WO2016069574A1 - Serpin fusion polypeptides and methods of use thereof - Google Patents

Serpin fusion polypeptides and methods of use thereof Download PDF

Info

Publication number
WO2016069574A1
WO2016069574A1 PCT/US2015/057533 US2015057533W WO2016069574A1 WO 2016069574 A1 WO2016069574 A1 WO 2016069574A1 US 2015057533 W US2015057533 W US 2015057533W WO 2016069574 A1 WO2016069574 A1 WO 2016069574A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
fusion protein
aat
seq
amino acid
Prior art date
Application number
PCT/US2015/057533
Other languages
English (en)
French (fr)
Inventor
Brendan P. Eckelman
John C. Timmer
Quinn Deveraux
Original Assignee
Inhibrx Lp
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
Priority claimed from US14/524,832 external-priority patent/US10400029B2/en
Priority to KR1020177014414A priority Critical patent/KR20170091096A/ko
Priority to IL251799A priority patent/IL251799B2/en
Priority to MX2017005467A priority patent/MX2017005467A/es
Priority to BR112017008525-9A priority patent/BR112017008525A2/pt
Priority to UAA201705128A priority patent/UA127305C2/uk
Priority to RU2017118325A priority patent/RU2746550C2/ru
Priority to AU2015339507A priority patent/AU2015339507B2/en
Priority to CN201580071331.7A priority patent/CN107206257A/zh
Priority to EP15854670.5A priority patent/EP3212290A4/en
Priority to CA2965151A priority patent/CA2965151A1/en
Priority to CN202111439584.9A priority patent/CN114316068A/zh
Priority to SG11201703390SA priority patent/SG11201703390SA/en
Priority to KR1020237043666A priority patent/KR20240005109A/ko
Priority to IL308589A priority patent/IL308589A/en
Priority to JP2017522654A priority patent/JP6737781B2/ja
Application filed by Inhibrx Lp filed Critical Inhibrx Lp
Publication of WO2016069574A1 publication Critical patent/WO2016069574A1/en
Priority to HK18103148.5A priority patent/HK1244460A1/zh
Priority to AU2021240153A priority patent/AU2021240153A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/10—Antimycotics
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • This invention relates to molecules, particularly polypeptides, more particularly fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptides and a second polypeptide. Additionally, the invention relates to fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from serpin polypeptides, a second polypeptide, and a third polypeptide.
  • this invention relates to fusion proteins that include at least one serpin polypeptide and a second polypeptide or fusion proteins that include at least one serpin polypeptide, a second polypeptide, and a third polypeptide, where the second and third polypeptides of the fusion proteins of the invention can be at least one the following: an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide; a cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide; a WAP domain containing polypeptide or a sequence derived from a WAP containing polypeptide; or an albumin polypeptide or an amino acid sequence that is derived from a serum albumin polypeptide.
  • This invention also relates to methods of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing such molecules.
  • the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide (Polypeptide 1) and second polypeptide (Polypeptide 2). Additionally, the fusion proteins described herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide (Polypeptide 1), a second polypeptide (Polypeptide 2), and a third polypeptide (Polypeptide 3).
  • fusion protein and “fusion polypeptide” refer to a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide operably linked to at least a second polypeptide or an amino acid sequence derived from at least a second polypeptide.
  • the individualized elements of the fusion protein can be linked in any of a variety of ways, including for example, direct attachment, the use of an intermediate or a spacer peptide, the use of a linker region, the use of a hinge region or the use of both a linker and a hinge region.
  • the linker region may fall within the sequence of the hinge region, or alternatively, the hinge region may fall within the sequence of the linker region.
  • the linker region is a peptide sequence.
  • the linker peptide includes anywhere from zero to 40 amino acids, e.g., from zero to 35 amino acids, from zero to 30 amino acids, from zero to 25 amino acids, or from zero to 20 amino acids.
  • the hinge region is a peptide sequence.
  • the hinge peptide includes anywhere from zero to 75 amino acids, e.g., from zero to 70 amino acids, from zero to 65 amino acids or from zero to 62 amino acids.
  • each of the linker region and the hinge region is a peptide sequence.
  • the hinge peptide and the linker peptide together include anywhere from zero to 90 amino acids, e.g., from zero to 85 amino acids or from zero to 82 amino acids.
  • the serpin polypeptide and the second polypeptide can be linked through an intermediate binding protein.
  • the serpin-based portion and second polypeptide portion of the fusion protein may be non-covalently linked.
  • fusion proteins according to the invention can have one of the following formulae, in an N-terminus to C-terminus direction or in a C-terminus to N-terminus direction:
  • polypeptides in the formulae also includes Polypeptide 3 (C) - Polypeptide l (a) - Polypeptide 2(b), Polypeptide 2(b) - Polypeptide 3( C )- Polypeptide l( a ), or any variation or combination thereof.
  • the Polypeptide 1 sequence includes a serpin polypeptide.
  • Serpins are a group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases.
  • Serpin proteins suitable for use in the fusion proteins provided herein include, by way of non-limiting example, alpha- 1 antitrypsin (AAT), antitrypsin-related protein (SERPINA2), alpha 1-antichymotrypsin (SERPINA3), kallistatin (SERPINA4), monocyte neutrophil elastase inhibitor (SERPINB1), PI-6
  • the Polypeptide 1 sequence includes an alpha- 1 antitrypsin (AAT) polypeptide sequence or an amino acid sequence that is derived from AAT.
  • AAT alpha- 1 antitrypsin
  • the Polypeptide 1 sequence includes a portion of the AAT protein.
  • the Polypeptide 1 sequence includes at least the reactive site loop portion of the AAT protein.
  • the reactive site loop portion of the AAT protein includes at least the amino acid sequence:
  • the AAT polypeptide sequence or an amino acid sequence that is derived from AAT is or is derived from a human AAT polypeptide sequence.
  • the fusion protein includes a full-length human AAT polypeptide sequence having the following amino acid sequence:
  • the fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2.
  • the AAT polypeptide sequence is, or the amino acid sequence derived from an AAT polypeptide is derived from, one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1,
  • NP_001 121172.1 and/or AA A51547.1.
  • the fusion proteins contain one or more mutations.
  • the fusion protein contains at least one mutation at a methionine (Met) residue in the serpin portion of the fusion protein.
  • the Met residue can be substituted with any amino acid.
  • the Met residue can be substituted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L).
  • the Met mutation(s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the invention.
  • the Met residue can be substituted with a charged residue, such as, for example, glutamate (Glu, E).
  • the Met mutation is at position 358 of an AAT polypeptide.
  • the Met mutation is Met358Leu (M358L).
  • the Met mutation is at position 351 of an AAT polypeptide.
  • the Met mutation is Met35 lGlu (M35 IE).
  • the Met mutation is at position 351 and at position 358 of an AAT polypeptide, for example, the Met mutation is Met351Glu (M351E) and Met358Leu (M358L).
  • the reactive site loop of this variant of the fusion AAT polypeptide has the following sequence:
  • the Met mutation is at position 351 and at position 358 of an AAT polypeptide, for example, the Met mutation is Met351Leu (M351L) and Met358Leu (M358L).
  • the reactive site loop of this variant of the fusion AAT polypeptide has the following sequence:
  • the fusion protein includes a full-length human AAT polypeptide sequence containing a variant reactive site loop modified at M351 and M358, having the following amino acid sequence:
  • the fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 80.
  • the second polypeptide (Polypeptide 2) of the serpin fusion protein is an Fc polypeptide or derived from an Fc polypeptide.
  • serpin-Fc fusion proteins include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.
  • the serpin-Fc fusion protein includes a single serpin polypeptide.
  • the serpin-Fc fusion proteins includes more than one serpin polypeptide, and these embodiments are collectively referred to herein as "serpin (a') -Fc fusion protein,” wherein (a') is an integer of at least 2.
  • each serpin polypeptides in a serpin (a ' ) -Fc fusion protein includes the same amino acid sequence. In other embodiments, each serpin polypeptide in a serpin (a ' ) -Fc fusion protein includes serpin polypeptides with distinct amino acid sequences. The serpin polypeptides of serpin (a ' ) -Fc fusion proteins can be located at any position within the fusion protein.
  • the serpin polypeptide of the serpin-Fc fusion protein includes at least the amino acid sequence of the reactive site loop portion of the AAT protein. In some embodiments, the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein. In some embodiments, the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In some
  • the serpin polypeptide of the serpin-Fc fusion protein includes at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 80.
  • the serpin polypeptide of the serpin-Fc fusion protein includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.
  • the serpin polypeptide of the serpin-Fc fusion protein includes the AAT polypeptide sequence is or the amino acid sequence derived from an AAT polypeptide is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1,
  • NP_000286.3 NP_001121 179.1, NP_001121 178.1, NP_001 121177.1, NP_001 121 176.16, NP_001 121175.1, NP_001 121174.1 , NP_001121 172.1, and/or AAA51547.1.
  • the Fc polypeptide of the fusion protein is a human Fc polypeptide, for example, a human IgG Fc polypeptide sequence or an amino acid sequence that is derived from a human IgG Fc polypeptide sequence.
  • the Fc polypeptide is a human IgGl Fc polypeptide or an amino acid sequence that is derived from a human IgGl Fc polypeptide sequence.
  • the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence that is derived from a human IgG2 Fc polypeptide sequence.
  • the Fc polypeptide is a human IgG3 Fc polypeptide or an amino acid sequence that is derived from a human IgG3 Fc polypeptide sequence.
  • the Fc polypeptide is a human IgG4 Fc polypeptide or an amino acid sequence that is derived from a human IgG4 Fc polypeptide sequence.
  • the Fc polypeptide is a human IgM Fc polypeptide or an amino acid sequence that is derived from a human IgM Fc polypeptide sequence.
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgGl Fc polypeptide sequence having the following amino acid sequence:
  • the fusion protein of the invention includes an
  • the fusion protein includes a hinge region coupled to the N-terminus of the Fc polypeptide of the fusion protein, where the Fc polypeptide includes a human IgGl Fc polypeptide sequence having the following amino acid sequence:
  • fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgGl Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3 or 43.
  • the fusion protein of the invention includes an
  • the Fc polypeptide is mutated or modified to enhance FcRn binding.
  • the mutated or modified Fc polypeptide includes the following mutations: Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) or Met428Leu and Asn434Ser (M428L, N434S) or Met428Val and Asn434Ser (M428V, N434S) using the Kabat numbering system.
  • the mutated or modified Fc polypeptide includes one or more mutations selected from the group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser (N434S), and combinations thereof.
  • the Fc polypeptide portion is mutated or otherwise modified so as to disrupt Fc-mediated dimerization.
  • the fusion protein is monomeric in nature.
  • the fusion protein of the invention includes an
  • the Fc polypeptide is mutated or modified.
  • the mutated or modified Fc polypeptide includes one or more mutations at a position selected from M252, T246, M428, and combinations thereof.
  • the mutated or modified Fc polypeptide includes the following mutations: Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L) using the Kabat numbering system.
  • the modified IgGl Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 198 of SEQ ID NO: 3 or residues 32, 36, and 208 of SEQ ID NO: 43 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgGl Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 198 of SEQ ID NO: 3
  • the modified IgGl Fc polypeptide of the fusion protein includes a modified human IgGl Fc polypeptide sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 shown above, is deleted and has the following amino acid sequence:
  • VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK SEQ ID NO: 46
  • the modified IgGl Fc polypeptide of the fusion protein includes a modified human IgGl Fc polypeptide sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 shown above, is deleted, and the fusion protein includes at least the following amino acid sequence:
  • VEVHNAKTKP REEQYNSTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
