a 1 ⁇ 2 volume of lysis buffer that is equal to the volume of culture medium in each well was added, and samples were incubated for at least 3 minutes to lyse cells. The luminescence was then read using a SpectraMax iD3 instrument. The luciferase activity was calculated, and results were displayed using GraphPad-Prism.
  • Example 6 Measuring inflammatory response in Human Schwann cells (HSCs) following AAT-Fc treatment
  • Human SCs were plated in a 24-well plate at a density of 70000 cells per well. Two days after seeding, AAT-Fc or pdAAT pre-treatment was initiated for 1 day at 3 ⁇ M with or without LPS 1 ⁇ g/mL or TNF ⁇ 10ng/mL. Then, the medium was replaced with fresh medium containing 3 ⁇ M AAT-Fc or pdAAT for 2 days with or without LPS 10 ⁇ g/mL or TNF ⁇ 10ng/mL. Subsequently, the cells were harvested for RNA extraction.
  • HSCs express the Fc ⁇ R and elicit upregulation in pro-inflammatory cytokine mRNA in response to stimulation.
  • IL-1 ⁇ shows lowest levels with AGX-101R and AGX-101SY.
  • TNF ⁇ levels shows no decrease with all compounds.
  • IL-6 levels is decreased with AGX-101SY but not with the other compounds.
  • IL-4 levels are decreased with AGX-SY and AGX-101T compounds but not with the other compounds.
  • AGX-101R and AGX-101SY compounds exhibit the least immunogenicity with lower levels of IL-1 ⁇ and IL-6.
  • Example 7 ADAM-17 activity inhibition by AAT-Fc and IC50 values for AAT-FC
  • Human recombinant ADAM-17 protein (930-ADB-010, R&D system) and fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (ES003 R&D system) were used for the assay.
  • Hr-ADAM-17 protein was reconstituted in sterile water at 100ug/ml.
  • Peptide substrate was diluted to 2mM in DMSO.
  • 50ug of AAT-Fc SEQ ID NOs: 17 to 21 and a dimer of SEQ ID NOs: 22 and 23
  • Control negative contains 50 ⁇ l of assay buffer and 50ul of peptide substrate. Fluorescence was measured immediately using a SpectraMax iD3 instrument using the following parameters: excitation wavelength 320 nm, emission wavelength 405 nm, kinetic mode for five minutes. The blank values were subtracted from the other values and the analysis was performed using GraphPad-Prism. Results ADAM17 activity inhibition was tested using different AAT-Fc derivatives AGX-101R, AGX-101S, AGX101C, AGX-101A and AGX-101SY.
  • Example 8 Expression and purification of AAT-Fc constructs Materials and methods AAT-Fc constructs were expressed in CHO K1 cells cultured in shake flasks (see Table 2).
  • Cell culture supernatants were collected and purified by Affinity Chromatography (MabSelect SuRe resin) followed by Size Exclusion Chromatography (Superdex 200 pg resin). After chromatography purification, the proteins were dialyzed into water, and sucrose was added to a final concentration of 8% (w/v) before lyophilization. The final product was in the form of the lyophilized powder.
  • the QC testing panel included protein concentration by A280, non- reduced/reduced SDS-PAGE, SEC-HPLC, endotoxin level testing, and LC-MS.
  • the ⁇ KTA M150 system was employed for column chromatography.
  • the Affinity Chromatography was performed in bind- elute mode, with the bound protein eluted using acid elution.
  • Superdex 200 pg resin was used for size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • the sample was directly loaded onto a 320 mL SEC column.
  • PBS at pH 7.4 was used as the running buffer for SEC.
  • Slide-A-LyzerTM Dialysis Cassettes, 3.5K MWCO was used for Dialysis.
  • the dialysis cassette is first hydrated according to the manufacturer's instructions. After hydration, the sample is loaded into the dialysis cassette. The cassette is then placed in 50 times the sample volume of dialysis buffer (sterile water).
  • the dialysis buffer is changed every 3 hours, for a total of 3 times.
  • the sample was pre-frozen at -80°C for 6 hours before lyophilization.
  • the frozen sample was then placed in the freeze dryer to begin the vacuum freeze-drying process.
  • the entire process includes three stages: pre-freezing (4 hours), primary drying (sublimation, 12 hours), and secondary drying (desorption, 12 hours), following the manufacturer's built-in programs of the instrument.
  • After lyophilization, the sample was sealed for storage at -80°C.
  • the final products were subjected to quality control testing, including SDS-PAGE, SEC- HPLC, A280, endotoxin level testing, and LC-MS. Results Yields for each construct are shown in Table 4.
  • Example 9 Neutrophil Elastase activity inhibition by AAT-Fc and IC50 values for AAT-FC Materials and methods Neutrophil Elastase Inhibition Assay.
  • Substrate MeOSuc-AAPV-pNA l00 ⁇ M, purified human neutrophil elastase (ELA2) 2.2 ⁇ U/ ⁇ L and several concentrations of AAT-Fc (100, 30, 10 , 5, 3, 2 nM) were incubated together for 30min. Absorbance was measured every minute in a spectrophotometer reader (A405nm). Vmax value was determined with the slope of the trendline during 10 min. The Experiment was performed in duplicate. Error bars indicate the standard deviation.
  • SC sub-cutaneous
  • the samples were added to the plate and after a first incubation, the secondary antibody (goat anti- human IgG-biotin) was added to the wells. Following incubation, streptavidin-horseradish peroxidase (HRP) and tetramethylbenzidine (TMB) were added, and then absorbance was measured at 450 nm. Results The PK characteristics of each molecule were calculated using Phoenix WinNonlin 8.0 and are summarized in the table below.
  • a fusion protein comprising: (i) an immunoglobulin IgG Fc domain, and (ii) an alpha1-antitrypsin (AAT) polypeptide, wherein the AAT polypeptide is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain, and wherein: (a) the Fc domain comprises mutations corresponding to L234A or F234A, L235A, and P329G; (b) the Fc domain comprises mutations corresponding to M252Y, S254T, and T256E; and/or (c) the fusion protein comprises a linker between the Fc domain and the AAT polypeptide, wherein the linker comprises at least 5 amino acids, optionally wherein the linker is a glycine- serine linker and/or comprises the amino acid sequence of SEQ ID NO: 24.
  • AAT alpha1-antitrypsin
  • the fusion protein of any one of the preceding embodiments, wherein the Fc domain comprises or consists of an amino acid sequence that is identical to the amino acid sequence of any one of SEQ ID NOs: 29 to 33.
  • the fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of any one of SEQ ID NOs: 17 to 19, 21, or 22.
  • 10. The fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises or consists of an amino acid sequence that is identical to the amino acid sequence of any one of SEQ ID NOs: 17 to 19, 21, or 22. 11.
  • a dimer comprising the fusion protein of any one of the preceding embodiments and a second protein.
  • the dimer of embodiment 11 or embodiment 13, wherein the second protein does not comprise an AAT polypeptide.
  • the dimer of embodiment 14, wherein the second protein comprises or consists of an amino acid sequence of SEQ ID NO: 23. 16.
  • fusion protein or the dimer of any one of the preceding embodiments wherein the fusion protein or the dimer has a lower immunogenicity than: (a) a fusion protein consisting of the amino acid sequence of SEQ ID NO: 20 or 34, or a dimer consisting of two fusion proteins consisting of the amino acid sequence of SEQ ID NO: 20 or 34; (b) an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, and P329G; (c) an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E; (d) an equivalent fusion protein not comprising mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; and/or (e) an equivalent fusion protein not comprising mutations corresponding to M252Y, S254T, and T256E and not comprising a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO:
  • the fusion protein or the dimer of embodiment 16 wherein the immunogenicity is measured by contacting human Schwann cells with the fusion protein or the dimer, harvesting the cells, extracting RNA, and analysing the expression of at least one cytokine.