  • the modified IgGl Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 of SEQ ID NO: 43 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgGl Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 of SEQ ID NO: 43 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgGl Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234 and L235, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgGl Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234 and L235, and has the following amino acid sequence, where the mutated amino acid residues are boxed: l DKTHTCPPCP APE
  • VEVHNAKTKP REEQYNSTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 181 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 51)
  • the modified IgGl Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgGl Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • VEVHNAKTKP REEQYNSTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 181 GSFFLYSKLT VDKSRWQQGN VFSCSV
  • the modified IgGl Fc polypeptide of the fusion protein includes a modified human IgGl Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53.
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgG2 Fc polypeptide sequence having the following amino acid sequence:
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4.
  • the modified IgG2 Fc polypeptide of the fusion protein includes a modified human IgG2 Fc polypeptide sequence having the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG2 Fc polypeptide of the fusion protein includes a modified human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 54.
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgG3 Fc polypeptide sequence having the following amino acid sequence:
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 5.
  • the modified IgG3 Fc polypeptide of the fusion protein includes a modified human IgG3 Fc polypeptide sequence having the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG3 Fc polypeptide of the fusion protein includes a modified human IgG3 Fc polypeptide sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 5 shown above, is deleted and has the following amino acid sequence:
  • the modified IgG3 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 5 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG3 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234 and L235 and has the following amino acid sequence:
  • the modified IgG3 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and has the following amino acid sequence:
  • the modified IgG3 Fc polypeptide of the fusion protein includes a modified human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 55, 56, 57, 58, or 59.
  • the human IgG3 Fc region is modified at amino acid
  • the human IgG3 Fc region is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H).
  • the human IgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide sequence having the following amino acid sequence:
  • the Fc polypeptide of the fusion protein includes a hinge region coupled to the N-terminus of the Fc polypeptide of the fusion protein, where the Fc polypeptide includes a human IgG4 Fc polypeptide sequence having the following amino acid sequence:
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 60.
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 19 of SEQ ID NO: 6 or residues 34, 38, and 210 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 197 of SEQ ID NO: 6 or residues 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTL[I]I SR
  • the modified IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc polypeptide sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 shown above, is deleted and has the following amino acid sequence:
  • the modified IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc polypeptide sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 shown above, is deleted, and the fusion protein includes at least the following amino acid sequence:
  • the modified IgG4 Fc polypeptide of the fusion protein includes a mutation at residue L235, which corresponds to residue 5 of SEQ ID NO: 6 or residue 17 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residue is boxed:
  • the fusion protein of the invention includes a hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide
  • the modified IgG4 Fc polypeptide of the fusion protein includes a mutation at residue L235, which corresponds to residue 5 of SEQ ID NO: 6 or residue 17 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residue is boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 6 or residues 16 and 17 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 6 or residues 16 and 17 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes a mutation at residue S228, which corresponds to residue 10 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residue is boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues S228 and L235, which correspond to residues 10 and 17 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L235, M252, T256, and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO: 6 or residues 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L235, M252, T256, and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO: 6 or residues 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues S228, L235, M252, T256, and M428, which correspond to residues 10, 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed:
  • the modified IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the fusion protein of the invention includes an
  • the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence having the following amino acid sequence:
  • the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7.
  • the serpin-Fc fusion protein includes at least the amino acid sequence of the reactive site loop portion of the AAT protein operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1.
  • the serpin polypeptide and the Fc polypeptide are operably linked via a linker region, for example, a glycine-serine linker or glycine-serine based linker. In some embodiments, the serpin polypeptide and the Fc polypeptide are operably linked via a hinge region. In some embodiments, the serpin polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.
  • the serpin-Fc fusion protein includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33.
  • the serpin polypeptide and the Fc polypeptide are operably linked via a linker region, for example, a glycine-serine linker or glycine-serine based linker.
  • the serpin polypeptide and the Fc polypeptide are operably linked via a hinge region.
  • the serpin polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.
  • the serpin-Fc fusion protein includes at least the full- length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2 operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the serpin-Fc fusion protein includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
  • the serpin polypeptide and the Fc polypeptide are operably linked via a linker region, for example, a glycine-serine linker or glycine-serine based linker. In some embodiments, the serpin polypeptide and the Fc polypeptide are operably linked via a hinge region. In some embodiments, the serpin polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.
  • the serpin-Fc fusion protein includes at least the full- length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 80 operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the serpin-Fc fusion protein includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 80 operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.
  • the serpin polypeptide and the Fc polypeptide are operably linked via a linker region, for example, a glycine-serine linker or glycine-serine based linker. In some embodiments, the serpin polypeptide and the Fc polypeptide are operably linked via a hinge region. In some embodiments, the serpin polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.
  • the second polypeptide (Polypeptide 2) of the serpin fusion protein is a cytokine targeting polypeptide or derived from a cytokine targeting polypeptide.
  • the serpin- cytokine targeting polypeptide fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and a cytokine targeting polypeptide, or derivation thereof.
  • the serpin- cytokine targeting polypeptide fusion protein includes a single serpin polypeptide.
  • the serpin-cytokine targeting polypeptide fusion protein includes more than one serpin polypeptide, and these embodiments are collectively referred to herein as "serpin (a ' ) -cytokine targeting polypeptide fusion proteins," wherein (a') is an integer of at least 2.
  • each serpin polypeptide in a serpin (a ' ) -cytokine targeting polypeptide fusion protein includes the same amino acid sequence.
  • each serpin polypeptide of a serpin (a) -cytokine targeting polypeptide fusion protein includes serpin polypeptides with distinct amino acid sequences.
  • the cytokine targeting polypeptide of the serpin- cytokine targeting polypeptide fusion protein is a cytokine receptor or derived from a cytokine receptor.
  • the cytokine targeting polypeptide or an amino acid sequence that is derived from the cytokine receptor is or is derived from a human cytokine receptor sequence.
  • the cytokine targeting polypeptide is an antibody or an antibody fragment, for example an anti-cytokine antibody or anti-cytokine antibody fragment.
  • the cytokine targeting polypeptide or an amino acid sequence that is derived from the antibody or antibody fragment is derived from a chimeric, humanized, or fully human antibody sequence.
  • the term antibody fragment includes single chain, Fab fragment, a F(ab') 2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • the cytokine targeting polypeptide binds a cytokine receptor and prevents binding of a cytokine to the receptor.
  • the cytokine targeting polypeptide is an antibody or an antibody fragment, for example an anti- cytokine receptor antibody or anti-cytokine receptor antibody fragment.
  • the serpin polypeptide of the serpin-cytokine targeting polypeptide fusion proteins includes at least the amino acid sequence of the reactive site loop portion of the AAT protein. In some embodiments, the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: l . In some embodiments, the serpin polypeptide of the serpin-cytokine targeting fusion proteins includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein. In some embodiments, the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33.
  • the serpin polypeptide of the serpin-cytokine targeting fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In some embodiments, the serpin polypeptide of the serpin-cytokine targeting fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 80.
  • the serpin polypeptide of the serpin-cytokine targeting fusion protein includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.
  • the serpin polypeptide of the serpin-cytokine targeting fusion protein includes an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide that is or is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos.
  • the serpin-cytokine targeting polypeptide fusion protein can incorporate a portion of the serpin-Fc fusion protein.
  • an antibody contains an Fc polypeptide. Therefore, in some embodiments where the cytokine targeting polypeptide is a cytokine-targeting antibody, the serpin-cytokine targeting polypeptide fusion protein will incorporate a portion of the serpin-Fc fusion protein.
  • most receptor fusion proteins that are of therapeutic utility are Fc fusion proteins.
  • the serpin-cytokine targeting polypeptide fusion protein may incorporate an Fc polypeptide in addition to the serpin portion and the cytokine receptor portion.
  • the serpin-cytokine targeting polypeptide fusion protein includes an Fc polypeptide sequence
  • the Fc polypeptide sequence includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45,
  • the serpin-cytokine targeting fusion protein includes an Fc polypeptide sequence
  • the Fc polypeptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46,
  • the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a linker region, for example, a glycine-serine linker or glycine-serine based linker. In some embodiments, the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a hinge region. In some embodiments, the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the cytokine targeting polypeptide are directly attached.
  • the second polypeptide (Polypeptide 2) of the serpin fusion protein is a whey acidic protein (WAP) domain containing polypeptide, or an amino acid sequence that is derived from a WAP domain containing polypeptide.
  • WAP whey acidic protein
  • the serpin-WAP domain fusion proteins include at least a serpin polypeptide or at least an amino acid sequence that is derived from a serpin, a WAP domain-containing polypeptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide.
  • the serpin-WAP domain fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-WAP targeting polypeptide fusion protein includes more than one serpin polypeptide. These embodiments are collectively referred to herein as "serpin (a ' ) -WAP domain fusion proteins," wherein (a') is an integer of at least 2. In some embodiments, serpin polypeptides of the serpin (a') -WAP domain fusion protein includes the same amino acid sequence. In other embodiments, the serpin polypeptides of the serpin (a ' ) -cytokine targeting polypeptide fusion protein, includes serpin polypeptides with distinct amino acid sequences.
  • These serpin-WAP domain fusion proteins include a WAP domain containing polypeptide or polypeptide sequence that is or is derived from a WAP domain containing polypeptide.