  • the at least one cytokine is selected from IL-1 ⁇ , TNF ⁇ , IL-6, and IL-4. 19.
  • fusion protein or the dimer of any one of the preceding embodiments wherein the fusion protein or the dimer can inhibit and/or impair the activity of ADAM metallopeptidase domain 17 (ADAM-17).
  • ADAM-17 ADAM metallopeptidase domain 17
  • 21. The fusion protein or the dimer of embodiment 20, wherein inhibition of ADAM-17 is measured by incubating ADAM-17 with increasing concentrations of the fusion protein or the dimer and a fluorogenic ADAM-17 substrate, measuring the fluorescence, and calculating an IC50 value for the fusion protein or the dimer. 22.
  • the fusion protein or the dimer of embodiment 26 wherein the serum half-life is measured by administering the fusion protein to a mouse, taking blood samples from the mouse at various time points, and measuring the concentration of the fusion protein in the samples using an ELISA.
  • a vector comprising a nucleic acid sequence encoding the fusion protein or the dimer of any one of the preceding embodiments. 30.
  • a host cell comprising, or expressing, the fusion protein or the dimer of any one of embodiments 1 to 28 or the vector of embodiment 29.
  • a pharmaceutical composition comprising the fusion protein or the dimer of any one of embodiments 1 to 28, the vector of embodiment 29, or the host cell of embodiment 30 and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • a method of treating or preventing a disease or disorder comprising administering the fusion protein or the dimer of any one of embodiments 1 to 28, the vector of embodiment 29, the host cell of embodiment 30, or the pharmaceutical composition of embodiment 31 or embodiment 32 to a subject.
  • PNS peripheral nervous system
  • CMT Charcot–Marie–Tooth
  • GBS Guillain-Barré Syndrome
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • fusion protein, dimer, vector, host cell or pharmaceutical composition for use, method, or use of embodiment 37, wherein CMT is selected from the group comprising CMT1A, CMT1B, CMT1X, CMT2A, CMT3, and CMT4.
  • CMT is selected from the group comprising CMT1A, CMT1B, CMT1X, CMT2A, CMT3, and CMT4.
  • fusion protein, dimer, vector, host cell or pharmaceutical composition for use according to embodiment 33, the method of treatment of embodiment 34, or the use of embodiment 35, wherein the disease or disorder is AAT deficiency (AATD) or Chronic Obstructive Pulmonary Disease (COPD). 41.
  • AATD AAT deficiency
  • COPD Chronic Obstructive Pulmonary Disease
  • the fusion protein, dimer, vector, host cell or pharmaceutical composition for use, method, or use of any one of embodiments 36 to 40, wherein the fusion protein, dimer, vector, host cell or pharmaceutical composition is administered by a route of administration selected from the group comprising inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, intranasal, intracerebral, parenteral, pulmonary, instillation, subcutaneous, ex vivo gene therapy or ex vivo cell-therapy. 42.
  • a method of inhibiting ADAM-17 in a cell comprising contacting the cell with the fusion protein or the dimer of any one of embodiments 1 to 28, the vector of embodiment 29, the host cell of embodiment 30, or the pharmaceutical composition of embodiment 31.
  • a method of promoting remyelination and/ or arresting demyelination of the axons of the PNS in a subject comprising administering to the subject the fusion protein or the dimer of any one of embodiments 1 to 28, the vector of embodiment 29, the host cell of embodiment 30, or the pharmaceutical composition of embodiment 31.
  • 44. A method of producing a fusion protein or a dimer according to any one of embodiments 1 to 28.
  • 45. wherein the method comprises expressing the fusion protein or the dimer in a cell, optionally wherein the cell is a CHO cell, optionally a CHO HK1 cell. 46.
  • expressing the fusion protein or the dimer comprises transfecting the cell with a nucleic acid or vector encoding the fusion protein or the dimer.
  • 47. A method of purifying a fusion protein or a dimer according to any one of embodiments 1 to 28. 48. The method of embodiment 47, wherein the method comprises a step of affinity chromatography. 49. The method of embodiment 48, wherein the step of affinity chromatography uses a column comprising protein A, optionally wherein the protein A comprises a Z domain. 50.
  • the fusion protein or the dimer comprises an IgG Fc domain comprising: (i) mutations corresponding to M252Y, S254T, and T256E; (ii) mutations corresponding to L234A, L235A, M252Y, S254T, T256E, and P329G; or (iii) mutations corresponding to M252Y, S254T, and T256E and a wild-type hinge region, e.g. a hinge region comprising or consisting of the amino acid sequence of SEQ ID NO: 25. 51.
  • the method of any one of embodiments 47 to 50, wherein the method comprises a step of size exclusion chromatography. 52.
  • a fusion protein for use in the prevention and/or treatment of a disease or disorder of the nervous system comprising (i) an immunoglobulin IgG Fc domain, a variant or a fragment thereof, and (ii) an alpha1-antitrypsin (AAT) polypeptide, a variant, an isoform and/or a fragment thereof, wherein the AAT polypeptide, variant, isoform and/or fragment thereof, is covalently fused to the N-terminus or the C-terminus of the IgG Fc domain, variant or fragment thereof.
  • the fusion protein for use of embodiment 52 wherein the disease or disorder of the nervous system is a disease or disorder of the peripheral nervous system (PNS), preferably a demyelinating and/or dysmyelinating PNS disease or associated disorder.
  • PNS peripheral nervous system
  • the fusion protein for use of embodiment 52 or 53 wherein the PNS disease or disorder is selected from the group comprising Charcot–Marie–Tooth (CMT), Guillain-Barré Syndrome (GBS), neuropathic pain and chronic inflammatory demyelinating polyneuropathy (CIDP).
  • CMT Charcot–Marie–Tooth
  • GBS Guillain-Barré Syndrome
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • CMT Charcot–Marie–Tooth
  • GSS Guillain-Barré Syndrome
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • CMT is selected from the group comprising CMT1A, CMT1B, CMT2A, CMT3 and CMTX1.
  • a vector comprising a nucleic acid sequence encoding a fusion protein for use of anyone of embodiments 52 to 56.
  • a host cell comprising, or expressing, a fusion protein for use of anyone of embodiments 52 to 56 or a vector of embodiment 57. 59.
  • a pharmaceutical composition comprising a therapeutically effective amount of i) a fusion protein for use of any one of embodiments 52-56, ii) a vector of embodiment57 or iii) a host cell of embodiment 58, and at least one pharmaceutically acceptable excipient, diluent, carrier, salt and/or additive.
  • the carrier is a blood-brain barrier permeability enhancer.
  • a route of administration selected from the group comprising inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, intranasal, intracerebral, parenteral, pulmonary, instillation, subcutaneous, ex vivo gene therapy or ex vivo cell-therapy.
  • a route of administration selected from the group comprising inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, intranasal, intracerebral, parenteral, pulmonary, instillation, subcutaneous, ex vivo gene therapy or
  • 64. A method of treatment and/or prevention of a disease or disorder of the nervous system, comprising administering a fusion protein of embodiments 52 to 56 or a pharmaceutical composition of embodiments 59 to 61, to a subject in need thereof.
  • the method of treatment and/or prevention of embodiment 64, wherein the disease or disorder of the nervous system is a disease or disorder of the peripheral nervous system (PNS), preferably a demyelinating and/or dysmyelinating PNS disease or associated disorder.
  • PNS peripheral nervous system