  • the WAP domain is an evolutionarily conserved sequence motif of 50 amino acids containing eight cysteines found in a characteristic 4-disulfide core arrangement (also called a four-disulfide core motif).
  • the WAP domain sequence motif is a functional motif characterized by serine protease inhibition activity in a number of proteins.
  • WAP domain-containing polypeptides suitable for use in the fusion proteins provided herein include, by way of non-limiting example, secretory leukocyte protease inhibitor (SLPI), Elafin, and Eppin.
  • SLPI secretory leukocyte protease inhibitor
  • Elafin Elafin
  • Eppin Eppin
  • the WAP domain-containing polypeptide sequence of the fusion protein includes a secretory leukocyte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence that is derived from SLPI.
  • SLPI secretory leukocyte protease inhibitor
  • these embodiments are referred to herein as "serpin-SLPI-derived fusion proteins.”
  • the SLPI polypeptide sequence comprises a portion of the SLPI protein, such as for example, the WAP2 domain or a sub-portion thereof.
  • the SLPI polypeptide sequence or an amino acid sequence that is derived from SLPI is or is derived from a human SLPI polypeptide sequence.
  • SLPI sequence or a SLPI-derived sequence of the fusion protein includes a full-length human SLPI polypeptide sequence having the following amino acid sequence:
  • the serpin-SLPI fusion protein of the invention In some embodiments of the serpin-SLPI fusion protein of the invention, the
  • SLPI sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8.
  • SLPI sequence or a SLPI-derived sequence of the fusion protein includes a portion of the full-length human SLPI polypeptide sequence, where the portion has the following amino acid sequence: 1 SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK 61 PGKCPVTYGQ CLMLNPPNFC EMDGQCKRDL KCCMGMCGKS CVSPVKA (SEQ ID NO: 9)
  • SLPI sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9.
  • SLPI sequence or a SLPI-derived sequence of the fusion protein includes the WAP2 domain of the full-length human SLPI polypeptide sequence, where the WAP2 domain has the following amino acid sequence:
  • SLPI sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10.
  • the SLPI polypeptide sequence or the amino acid sequence derived from an SLPI polypeptide is or is derived from, one or more of the human SLPI polypeptide sequences shown in GenBank Accession Nos. CAA28187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, and/or BAG35125.1.
  • the SLPI polypeptide sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is modified at a Methionine (Met) residue.
  • Met residue can be substituted with any amino acid.
  • the Met residue can be substituted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L) or valine (Val, V).
  • the Met mutation(s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the invention.
  • the Met mutation is at position 98 of an SLPI polypeptide.
  • the modified SLPI polypeptide sequence of the serpin-SLPI includes mutations M98L or M98V in SEQ ID NO: 8.
  • the WAP domain-containing polypeptide sequence of the fusion protein includes an elafin polypeptide sequence or an amino acid sequence that is derived from elafin. These embodiments are referred to herein as "serpin-elafin fusion proteins.
  • the elafin polypeptide sequence includes a portion of the elafin protein, such as for example, the WAP domain or a sub-portion thereof.
  • the elafin polypeptide sequence or an amino acid sequence that is derived from elafin is or is derived from a human elafin polypeptide sequence.
  • the fusion protein includes a full-length human elafin polypeptide sequence having the following amino acid sequence:
  • the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11.
  • the fusion protein includes a portion of the full-length human elafin polypeptide sequence, where the portion has the following amino acid sequence:
  • the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.
  • the fusion protein includes the WAP domain of the full-length human elafin polypeptide sequence, where the WAP domain has the following amino acid sequence: 1 VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ (SEQ ID NO: 13)
  • the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13.
  • the elafin polypeptide sequence or the amino acid sequence derived from an elafin polypeptide is derived from one or more of the human elafin polypeptide sequences shown in GenBank Accession Nos. P19957.3, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, Q8IUB2.1, and/or NP_542181.1.
  • the WAP domain-containing polypeptide sequence of the fusion protein includes an Eppin polypeptide sequence or an amino acid sequence that is derived from Eppin. These embodiments are referred to herein as "serpin ⁇ -Eppin fusion proteins.
  • the Eppin polypeptide sequence of the serpin-Eppin fusion protein includes a portion of the Eppin protein, such as for example, the WAP domain or a sub-portion thereof.
  • the Eppin polypeptide sequence or an amino acid sequence that is derived from Eppin is or is derived from a human Eppin polypeptide sequence.
  • the Eppin polypeptide sequence or amino acid sequence derived from an Eppin polypeptide is or is derived from one or more of the human Eppin polypeptide sequences shown in GenBank Accession Nos. 095925.1, NP_065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and/or AAG00546.1.
  • the serpin polypeptide of the serpin-WAP domain fusion protein includes at least the amino acid sequence of the reactive site loop portion of the AAT protein.
  • the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1.
  • the serpin polypeptide of the serpin-WAP fusion protein includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein.
  • the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33.
  • the serpin polypeptide of the serpin-WAP domain fusion protein includes at least the full- length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In some embodiments, the serpin polypeptide of the serpin-cytokine targeting fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 80.
  • the serpin polypeptide of the serpin-WAP domain fusion protein includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.
  • the serpin polypeptide of the serpin-WAP domain fusion protein includes the AAT polypeptide sequence is, or the amino acid sequence derived from an AAT polypeptide is derived from, one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos.
  • the serpin-WAP domain fusion protein can also include an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide. These embodiments are referred to collectively herein as "serpin-Fc-WAP domain fusion proteins.” In these embodiments, no particular order is to be construed by this terminology. For example, the order of the fusion protein can be serpin-Fc-WAP domain, serpin-WAP domain-Fc, or any variation combination thereof.
  • the serpin-Fc-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin, WAP domain-containing polypeptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide, and an
  • Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.
  • the serpin-WAP domain fusion protein includes an Fc polypeptide sequence
  • the Fc polypeptide sequence can have the amino acid sequence of SEQ ID NO: 3-7 and 43-73.
  • the serpin-WAP domain fusion protein includes an Fc polypeptide sequence
  • the Fc polypeptide sequence can have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NOs. 3-7 and 43- 73.
  • the serpin-WAP domain fusion protein can also include an albumin polypeptide, or an amino acid sequence that is derived from an albumin polypeptide. These embodiments are referred to collectively herein as "serpin-albumin- WAP domain fusion proteins.” In these embodiments, no particular order is to be construed by this terminology. For example, the order of the fusion protein can be serpin-albumin- WAP domain, serpin-WAP domain-albumin, or any variation combination thereof.
  • the serpin-albumin-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin, WAP domain- containing polypeptide, or an amino acid sequence that is derived from a WAP domain- containing polypeptide, and an albumin polypeptide, or an amino acid sequence that is derived from an albumin polypeptide.
  • the albumin polypeptide sequence includes the amino acid sequence of SEQ ID NO: 14-15, described herein.
  • the albumin polypeptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the any one of the amino acid sequences having SEQ ID NO: 14 or 15.
  • the second polypeptide (Polypeptide 2) of the serpin fusion protein is an albumin polypeptide or is derived from an albumin polypeptide.
  • serpin (a ' ) -albumin fusion proteins include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and an albumin polypeptide or an amino acid sequence that is derived from an albumin polypeptide.
  • this invention relates to serpin albumin binding polypeptide fusion proteins, wherein the albumin is operably linked to the serpin via an intermediate binding molecule.
  • the serpin is non- covalently or covalently bound to human serum albumin.
  • the albumin polypeptide sequence of the fusion protein is a human serum albumin (HSA) polypeptide or an amino acid sequence derived from HSA.
  • HSA human serum albumin
  • the fusion protein includes a HSA polypeptide sequence having the following amino acid sequence: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV MCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLP
  • the albumin polypeptide sequence of the fusion protein includes a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14.
  • the albumin polypeptide sequence of the fusion protein fusion protein includes a domain 3 of human serum albumin polypeptide sequence having the following amino acid sequence:
  • the albumin polypeptide sequence of the fusion protein includes a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15.
  • the fusion protein of the invention includes an albumin polypeptide sequence
  • the fusion protein is linked to the human serum albumin via an intermediate albumin binding polypeptide.
  • the albumin binding polypeptide can be an antibody or an antibody fragment or derived from an antibody or antibody fragment.
  • the albumin binding polypeptide or an amino acid sequence that is derived from the antibody or antibody fragment is derived from a chimeric, humanized, or fully human antibody sequence.
  • the term antibody fragment includes single chain, Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • albumin binding polypeptide can be an albumin binding peptide.
  • Another embodiment of the invention is a serpin albumin binding polypeptide fusion, wherein the albumin binding polypeptide is domain 3 of Streptococcal protein G or a sequence derived from domain 3 of Streptococcal protein G.
  • the serpin polypeptide of the serpin (a ' ) -albumin fusion proteins includes at least the amino acid sequence of the reactive site loop portion of the AAT protein.
  • the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1.
  • the serpin polypeptide of the serpin-albumin fusion protein includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein.
  • the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33.
  • the serpin polypeptide of the serpin-albumin fusion proteins includes at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In some embodiments, the serpin polypeptide of the serpin-cytokine targeting fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 80.
  • the serpin polypeptide of the serpin-albumin fusion proteins includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.
  • the serpin polypeptide of the serpin-albumin fusion proteins includes the AAT polypeptide sequence or the amino acid sequence derived from an AAT polypeptide is or is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1,
  • the fusion proteins are modified to increase or otherwise inhibit proteolytic cleavage, for example, by mutating one or more proteolytic cleavage sites. In some embodiments, the fusion proteins are modified to alter or otherwise modulate an Fc effector function of the fusion protein, while simultaneously retaining binding and inhibitory function as compared to an unaltered fusion protein.
  • Fc effector functions include, by way of non-limiting examples, Fc receptor binding, prevention of proinflammatory mediator release upon binding to the Fc receptor, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity (ADCC), modified complement-dependent cytotoxicity (CDC), modified glycosylation at Asn297 residue (EU index of Kabat numbering, Kabat et al 1991 Sequences of Proteins of Immunological Interest) of the Fc polypeptide.
  • the fusion proteins are mutated or otherwise modified to influence Fc receptor binding.
  • the Fc polypeptide is modified to enhance FcRn binding.
  • Fc polypeptide mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol 281(33) 23514-23524), or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159). (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest) or Met428Val and Asn434Ser (M428V, N434S) using the Kabat numbering system.
  • the mutated or modified Fc polypeptide includes one or more mutations selected from the group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser ( 434S), and combinations thereof.
  • the Fc polypeptide portion is mutated or otherwise modified so as to disrupt Fc-mediated dimerization (Ying et al 2012 J. Biol Chem 287(23): 19399-19408).
  • the fusion protein is monomeric in nature.