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne une protéine de fusion comprenant un domaine Fc d'immunoglobuline IgG et un polypeptide alpha1-antitrypsine (AAT). La présente invention concerne également une méthode de traitement ou de prévention d'un trouble ou d'une maladie comprenant l'administration d'une protéine de fusion comprenant un domaine Fc d'immunoglobuline IgG et un polypeptide alpha1-antitrypsine (AAT) à un sujet.
PCT/EP2025/055585 2024-02-29 2025-02-28 Protéine de fusion aat-fc Pending WO2025083294A2 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP24160663.1 2024-02-29
EP24160663 2024-02-29
EP24166209 2024-03-26
EP24166209.7 2024-03-26
EP24196583.9 2024-08-27
EP24196583 2024-08-27

Publications (2)

Publication Number Publication Date
WO2025083294A2 true WO2025083294A2 (fr) 2025-04-24
WO2025083294A3 WO2025083294A3 (fr) 2025-05-30

Family

ID=94771306

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/055585 Pending WO2025083294A2 (fr) 2024-02-29 2025-02-28 Protéine de fusion aat-fc

Country Status (1)

Country Link
WO (1) WO2025083294A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013003641A2 (fr) 2011-06-28 2013-01-03 Inhibrx Llc Polypeptides de fusion à base de serpine et leurs procédés d'utilisation
WO2023247736A1 (fr) 2022-06-22 2023-12-28 Ageronix SA Alpha1-antitrypsine pour utilisation dans le traitement de maladies ou de troubles du système nerveux tels que la polyneuropathie inflammatoire démyélinisante chronique

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG10201602394QA (en) * 2011-03-29 2016-05-30 Roche Glycart Ag Antibody FC Variants
AU2012275295B2 (en) * 2011-06-28 2016-11-10 Inhibrx, Lp WAP domain fusion polypeptides and methods of use thereof
AU2013202648B2 (en) * 2012-01-10 2016-05-19 Konkuk University Compositions, methods and uses for alpha-1 antitrypsin fusion molecules
EP4073117A1 (fr) * 2019-12-10 2022-10-19 Institut Pasteur Nouvel anticorps bloquant fcgriiia et fcgriiib humain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013003641A2 (fr) 2011-06-28 2013-01-03 Inhibrx Llc Polypeptides de fusion à base de serpine et leurs procédés d'utilisation
WO2023247736A1 (fr) 2022-06-22 2023-12-28 Ageronix SA Alpha1-antitrypsine pour utilisation dans le traitement de maladies ou de troubles du système nerveux tels que la polyneuropathie inflammatoire démyélinisante chronique

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL SF ET AL., BIOINFORMATICS, vol. 21, no. 8, 2005, pages 1451 - 6
ALTSCHUL SF ET AL., NUCLEIC ACIDS RES., vol. 25, no. 17, 1997, pages 3389 - 402
BERGIN, D. A. ET AL., J CLIN INVEST, vol. 120, no. 12, 2010, pages 4236 - 4250
CHANG ET AL., CHARCOT-MARIE-TOOTH DISEASE, 2012
CROSSLEY ET AL.: "Alpha-1 Antitrypsin Deficiency and Accelerated Aging: A New Model for an Old Disease", DRUGS & AGING, vol. 36, June 2019 (2019-06-01), pages 823 - 840, XP009527792, DOI: 10.1007/s40266-019-00684-7
FLECK, D. ET AL., J BIOL CHEM, vol. 291, no. 1, 2016, pages 318 - 333
FRIDMAN, V.M. A. SAPORTA, NEUROTHERAPEUTICS, vol. 18, no. 4, 2021, pages 2236 - 2268
KO SPARK SSOHN MHJO MKO BJNA JHYOO HJEONG ALHA KWOO JR: "An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies", EXP MOL MED., vol. 54, no. 11, November 2022 (2022-11-01), pages 1850 - 1861, XP093198307, DOI: 10.1038/s12276-022-00870-5
LA MARCA, R., NAT NEUROSCI, vol. 14, no. 7, 2011, pages 857 - 865
LAI PKGHAG GYU YJUAN VFAYADAT-DILMAN LTROUT BL: "Differences in human IgG1 and IgG4 S228P monoclonal antibodies viscosity and self-interactions: Experimental assessment and computational predictions of domain interactions", MABS, vol. 13, no. 1, January 2021 (2021-01-01), pages 1991256
LEE SLEE YHONG KHONG JBAE SCHOI JJHUN HKWAK AKIM EJO S: "Effect of recombinant al-antitrypsin Fc-fused (AAT-Fc) protein on the inhibition of inflammatory cytokine production and streptozotocin-induced diabetes", MOL MED., vol. 19, no. 1, 20 May 2013 (2013-05-20), pages 65 - 71
MATSUDA, E.ISHIZAKI, R.TAIRA, T.IGUCHI-ARIGA, S. M.ARIGA, H., BIOLOGICAL & PHARMACEUTICAL BULLETIN, vol. 28, no. 5, 2005, pages 898 - 901
NEEDLEMANWUNSCH, J MOL BIOL., vol. 48, no. 3, 1970, pages 443 - 53
RISPENS, T.HUIJBERS, M.G.: "The unique properties of IgG4 and its roles in health and disease", NAT REV IMMUNOL, vol. 23, 2023, pages 763 - 778, XP093150085, DOI: 10.1038/s41577-023-00871-z
SMITHWATERMAN, J THEOR BIOL., vol. 91, no. 2, 1981, pages 379 - 80
TAVEGGIA, C. ET AL., NEURON, vol. 47, no. 5, 2005, pages 681 - 694
VAN'T WOUT E. F. ET AL., HUM MOL GENET., vol. 23, no. 4, 2014, pages 929 - 4
WANG WMALIEPAARD JCLDAMELANG TVIDARSSON GHECK AJRREIDING KR: "Human IgG Subclasses Differ in the Structural Elements of Their N-Glycosylation", ACS CENT SCI., vol. 10, no. 11, 10 October 2024 (2024-10-10), pages 2048 - 2058