  • the fusion proteins and variants thereof provided herein exhibit inhibitory activity, for example by inhibiting a serine protease such as human neutrophil elastase (NE), a chemotrypsin-fold serine protease that is secreted by neutrophils during an inflammatory response.
  • a serine protease such as human neutrophil elastase (NE)
  • NE neutrophil elastase
  • the fusion proteins provided herein completely or partially reduce or otherwise modulate serine protease expression or activity upon binding to, or otherwise interacting with, a serine protease, e.g., a human serine protease.
  • the reduction or modulation of a biological function of a serine protease is complete or partial upon interaction between the fusion proteins and the human serine protease protein, polypeptide and/or peptide.
  • the fusion proteins are considered to completely inhibit serine protease expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of serine protease expression or activity in the absence of interaction, e.g., binding, with a fusion protein described herein.
  • the fusion proteins are considered to partially inhibit serine protease expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of serine protease expression or activity in the absence of interaction, e.g., binding, with a fusion protein described herein.
  • the fusion proteins described herein are useful in a variety of therapeutic, diagnostic and prophylactic indications.
  • the fusion proteins are useful in treating a variety of diseases and disorders in a subject.
  • the serpin fusion proteins including, fusion proteins described herein, are useful in treating, alleviating a symptom of, ameliorating and/or delaying the progression of a disease or disorder in a subject suffering from or identified as being at risk for a disease or disorder selected from alpha- 1 -antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia-reperfusion injury, including, e.g., ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis
  • AAT alpha- 1
  • compositions according to the invention include a fusion protein of the invention, including modified fusion proteins and other variants, along with a suitable carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits. Brief Description of the Drawings
  • Figure 1A is a schematic representation of some embodiments of serpin-Fc fusion proteins according to the invention.
  • the serpin can be located at any position within the fusion protein.
  • Serpin-Fc fusion protein incorporating more than one serpin polypeptide are also represented.
  • Figure IB is a photograph of a SDS-PAGE gel showing serum derived AAT (lane 1), AAT-Fc 1 (lane 2, human IgGl Fc), and AAT-EL-Fcl (lane 3, Met351Glu, Met358Leu mutations within AAT, human IgGl Fc).
  • Figure 1C is a graph showing the inhibition of neutrophil elastase activity by AAT-Fc fusion proteins.
  • Figure ID is a photograph of a SDS-PAGE gel showing tetravalent AAT-Fc-AAT, having two AAT polypeptides per Fc polypeptide.
  • Figure IE is a graph showing the inhibition of neutrophil elastase activity by a tetravalent AAT-Fc-AAT fusion protein.
  • Figure IF is a graphing demonstrating the effect of low pH elution from protein A resin, wherein the NE inhibiting capacity of the AAT-Fc fusion protein eluted at low pH is drastically reduced.
  • Figure 1G is a graph showing that the double mutant, AAT-EL-Fc (Met351Glu, Met358Leu mutations) is resistant to H2O2 inactivation (cone), compared to wild type AAT and the single mutant AAT-EM-Fc (Met351Glu).
  • Figure 1H is a graph depicting the serum clearance rates of serum derived AAT (sdAAT) compared to AAT-Fc in rats dosed with lOmg/kg protein (3 rats/test protein). The half-life of AAT-Fc is substantially longer than that of sdAAT.
  • FIG. 2A is a schematic representation of some embodiments of the serpin- cytokine targeting fusion proteins of the invention.
  • the serpin can be fused to either the heavy chain, the light chain, or both of an antibody.
  • Serpin-cytokine receptor fusion proteins are also depicted.
  • Figure 2B is a photograph of a SDS-PAGE gel showing the D2E7 antibody (lane 1), and the D2E7 antibody with-AAT fused to heavy chain (lane 2).
  • Figure 2C is a graph showing the inhibition of neutrophil elastase activity by a D2E7 antibody fused to AAT. Serum derived AAT is shown as a positive control, whereas the D2E7 antibody alone is shown as a negative control for NE inhibition.
  • Figure 3A is a schematic representation of some embodiments of the serpin- Fc-WAP fusion proteins.
  • Figure 3B is a photograph of a SDS-PAGE gel showing AAT-Fc- ELAFIN (lane 1) and AAT-Fc-SLPI (lane 2).
  • Figure 3C is a graph showing the inhibition of neutrophil elastase activity by an AAT-Fc-ELAFIN fusion protein and an AAT-Fc-SLPI fusion protein.
  • An AAT-Fc fusion protein and serum derived AAT are included for comparison.
  • Figure 4A is a schematic representation of some embodiments of the AAT- HSA fusion proteins.
  • Figure 4B is a photograph of a SDS-PAGE gel showing an AAT- HSA fusion.
  • Figure 4C is a graph showing the inhibition of neutrophil elastase activity by an AAT-HSA compared to serum derived AAT.
  • NE Human neutrophil elastase
  • AAT alpha 1 -antitrypsin
  • AAT a deficiency of AAT, usually as a result of a point mutation that causes ATT to aggregate and accumulate in the liver, leaves the lungs exposed to unchecked NE activity.
  • Individuals with AAT deficiencies are at increased the risk of emphysema, COPD, liver disease, and numerous other conditions.
  • AAT deficiency affects approximately 100,000 Americans (according to estimates from the Alpha-1 Foundation), and many of the afflicted people die in their 30's and 40's.
  • There are currently only a few FDA-approved drugs for treatment of ATT deficiency Prolastin®, AralastTM, Zemaira®, GlassiaTM.
  • Each drug is the natural AAT derived from pooled human plasma, which appears to be insufficient to meet the anticipated clinical demand.
  • these products have short serum half-lives (T 2 of approximately 5 days) and require high dose (60 mg/kg body weight) weekly infusions.
  • the current market for these drugs is estimated at approximately $400 million.
  • AAT-like drugs are likely substantially larger, based on the estimation that as many as 95% of individuals with AAT-deficiencies go undiagnosed, and the fact that these drugs have the potential to be effective therapies for pathologies characterized by enhanced NE activity in individuals that are not AAT-deficient (e.g., cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), smoking-induced emphysema and/or COPD).
  • CF cystic fibrosis
  • ARDS acute respiratory distress syndrome
  • COPD smoking-induced emphysema and/or COPD
  • AAT has been suggested to have broad spectrum anti-inflammatory activity (Tilg et al 1993 J Exp Med 178: 1629-1636, Libert et al 1996 Immunol 157:5126-5129, Pott et al, Journal of Leukocyte Biology 85 2009, Janciauskiene et al 2007 J. Biol Chem
  • AAT may be useful in treating numerous human pathologies, outside of the commonly suggested inflammatory pulmonary conditions.
  • Human AAT has shown to protect mice from clinical and histopathological signs of experimental autoimmune encephalomyelitis (EAE), suggesting it could be a potential treatment of autoimmune diseases, such as multiple sclerosis or systemic lupus erythematosus (SLE) (Subramanian et a 201 i Metab Brain Dis 26: 107-1 13 ).
  • Serum AAT has shown activity in rodent models of Graft Versus Host Disease (GVHD) (Tawara et al 201 1 Proc. Natl. Acad. Sci. USA 109: 564-569, Marcondes et al 2011 5/ooi/ Nov 3; 1 18(18):5031-9), which has led to a human clinical trial using AAT to treat individuals with Steroid Non-responsive Acute GVHD (NCT01523821).
  • GVHD Graft Versus Host Disease
  • AAT has been effective in animal models of type I and type II diabetes, dampening inflammation, protecting islet cells from apoptosis and enabling durable islet cell allograft (Zhang et al 2007 Diabetes 56: 1316- 1323, Lewis et al 2005 Proc Natl Acad Sci USA 102: 12153-12158, Lewis et al 2008 Proc Natl Acad Sci USA 105: 16236-16241, Kalis et al 2010 Islets 2: 185-189).
  • the fusion proteins of the present invention have enhanced functionalities compared to the unmodified AAT molecule.
  • the fusion of an AAT polypeptide with a second polypeptide that interacts with the neonatal Fc receptor (FcRn) serves to increase the serum half-life, providing a much needed dosing benefit for patients.
  • FcRn interacting polypeptides of the fusion protein include immunoglobulin (Ig) Fc polypeptides derived from human IgGl, IgG2, IgG3, IgG4, or IgM, and derivatives of human albumin.
  • the fusion protein incorporates mutations with the AAT portion that render the molecule more resistant to inactivation by oxidation.
  • AAT is a natural anti-inflammatory protein
  • some embodiments of the invention provide enhanced inflammation dampening capacity through the fusion of an AAT polypeptide and a cytokine targeting polypeptide.
  • the coupling of dual antiinflammatory functionalities from AAT and a second polypeptide will provide more a potent therapeutic protein than either polypeptide on their own. Additionally, the coupling the anti-infective activity of AAT will mitigate the infection risk of most cytokine targeting biologies.
  • Some embodiments provide for more potent anti-inflammatory and anti-infective proteins through the fusion an AAT -polypeptide and WAP domain contain polypeptide.
  • the fusion proteins of the present invention are expected to be a great therapeutic utility and be superior to the current serum derived AAT products.
  • the fusion proteins of the present invention are shown to be potent inhibitors of NE, have extended serum half-lives, and in some embodiments resistant to oxidation. In other embodiments, the fusion proteins described herein have distinct properties by the incorporation of other functional polypeptides, including cytokine targeting polypeptides, and WAP domain containing polypeptides.
  • oxidation of an Fc region at Met252 and Met428 has been shown to reduce FcRn binding and subsequently reduce the serum half-life of the Fc containing protein.
  • Mutation of Met252 and Met428 reduces oxidation of the Fc region.
  • the present invention discloses and optimized AAT-Fc fusion protein, wherein it is resistant to oxidation-inactivation within the AAT portion of the fusion protein through mutations at M351 and M358; and oxidative disruption of the FcRn interaction through mutations at M252 and M428 and has an extended half-life through the set mutation of M252I, T256D, and M428L.
  • the Fc polypeptide is mutated or modified to enhance FcRn binding.
  • the mutated or modified Fc polypeptide includes the following mutations: Met252Ile, Thr256Asp and Met428Leu (M252I, T256D, M428L) using the Kabat numbering system.
  • the Fc polypeptide is a modified IgGl Fc polypeptide, and the fusion protein includes at least the amino acid sequence of SEQ ID NO: 53.
  • the Fc polypeptide is a modified IgGl Fc polypeptide, and the fusion protein includes at least the amino acid sequence of SEQ ID NO: 73.
  • the Fc polypeptide is mutated or modified to reduce binding to Fc-gamma receptors (FcgRs).
  • FcgRs Fc-gamma receptors
  • reduced FcgRs binding can be achieved by modification of Fc glycosylation at Asn297.
  • reduced FcgRs binding is achieved by modification of the lower hinge region of Fc.
  • the Fc polypeptide is derived from human IgGl .
  • lower hinge region is modified to mimic that of IgG2, through mutation of Leu234Val and Leu235Ala (L235V/L235A) and deletion of Gly236 (AG236) using the Kabat numbering system:
  • IgGl -hinge wt DKTHTCPPCPAPE
  • IgGl-VAAG Hinge DKTHTCPPCPAPE
  • the fusion protein includes at least the amino acid sequence of SEQ ID NO: 51.
  • the Fc polypeptide is derived from human IgG4.
  • the lower hinge region is modified by mutation at Leu235Glu (L235E).
  • the hinge region is modified through the stabilizing mutation Ser228Pro (S228P) using the Kabat numbering system:
  • IgG4-hinge wt ESKYGPPCP
  • IgG4-hinge PE ESKYGPPCP
  • the fusion protein includes at least the amino acid sequence of SEQ ID NO: 70.
  • the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and a second polypeptide.
  • the invention provides a serpin polypeptide fused to human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA derivatives.
  • the serpin- fusion described herein are expected to be useful in treating a variety of indications, including, by way of non- limiting example, alpha- 1 -antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia-reperfusion injury, including, e.g., ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft versus host disease (GVHD), wound healing, Systemic lupus erythematosus, and Multiple sclerosis.
  • AAT alpha- 1
  • the fusion proteins described herein include at least an alpha- 1 -antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and second polypeptide.
  • AAT alpha- 1 -antitrypsin
  • the invention provides alpha- 1 -antitrypsin (AAT) fused to human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA derivatives.
  • the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and a cytokine targeting polypeptide or an amino acid sequence that is derived from a cytokine targeting polypeptide.
  • the invention provides serpin polypeptide or a sequence derived from a serpin polypeptide fused to a human cytokine receptor or derivative thereof.
  • Another embodiment of the invention provides serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine targeting antibody, e.g., an anti-cytokine antibody, or a sequence derived from of a cytokine targeting antibody, e.g., an anti-cytokine antibody, or sequence derived from a fragment of cytokine targeting antibody, e.g., a fragment of an anti-cytokine antibody.
  • a cytokine targeting antibody e.g., an anti-cytokine antibody
  • a sequence derived from of a cytokine targeting antibody e.g., an anti-cytokine antibody
  • sequence derived from a fragment of cytokine targeting antibody e.g., a fragment of an anti-cytokine antibody.
  • the invention provides a serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFa, IgE, IL-12, IL-23, IL-6, IL-la, IL- ⁇ , IL-17, IL-13, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.
  • the cytokine targeting polypeptide targets TNFa and includes any of the following TNFa-targeting polypeptide or sequences derived from the following TNFa-targeting polypeptides: Remicade®, Humira®,
  • the cytokine targeting polypeptide targets IgE and includes any of the following IgE-targeting polypeptide or sequences derived from the following IgE-targeting polypeptides: Xolair or FceRI.
  • the cytokine targeting polypeptide targets the shared p40 subunit of IL-12 and IL-23 and includes the Stelara® polypeptide or sequences derived from the Stelara® polypeptide.
  • the cytokine targeting polypeptide targets IL-13 and includes the CDP7766 polypeptide or sequences derived from the CDP7766 polypeptide.
  • the fusion proteins described herein include at least a alpha- 1 -antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a cytokine targeting polypeptide or an amino acid sequence that is derived from a cytokine targeting polypeptide.
  • AAT alpha- 1 -antitrypsin
  • the invention provides alpha- 1 -antitrypsin inhibitor (AAT) fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFa, IgE, IL-6, IL-la, IL- ⁇ , IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.
  • AAT alpha- 1 -antitrypsin inhibitor
  • the cytokine targeting polypeptide binds a cytokine receptor and prevents binding of the cytokine.
  • the present invention includes a serpin fused to a cytokine receptor targeting antibody.
  • the invention provides alpha- 1 -antitrypsin inhibitor (AAT) fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds the receptor of any of the following human cytokines: TNFa, IgE, IL-6, IL-la, IL- ⁇ ⁇ , IL-12, IL-17, IL-13, IL-23, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.
  • AAT alpha- 1 -antitrypsin inhibitor
  • the cytokine targeting polypeptide targets the IL-6 receptor and includes the Actemra® polypeptide (as described in patent publication EP0628639), or the ALX-0061 polypeptide (as described in WO2010/1 15998), or sequences derived from the Actemra® polypeptide, or ALX-0061 polypeptide.
  • Actemra® the cytokine targeting polypeptide targets the IL-6 receptor and includes the tocilizumab polypeptide or sequences derived from the tocilizumab polypeptide.
  • cytokine targeting agents The most common proteins used as cytokine targeting agents are the soluble cytokine receptors and monoclonal antibodies and fragments thereof.
  • a significant drawback with targeting cytokines is the increased risk of infection in these patients, as evidenced by the TNFa targeting biologies, Remicade®, Humira®, Simponi®, Cimiza®, and Enbrel®, and the IL- 12/23 p40 targeting antibody, Stelara®. This is likely to be a common problem of targeting inflammatory cytokines leading to immune suppression in patients.
  • AAT and other serpin proteins are interesting in that they demonstrate both anti- infective and anti-inflammatory activities.
  • the serpin-cytokine targeting polypeptide fusion proteins of this invention can dampen aberrant cytokine activities while alleviating the risk of infections.
  • the fusion proteins described herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin, a WAP domain- containing polypeptide or an amino acid sequence that is derived from a WAP domain- containing polypeptide, and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.
  • the invention provides a serpin polypeptide, a WAP domain-containing polypeptide and human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc or IgM-Fc derivatives operably linked together in any functional combination.
  • the WAP domain containing protein is human SLPI or derived from human SLPI. In other embodiments, the WAP domain containing protein is human ELAFIN or derived from human ELAFIN.
  • the fusion proteins described herein include at least an alpha- 1 -antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a SLPI polypeptide or an amino acid sequence that is derived from SLPI. In some embodiments, the fusion proteins described herein include at least an AAT polypeptide or an amino acid sequence that is derived from AAT and an ELAFIN polypeptide or an amino acid sequence that is derived from Elafin.
  • AAT alpha- 1 -antitrypsin
  • SLPI and Elafin are WAP domain containing proteins that display serine protease inhibitory activity. Both of these proteins are anti-inflammatory in function. In addition these proteins possess broad anti-infective capacities toward numerous strains of bacteria, viruses, and fungi.
  • the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and a human serum albumin (HSA) polypeptide or an amino acid sequence that is derived from a HSA polypeptide.
  • HSA human serum albumin
  • Further embodiments of invention include serpin-albumin binding polypeptide fusion proteins, wherein the albumin binding polypeptide is responsible for the association of the serpin and HSA.
  • the invention includes both covalent and non-covalent linkages of the serpin polypeptide and the HSA polypeptide or sequences derived from the serpin polypeptide or a HSA polypeptide.
  • the invention provides a serpin polypeptide fused to human HSA, or HSA derivatives, or HSA binding peptide or polypeptides.
  • the fusion proteins described herein include at least an alpha- 1 -antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide.
  • AAT alpha- 1 -antitrypsin
  • the fusion proteins described herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin, a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide, and a WAP domain- containing polypeptide or an amino acid sequence that is derived from a WAP domain- containing polypeptide.
  • the fusion proteins described herein include at least an alpha- 1 -antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide, and a SLPI polypeptide or amino acid sequence derived from SLPI.
  • AAT alpha- 1 -antitrypsin
  • the fusion proteins described herein include at least an alpha- 1 -antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide, and an Elafin polypeptide or amino acid sequence derived from Elafin.
  • AAT alpha- 1 -antitrypsin
  • HSA HSA polypeptide
  • Elafin polypeptide or amino acid sequence derived from Elafin an Elafin polypeptide or amino acid sequence derived from Elafin.
  • the fusion proteins of the present invention can be readily produced in mammalian cell expression systems.
  • mammalian cell expression systems For example Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, COS cells, PER.C6®, NS0 cells, SP2/0, YB2/0 can readily be used for the expression of the serpin fusion proteins described herein.
  • mammalian cell expression systems produce proteins that are generally more optimal for therapeutic use.
  • mammalian cell expression systems yield proteins with glycosylation patterns that are similar or the same as those found in natural human proteins.
  • Proper glycosylation of a protein can greatly influence serum stability, pharmacokinetics, biodistribution, protein folding, and functionality. Therefore, the ability to produce therapeutic proteins in mammalian expression systems has distinct advantages over other systems. Furthermore, most of the mammalian cell expression systems (e.g., CHO, NS0, PER.C6® cells) can be readily scaled in commercial manufacturing facilities to produce therapeutic proteins to meet clinical demands.
  • the fusion proteins described herein have enhanced functionalities over the natural form of AAT and can be produced in mammalian expression systems for clinical and commercial supply.
  • Some embodiments of the invention include a purification system that enables the isolation of serpin fusion proteins that retain their ability to inhibit NE. Importantly, the purification process of the present invention can be readily incorporated into today's commercial mammalian cell-based manufacturing processes.
  • patient includes human and veterinary subjects.
  • LipofectinTM DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semi-solid mixtures containing carbowax.
  • Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W.
  • Therapeutic formulations of the invention which include a fusion protein of the invention, are used to treat or alleviate a symptom associated with a disease or disorder associated with aberrant serine protease activity in a subject.
  • the present invention also provides methods of treating or alleviating a symptom associated with a disease or disorder associated with aberrant serine protease activity in a subject.
  • a therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant serine protease activity, using standard methods, including any of a variety of clinical and/or laboratory procedures.
  • patient includes human and veterinary subjects.
  • subject includes humans and other mammals.
  • Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disease or disorder associated with aberrant serine protease activity. Alleviation of one or more symptoms of the disease or disorder associated with aberrant serine protease activity indicates that the fusion protein confers a clinical benefit.
  • Methods for the screening of fusion proteins that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzymatic assays, flow cytometry, and other immunologically mediated techniques known within the art.
  • ELISA enzyme linked immunosorbent assay
  • enzymatic assays enzyme linked immunosorbent assays
  • flow cytometry flow cytometry
  • other immunologically mediated techniques known within the art.
  • the fusion proteins described herein may be used in methods known within the art relating to the localization and/or quantitation of a target such as a serine protease, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • a target such as a serine protease
  • the terms "physiological sample” and “biological sample,” used interchangeably, herein are intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the terms "physiological sample” and “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph.
  • fusion proteins specific for a given target, or derivative, fragment, analog or homolog thereof, that contain the target-binding domain are utilized as pharmacologically active compounds (referred to hereinafter as "Therapeutics").
  • a fusion protein of the invention can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or
  • Detection can be facilitated by coupling (i.e., physically linking) the fusion protein to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
  • luminescent material includes luminol
  • bioluminescent materials include luciferase, luciferin, and aequorin
  • suitable radioactive material include I, I, S or H.
  • a therapeutically effective amount of a fusion protein of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the fusion protein and its target that, in certain cases, interferes with the functioning of the target.
  • the amount required to be administered will furthermore depend on the binding affinity of the fusion protein for its specific target, and will also depend on the rate at which an administered fusion protein is depleted from the free volume other subject to which it is administered.
  • Common ranges for therapeutically effective dosing of an fusion protein or fragment thereof invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 250 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a month.
  • fusion protein fragments are used, the smallest inhibitory fragment that specifically binds to the target is preferred.
  • peptide molecules can be designed that retain the ability to bind the target.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
  • the formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, growth-inhibitory agent, an anti-inflammatory agent or anti-infective agent.
  • an agent that enhances its function such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, growth-inhibitory agent, an anti-inflammatory agent or anti-infective agent.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fusion protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L- glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water,
  • Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • Exemplary, but non-limiting examples of AAT-Fc fusion proteins according to the invention include the following sequences. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge sequence and/or a linker sequence suitable in length and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence. For example, the polypeptide components can be directly attached.
  • An exemplary AAT-Fc fusion protein is the AAT-hFcl (human IgGl Fc) described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3).
  • AAT-hFcl human IgGl Fc
  • AAT-hFc2 human IgG2 Fc
  • An exemplary AAT-Fc fusion protein is the AAT-MM-EL-hFc 1 (human IgGl Fc, Met351Glu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), and the Met351Glu mutation is boxed, and the Met358Leu mutation is shaded in grey.
  • AAT -MM-EL-hFcl human IgGl Fc , Met351Glu/Met358Leu
  • An exemplary AAT-Fc fusion protein is the AAT-MM-EL-hFc2 (human IgG2 Fc, Met351Glu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Glu mutation is boxed, and the Met358Leu mutation is shaded in grey.