Also Published As

Publication number Publication date
WO2025083294A3 (fr) 2025-05-30

Similar Documents

Publication Publication Date Title
EP3820906B1 (fr) Molécules de substitution de wnt multispécifiques et leurs utilisations
US11524985B2 (en) IL-37 fusion protein and methods of making and using same
EP3212663B1 (fr) Protéines de fusion de l'inhibiteur de la c1 estérase et leurs utilisations
KR20140062139A (ko) Ｗnt 조성물 및 당해 조성물의 치료학적 용도
US12384822B2 (en) TAM receptor-binding fusion molecule having non-inflammatory phagocytosis inducing activity
WO2021052349A1 (fr) Anticorps gipr et protéine de fusion entre celui-ci et la glp-1, et composition pharmaceutique et application associée
WO2017157325A1 (fr) Protéine de fusion comprenant un facteur de croissance nerveuse et procédé de préparation et utilisation correspondants
CA2888628A1 (fr) Nouvelle enzyme hautement fonctionnelle ayant une specificite de substrat s-hexosaminidase humaine modifiee, et presentant une resistance a la protease
JP2023521867A (ja) 改変インターロイキン２２ポリペプチド及びその使用
WO2019179424A1 (fr) Anticorps gipr et proteine de fusion glp-1 de ce dernier, et composition pharmaceutique et son application
CN106046171B (zh) 用于防治阿尔茨海默病的融合蛋白及其制备方法和应用
WO2023197930A9 (fr) Protéine hybride inhibant le complément
CA3251039A1 (fr) Nouvelles protéines de fusion cd200
CA3222463A1 (fr) Troubles retiniens
WO2025083294A2 (fr) Protéine de fusion aat-fc
US12221488B2 (en) APJ antibody, fusion protein thereof with Elabela, and pharmaceutical compositions and use thereofus01
RU2823919C1 (ru) Слитая молекула, способная индуцировать невоспалительный фагоцитоз
US20260116946A1 (en) Novel cd200 fusion proteins
US20260116951A1 (en) Novel cd200 fusion proteins
CN117750968A (zh) 用于预防或治疗非酒精性脂肪性肝病或非酒精性脂肪性肝炎的包括生长分化因子-15变体的组合物
CA3208936A1 (fr) Vecteurs retroviraux
HK40102449A (en) Fusion molecule having non-inflammatory phagocytosis inducing activity
CA3251037A1 (fr) Nouvelles protéines de fusion cd200
CN115551529A (zh) 增强房水流出并降低眼内压的方法
HK40045832A (en) C1 esterase inhibitor fusion proteins and uses thereof

Legal Events

Date Code Title Description
DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)