  • AAT-MM-EL-hFc2 human IgG2 Fc, Met351Glu/Met358Leu
  • An exemplary AAT-Fc fusion protein is the AAT-MM-LL-hFc 1 (human IgGl Fc, Met351Leu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), the Met351Leu mutation is shaded in black, and the Met358Leu mutation is shaded in grey.
  • AAT-MM-LL-hFcl human IgGl Fc, Met351Leu/Met358Leu
  • An exemplary AAT-Fc fusion protein is the AAT-MM:LL-hFc2(human IgG2 Fc, Met351Leu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Leu mutation is shaded in black, and the Met358Leu mutation is shaded in grey.
  • AAT-MM LL-hFc2 (human IgG2 Fc , Met351Leu/Met358Leu)
  • An exemplary AAT-Fc fusion protein is the AAT-hFcl-AAT (human IgGl), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3). AAT-hFcl-AAT (human IgGl)
  • AAT-EL-Fc-IgGl-DV,AG,IDL (AAT : Met351Glu/Met358Leu; Fc IgGl: Leu234Val/Leu235Ala, Deleted Gly236, Met252Ile, Thr256Asp, Met428Leu)
  • the gene encoding human AAT was PCR amplified from human liver cDNA (Zyagen). Specific point mutations within the gene encoding AAT or the Fc region were generated by overlapping PCR.
  • the AAT encoding gene was cloned in frame with a gene encoding the hinge region, followed by a CH2 domain, and a CH3 domain of human IgGl, IgG2, IgG3, IgG4, or IgM into a mammalian expression vector, containing a mammalian secretion signal sequence up stream of the AAT gene insertion site.
  • AAT-Fc fusion proteins were transfected into mammalian cells (specifically HEK293 or CHO cells) and grown for several days in 8% CO 2 at 37° C.
  • the recombinant AAT-Fc fusion proteins were purified from the expression cell supernatant by protein A chromatography.
  • a near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo Scientific) to elute the AAT-Fc fusion protein from the protein A resin.
  • the AAT-Fc fusion protein cannot be eluted from protein A resin using a standard low pH elution, as the ability of AAT to inhibit NE is compromised following low pH treatment, likely due to a low pH mediated oligomerization of AAT.
  • Figure IF shows the effects of low pH elution on the ability of AAT to inhibit neutrophil elastase.
  • AAT-Fc fusion protein can be purified either by protein A and a near neutral pH elution buffer, by CaptureSelect® Alpha- 1 Antitrypsin affinity matrix (BAC BV).
  • BAC BV CaptureSelect® Alpha- 1 Antitrypsin affinity matrix
  • FIG. 1 The purified AAT-Fc fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase (NE).
  • Figure IB and ID show a reducing SDS- PAGE gel of purified serum derived AAT (sdAAT) and AAT-Fc fusion proteins (Fig IB- lane 1 : sdAAT, lane 2: AAT-Fc (SEQ ID NO: 16), lane 3 : AAT-EL-Fc (SEQ ID NO: 18), Fig ID AAT-Fc-AAT (SEQ ID NO: 20).
  • the proteins were visualized by staining with Coomassie blue.
  • NE Neutrophil Elastase
  • This assay buffer is composed of 100 mM Tris pH 7.4, 500 mM NaCl, and 0.0005% Triton X-100.
  • Human NE is used at a final concentration of 5 nM (but can also be used from 1-20 nM).
  • the fluorescent peptide substrate AAVP- AMC is used at a final concentration of 100 ⁇ in the assay.
  • the Gemini EM plate reader from Molecular Devices is used to read the assay kinetics using excitation and emission wavelengths of 370 nm and 440 nm respectively, and a cutoff of 420 nm.
  • the assay is read for 10 min at room temperature scanning every 5 to 10 seconds.
  • the Vmax per second corresponds to the residual NE activity, which is plotted for each concentration of inhibitor.
  • the intercept with the x-axis indicates the concentration of inhibitor needed to fully inactivate the starting concentration of NE in the assay.
  • Human serum derived AAT (sdAAT) was used as a positive control in these assays.
  • the AAT-Fc fusion proteins display potent NE inhibitory activity as shown in Figure 1C.
  • AAT-Fc-AAT displays enhanced potency over both sdAAT and the AAT-Fc fusion protein comprising a single AAT polypeptide ( Figure IE).
  • Figure IF demonstrates the resistance of the AAT-EL-Fc (M351E, M358L) fusion protein to inactivation by oxidation.
  • AAT fusion proteins, AAT-Fc (wt), AAT-EL- Fc (M351E, M358L), and AAT-EM-Fc (M351E) were treated with 33mM H 2 0 2 and compared to untreated fusion proteins in the NE inhibition assays.
  • the inhibition of NE by AAT-EL-Fc was not comprised by oxidation, converse to the other proteins tested.
  • AAT-Fc fusion protein displayed a longer serum half-life in rats compared to serum derived AAT ( Figure 1H).
  • 3 rats per each test protein were injected I.V. with lOmg/kg of sdAAT or AAT-Fc.
  • Serum sample were taken at various time points over a 48 period.
  • the serum ATT concentration was using an ELISA.
  • the fusion proteins below include cytokine targeting polypeptide sequences that are from or are derived from (i) the anti-TNFa antibody D2E7 (also known as
  • Adalimumab or Humira® Adalimumab or Humira®
  • TNFR2-ECD Type 2 TNFa Receptor
  • the AAT polypeptide portion of the fusion protein is underlined, the antibody constant regions (CHl-hinge-CH2-CH3, or CL) are italicized, and D2E7-VH, D2E7-VK, and TNFR2-ECD are denoted in bold text. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge sequence and/or a linker sequence suitable in length and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence.
  • An exemplary AAT-TNFa fusion protein is D2E7-Light Chain-AAT (G 3 S) 2 Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is denoted in bold text (SEQ ID NO: 37), and the antibody constant regions are italicized (SEQ ID NO: 38) D2E7-Light Chain-AAT (G 3 S) 2 Linker
  • An exemplary AAT-TNFa fusion protein is D2E7-Light Chain-AAT ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is denoted in bold text (SEQ ID NO: 37), and the antibody constant regions is italicized (SEQ ID NO: 38)
  • An exemplary AAT-TNFa fusion protein is D2E7-Heavy Chain-AAT (G 3 S) 2 Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is denoted in bold text (SEQ ID NO: 39), and the antibody constant regions is italicized (SEQ ID NO: 40)
  • An exemplary AAT-TNFa fusion protein is D2E7-Heavy Chain-AAT ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is denoted in bold text (SEQ ID NO: 39), and the antibody constant regions is italicized (SEQ ID NO: 40)
  • An exemplary AAT-TNFa fusion protein is TNFR2-ECD-Fcl-AAT(G 3 S) 2
  • An exemplary AAT-TNFa fusion protein is TNFR2-ECD-Fcl-AAT ASTGS
  • variable heavy (VH) and variable kappa (VK) regions of the anti-TNFa antibody, D2E7 were generated by gene synthesis.
  • the D2E7- VH gene was cloned in frame with a gene encoding a human IgGl antibody heavy chain constant region, consisting of a CHI domain, a hinge domain, a CH2 domain, and a CH3 domain, into a mammalian expression vector, containing a mammalian secretion signal sequence up stream of the VH domain insertion site (D2E7-HC).
  • the D2E7-VK gene was cloned in frame with a human antibody kappa light chain constant (CL) domain, into a mammalian expression vector, containing a mammalian secretion signal sequence up stream of the VK domain insertion site (D2E7-LC).
  • the AAT encoding gene and the adjacent 5' linker sequence were cloned in frame into the 3 ' end of either, the CH3 domain of the D2E7 heavy chain gene (D2E7-HC-AAT), or the CL domain of the D2E7 light chain gene (D2E7- LC-AAT) coding sequences in the above described mammalian expression vectors.
  • the extracellular domain of the TNFa Receptor 2 was generated by gene synthesis and cloned in frame with a gene encoding the hinge region, followed by a CH2 domain and a CH3 domain of human IgGl (hFcl) into a mammalian expression, containing a mammalian secretion signal sequence up stream of the TNFR2-ECD insertion site.
  • the AAT encoding gene and the adjacent 5' linker sequence were cloned in frame into the 3 ' end of the gene encoding TNFR2-ECD-hFcl into a mammalian expression vector (TNFR2- ECD-hFcl-AAT).
  • the D2E7-HC-AAT expression vector was co-transfected with either the D2E7-LC or the D2E7-LC-AAT expression vector into mammalian cells (specifically HEK293 or CHO cells) to generate the D2E7 antibody with AAT fused to the C-terminus of the heavy chain or to the C-terminus of both the heavy chain and light chain, respectively.
  • the D2E7-LC-AAT was co-transfected with the D2E7-HC expression vector into mammalian cells to generate the D2E7 antibody with AAT fused to the C-terminus of the light chain.
  • the TNFR2-hFcl-AAT expression vector was transfected into mammalian cells. Transfected cells were grown for several days in 8% CO2 at 37° C.
  • AAT -TNFa targeting fusion proteins were purified from the expression cell supernatant by protein A chromatography.
  • a near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo Scientific) to elute the AAT -TNFa targeting fusion proteins from the protein A resin.
  • Figure 2B shows an SDS-PAGE gel of the D2E7 antibody alone (lane 1) and variant wherein AAT is fused to the heavy chain of D2E7 (lane 2).
  • the proteins were visualized by staining with Coomassie blue.
  • AAT -TNFa targeting molecule fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase.
  • Human serum derived AAT (sdAAT) was used as a positive control in these assays.
  • NE inhibitory assay were conducted as described above.
  • Figure 2C demonstrates relative to sdAAT, the AAT-TNFa targeting molecule fusion protein shows similar inhibition of neutrophil elastase, indicating that the inhibitory capacity of AAT has not been compromised by its fusion to an antibody.
  • Example 3 AAT-Fc-SLPI and AAT-Fc-Elafin
  • AAT derivatives comprising human AAT fused a WAP domain containing protein. These examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention.
  • the AAT polypeptide portion of the fusion protein is underlined, the Fc portion is italicized, and the WAP domain containing polypeptide is in bold font. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge sequence and/or a linker sequence suitable in length and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence. For example, the polypeptide components can be directly attached.
  • AAT-Fc-SLPI fusion protein is AAT-hFcl-SLPI (human IgGl Fc), described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the Fc portion is italicized (SEQ ID NO: 3), and the WAP domain containing polypeptide is in bold font (SEQ ID NO: 9)
  • AAT-hFcl-SLPI human IgGl Fc
  • the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the Fc portion is italicized (SEQ ID NO: 3), and the WAP domain containing polypeptide is in bold font (SEQ ID NO: 12)
  • the genes encoding the SLPI and Elafin were PCR amplified from human spleen cDNA (Zyagen). These genes were cloned into the mammalian expression vectors of example 1 , wherein the SLPI or Elafin gene was inserted in frame with the AAT-Fc gene. These expression vectors were transfected into mammalian cells (specifically HEK293 or CHO cells) and grown for several days in 8% CO 2 at 37° C. The recombinant AAT-Fc- WAP domain fusion proteins were purified from the expression cell supernatant by protein A chromatography. A near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo Scientific) to elute the AAT-Fc-WAP domain fusion protein from the protein A resin.
  • Figure 3B shows an SDS-PAGE gel of the AAT-Fc-WAP fusion proteins (lane 1 AAT-Fc-Elafin, lane 2 AAT-Fc-SLPI). The proteins were visualized by staining with Coomassie blue. The purified AAT-Fc-WAP domain fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. NE inhibitory assays were conducted as described above. Human serum derived AAT (sdAAT) and the AAT-Fc fusion protein were used as a positive control in these assays. Relative to sdAAT, the AAT- Fc-WAP targeting molecule fusion proteins display enhanced potency of NE inhibition of neutrophil elastase (Figure 3C).
  • sdAAT Human serum derived AAT
  • AAT-Fc-WAP targeting molecule fusion proteins display enhanced potency of NE inhibition of neutrophil elastase
  • AAT derivatives comprising human AAT fused an albumin polypeptide. These examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not and are not intended to limit the claimed invention.
  • the AAT portion is underlined and the albumin portion is italicized. For example, the polypeptide components can be directly attached.
  • AAT-Albumin fusion protein is AAT-HSA, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), and the albumin polypeptide is italicized (SEQ ID NO: 14)
  • AAT-Albumin fusion protein is AAT-HSA Domain 3, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), and the albumin polypeptide is italicized (SEQ ID NO: 15)
  • HSA human serum albumin
  • Figure 4B shows an SDS-PAGE gel of the AAT-HSA fusion protein
  • the proteins were visualized by staining with Coomassie blue.
  • the purified AAT-HSA fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase.
  • NE inhibitory assays were conducted as described above. Human serum derived AAT (sdAAT) was used as a positive control in these assays. Relative to sdAAT, the AAT-HS fusion protein displays similar potency of NE inhibition, demonstrating that the fusion to albumin does not dampen the capacity of AAT to inhibit NE ( Figure 4C.)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Diabetes (AREA)
  • Rheumatology (AREA)
  • Pulmonology (AREA)
  • Pain & Pain Management (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Neurosurgery (AREA)
  • Virology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Biomedical Technology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Emergency Medicine (AREA)
PCT/US2015/057533 2014-10-27 2015-10-27 Serpin fusion polypeptides and methods of use thereof WO2016069574A1 (en)

Priority Applications (17)

Application Number Priority Date Filing Date Title
IL251799A IL251799B2 (en) 2014-10-27 2015-10-27 Serpin-fused polypeptides and methods of using them
CA2965151A CA2965151A1 (en) 2014-10-27 2015-10-27 Serpin fusion polypeptides and methods of use thereof
EP15854670.5A EP3212290A4 (en) 2014-10-27 2015-10-27 SERPIN FUSION POLYPEPTIDES AND METHODS OF USE
BR112017008525-9A BR112017008525A2 (pt) 2014-10-27 2015-10-27 proteína de fusão isolada, e, métodos para tratamento ou alívio de um sintoma de uma doença ou distúrbio, para tratamento ou alívio de uma inflamação ou um sintoma de uma doença ou distúrbio inflamatório e para redução do risco de infecção em um indivíduo.
UAA201705128A UA127305C2 (uk) 2014-10-27 2015-10-27 Злитий серпіновий поліпептид і спосіб його застосування
RU2017118325A RU2746550C2 (ru) 2014-10-27 2015-10-27 Слитые серпиновые полипептиды и способы их применения
AU2015339507A AU2015339507B2 (en) 2014-10-27 2015-10-27 Serpin fusion polypeptides and methods of use thereof
CN201580071331.7A CN107206257A (zh) 2014-10-27 2015-10-27 丝氨酸蛋白酶抑制剂融合多肽和其使用方法
MX2017005467A MX2017005467A (es) 2014-10-27 2015-10-27 Polipéptidos de fusión de serpina y métodos para utilizar los mismos.
KR1020177014414A KR20170091096A (ko) 2014-10-27 2015-10-27 세르핀 융합 폴리펩티드 및 이의 사용 방법
SG11201703390SA SG11201703390SA (en) 2014-10-27 2015-10-27 Serpin fusion polypeptides and methods of use thereof
CN202111439584.9A CN114316068A (zh) 2014-10-27 2015-10-27 丝氨酸蛋白酶抑制剂融合多肽和其使用方法
KR1020237043666A KR20240005109A (ko) 2014-10-27 2015-10-27 세르핀 융합 폴리펩티드 및 이의 사용 방법
IL308589A IL308589A (en) 2014-10-27 2015-10-27 Serpin-fused polypeptides and methods of using them
JP2017522654A JP6737781B2 (ja) 2014-10-27 2015-10-27 セルピン融合ポリペプチド及びその使用方法
HK18103148.5A HK1244460A1 (zh) 2014-10-27 2018-03-05 絲氨酸融合多肽和其使用方法
AU2021240153A AU2021240153A1 (en) 2014-10-27 2021-09-28 Serpin fusion polypeptides and methods of use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14/524,832 US10400029B2 (en) 2011-06-28 2014-10-27 Serpin fusion polypeptides and methods of use thereof
US14/524,832 2014-10-27

Publications (1)

Publication Number Publication Date
WO2016069574A1 true WO2016069574A1 (en) 2016-05-06

Family

ID=55858243

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/057533 WO2016069574A1 (en) 2014-10-27 2015-10-27 Serpin fusion polypeptides and methods of use thereof

Country Status (14)

Country Link
EP (1) EP3212290A4 (pt)
JP (2) JP6737781B2 (pt)
KR (2) KR20240005109A (pt)
CN (2) CN107206257A (pt)
AU (2) AU2015339507B2 (pt)
BR (1) BR112017008525A2 (pt)
CA (1) CA2965151A1 (pt)
HK (1) HK1244460A1 (pt)
IL (2) IL251799B2 (pt)
MX (2) MX2017005467A (pt)
RU (1) RU2746550C2 (pt)
SG (2) SG10201903142RA (pt)
UA (1) UA127305C2 (pt)
WO (1) WO2016069574A1 (pt)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020092448A1 (en) * 2018-10-29 2020-05-07 Spin Therapeutics, Llc Compositions and methods for alpha-1-antitrypsin disorders
WO2022133521A1 (en) * 2020-12-21 2022-06-30 Macquarie University Treatment of glaucoma
WO2022178175A1 (en) 2021-02-17 2022-08-25 Arecor Limited Aqueous solution compositions for increasing stability of engineered dimeric proteins
WO2022184854A3 (en) * 2021-03-03 2022-10-20 Formycon Ag Formulations of ace2 fc fusion proteins
WO2023225513A1 (en) 2022-05-16 2023-11-23 Inhibrx, Inc. Effective dosage of recombinant serpin-fc fusion protein for use in a method of treating aat deficiency in a subject

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020097946A1 (zh) * 2018-11-18 2020-05-22 杭州博虎生物科技有限公司 一种重组人白细胞介素10融合蛋白及其应用
CN112315897A (zh) * 2020-11-04 2021-02-05 深圳前海鹰岗生物科技有限公司 一种抑制细胞炎症反应释放治疗痛风急性发作的聚合物微针及制备方法
CN113325181A (zh) * 2021-04-25 2021-08-31 苏州市立医院(北区) 丝氨酸蛋白酶抑制剂在用于脓毒症早期预警的标志物中的应用
CN114874333A (zh) * 2021-10-18 2022-08-09 深圳科兴药业有限公司 一种生长激素融合蛋白及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060173170A1 (en) * 2004-11-12 2006-08-03 Xencor, Inc. Fc variants with altered binding to FcRn
US20080171689A1 (en) * 2005-07-22 2008-07-17 Williams Lewis T Compositions and Methods of Treating Disease with Fgfr Fusion Proteins
US20130011398A1 (en) * 2011-06-28 2013-01-10 Inhibrx Llc Serpin Fusion Polypeptides and Methods of Use Thereof
US20130011386A1 (en) * 2010-12-23 2013-01-10 Randall Brezski Active protease-resistant antibody fc mutants
US8633305B2 (en) * 2003-08-26 2014-01-21 Leland Shapiro Compositions of, and methods for, alpha-1 anti trypsin Fc fusion molecules
US20140051834A1 (en) * 2012-06-21 2014-02-20 Hoffmann-La Roche, Inc. Incretin Receptor Ligand Polypeptide Fc-Region Fusion Polypeptides And Conjugates With Altered Fc-Effector Function

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3609300A (en) * 1999-03-01 2000-09-21 Human Genome Sciences, Inc. Human serpin proteins
US7317091B2 (en) * 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US7217797B2 (en) * 2002-10-15 2007-05-15 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
EP1646642A2 (en) * 2003-07-18 2006-04-19 Vertex Pharmaceuticals Incorporated Inhibitors of serine proteases, particularly hcv ns3-ns4a protease
US8802820B2 (en) * 2004-11-12 2014-08-12 Xencor, Inc. Fc variants with altered binding to FcRn
EP1926747A1 (en) * 2005-08-12 2008-06-04 Schering Corporation Mcp1 fusions
AU2009262199B2 (en) * 2008-06-27 2012-08-09 Amgen Inc. Ang-2 inhibition to treat multiple sclerosis
CN106432503B (zh) * 2008-12-19 2020-03-06 宏观基因有限公司 共价双抗体及其用途
EP3590965A1 (en) * 2011-03-29 2020-01-08 Roche Glycart AG Antibody fc variants
MX356433B (es) * 2011-06-28 2018-05-29 Inhibrx Lp Polipéptidos de fusión con domino de proteína ácida del suero, y métodos de uso de los mismos.
KR102348985B1 (ko) * 2012-01-10 2022-01-12 더 리젠츠 오브 더 유니버시티 오브 콜로라도, 어 바디 코포레이트 알파-1 안티트립신 융합 분자용 조성물, 방법 및 용도
WO2014001325A1 (en) * 2012-06-27 2014-01-03 F. Hoffmann-La Roche Ag Method for making antibody fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
DK2880170T3 (en) * 2012-08-02 2016-10-24 Hoffmann La Roche PROCEDURE FOR PREPARING SOLUBLE FcR AS Fc FUSION WITH INERT IMMUNOGLOBULIN Fc REGION AND APPLICATIONS THEREOF
EP2929045B1 (en) * 2012-12-05 2020-12-02 SOLA Biosciences LLC Protein expression enhancing polypeptides

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8633305B2 (en) * 2003-08-26 2014-01-21 Leland Shapiro Compositions of, and methods for, alpha-1 anti trypsin Fc fusion molecules
US20060173170A1 (en) * 2004-11-12 2006-08-03 Xencor, Inc. Fc variants with altered binding to FcRn
US20080171689A1 (en) * 2005-07-22 2008-07-17 Williams Lewis T Compositions and Methods of Treating Disease with Fgfr Fusion Proteins
US20130011386A1 (en) * 2010-12-23 2013-01-10 Randall Brezski Active protease-resistant antibody fc mutants
US20130011398A1 (en) * 2011-06-28 2013-01-10 Inhibrx Llc Serpin Fusion Polypeptides and Methods of Use Thereof
US20140051834A1 (en) * 2012-06-21 2014-02-20 Hoffmann-La Roche, Inc. Incretin Receptor Ligand Polypeptide Fc-Region Fusion Polypeptides And Conjugates With Altered Fc-Effector Function

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3212290A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020092448A1 (en) * 2018-10-29 2020-05-07 Spin Therapeutics, Llc Compositions and methods for alpha-1-antitrypsin disorders
CN113286816A (zh) * 2018-10-29 2021-08-20 斯宾疗法有限责任公司 用于α-1-抗胰蛋白酶病症的组合物和方法
WO2022133521A1 (en) * 2020-12-21 2022-06-30 Macquarie University Treatment of glaucoma
WO2022178175A1 (en) 2021-02-17 2022-08-25 Arecor Limited Aqueous solution compositions for increasing stability of engineered dimeric proteins
US20220265788A1 (en) * 2021-02-17 2022-08-25 Arecor Limited Aqueous solution compositions for increasing stability of engineered dimeric proteins
WO2022184854A3 (en) * 2021-03-03 2022-10-20 Formycon Ag Formulations of ace2 fc fusion proteins
WO2023225513A1 (en) 2022-05-16 2023-11-23 Inhibrx, Inc. Effective dosage of recombinant serpin-fc fusion protein for use in a method of treating aat deficiency in a subject

Also Published As

Publication number Publication date
RU2017118325A3 (pt) 2019-03-21
IL308589A (en) 2024-01-01
CN107206257A (zh) 2017-09-26
JP2020180157A (ja) 2020-11-05
IL251799B1 (en) 2023-12-01
IL251799A0 (en) 2017-06-29
MX2021012047A (es) 2021-11-03
KR20240005109A (ko) 2024-01-11
CN114316068A (zh) 2022-04-12
CA2965151A1 (en) 2016-05-06
EP3212290A4 (en) 2019-01-23
AU2015339507A1 (en) 2017-05-11
JP2017537888A (ja) 2017-12-21
SG10201903142RA (en) 2019-05-30
RU2017118325A (ru) 2018-11-29
JP6737781B2 (ja) 2020-08-12
EP3212290A1 (en) 2017-09-06
BR112017008525A2 (pt) 2018-01-30
IL251799B2 (en) 2024-04-01
MX2017005467A (es) 2017-11-30
UA127305C2 (uk) 2023-07-19
KR20170091096A (ko) 2017-08-08
HK1244460A1 (zh) 2018-08-10
AU2021240153A1 (en) 2021-10-28
SG11201703390SA (en) 2017-05-30
RU2746550C2 (ru) 2021-04-15
AU2015339507B2 (en) 2021-07-01

Similar Documents

Publication Publication Date Title
AU2021202131B2 (en) Serpin fusion polypeptides and methods of use thereof
AU2015339507B2 (en) Serpin fusion polypeptides and methods of use thereof
AU2012275295B2 (en) WAP domain fusion polypeptides and methods of use thereof
US11046752B2 (en) Serpin fusion polypeptides and methods of use thereof
NZ619023B2 (en) Serpin fusion polypeptides and methods of use thereof

Legal Events

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

Ref document number: 15854670

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2965151

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 251799

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 2017522654

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: MX/A/2017/005467

Country of ref document: MX

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 11201703390S

Country of ref document: SG

ENP Entry into the national phase

Ref document number: 2015339507

Country of ref document: AU

Date of ref document: 20151027

Kind code of ref document: A

REEP Request for entry into the european phase

Ref document number: 2015854670

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 20177014414

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: A201705128

Country of ref document: UA

ENP Entry into the national phase

Ref document number: 2017118325

Country of ref document: RU

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112017008525

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112017008525

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20170425