Serpin fusion polypeptides and methods of use thereof

Abstract

To provide more potent anti-inflammatory and anti-infective proteins.SOLUTION: The invention provides molecules, particularly polypeptides, more particularly fusion proteins that comprise a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide and a second polypeptide comprising at least one of: an Fc polypeptide or an amino acid sequence 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 derived from a serum albumin polypeptide.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 502,055, filed June 28, 2011; U.S. Provisional Application No. 61 / 570,394, filed December 14, 2011; and U.S. Provisional Application No. 61 / 577,204, filed December 19, 2011; and U.S. Provisional Application No. 61 / 638,168, filed April 25, 2012. The entire contents of each of these applications are incorporated herein by reference. FIELD OF THE PRESENT APPLICATION

[0002] The present invention relates to molecules, particularly polypeptides, more particularly fusion proteins comprising a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide and a second polypeptide. Furthermore, the present invention relates to fusion proteins comprising a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide, a second polypeptide, and a third polypeptide. In particular, the present invention relates to fusion proteins comprising at least one serpin polypeptide and a second polypeptide, or at least one serpin polypeptide, a second polypeptide, and a third polypeptide, wherein the second and third polypeptides of the fusion protein of the present invention are at least one of the following: an Fc polypeptide or an amino acid sequence 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 derived from a serum albumin polypeptide. The present invention also relates to methods of using such molecules for various therapeutic and diagnostic indications, and to methods of producing such molecules. [Background technology]

[0003] Aberrant serine protease activity or an imbalance of proteases versus protease inhibitors leads to protease-mediated tissue destruction and inflammatory responses. Thus, there is a need for therapeutic agents and therapies that target aberrant serine protease activity and / or an imbalance of proteases versus protease inhibitors. Furthermore, enhanced therapeutic effects can be obtained by reducing aberrant cytokine signals and serine protease activity. In addition, serpin proteins have demonstrated anti-infective drug activity, while targeting inflammatory cytokines has been shown to enhance the risk of infection. The fusion proteins of the present invention have the potential to suppress inflammatory cytokine activity and limit the risk of infection. Summary of the Invention

[0004] The fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide (polypeptide 1) and a second polypeptide (polypeptide 2). Additionally, the fusion proteins described herein include a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide (polypeptide 1), a second polypeptide (polypeptide 2), and a third polypeptide (polypeptide 3). As used interchangeably herein, the terms "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 conjugation, use of an intermediate or spacer peptide, use of a linker region, use of a hinge region, or use of both a linker and a hinge region. In some embodiments, the linker region can be included within the sequence of the hinge region, or alternatively, the hinge region can be included within the sequence of the linker region. Preferably, the linker region is a peptide sequence. For example, the linker peptide is in the range of 0 to 40 amino acids, for example, 0 to 35 amino acids, 0 to 30 amino acids, 0 to 25 amino acids, or 0 to 20 amino acids. Preferably, the hinge region is a peptide sequence. For example, the hinge peptide is in the range of 0 to 75 amino acids, for example, 0 to 70 amino acids, 0 to 65 amino acids, or 0 to 62 amino acids. In embodiments in which the fusion protein comprises both a linker region and a hinge region, preferably each of the linker region and the hinge region is a peptide sequence. In these embodiments, the hinge peptide and the linker peptide together are in the range of 0 to 90 amino acids, for example, 0 to 85 amino acids or 0 to 82 amino acids.

[0005] In some embodiments, the serpin polypeptide and the second polypeptide are linked through an intermediate binding protein. In some embodiments, the serpin-based portion and the second polypeptide portion of the fusion protein may be linked non-covalently.

[0006] In some embodiments, a fusion protein according to the invention has, from N-terminal to C-terminal or C-terminal to N-terminal, one of the following formulas: Polypeptide 1 (a) -Hinges m -Polypeptide 2 (b) Polypeptide 1 (a) -Linker n -Polypeptide 2 (b) Polypeptide 1 (a) -Linker n -Hinges m -Polypeptide 2 (b) Polypeptide 1 (a) -Hinges m -Linker n -Polypeptide 2 (b) Polypeptide 1 (a) -Polypeptide 2 (b) -Polypeptide 3 (c) Polypeptide 1 (a) -Hinges m -Polypeptide 2 (b) -Hinges m -Polypeptide 3 (c) Polypeptide 1 (a) -Linker n -Polypeptide 2 (b) -Linker n -Polypeptide 3 (c) Polypeptide 1 (a) -Hinges m -Linker n -Polypeptide 2 (b) -Hinges m -Linker n-Polypeptide 3 (c) Polypeptide 1 (a) -Linker n -Hinges m -Polypeptide 2 (b) -Linker n -Hinges m -Polypeptide 3 (c) wherein n is an integer between 0 and 20, m is an integer between 1 and 62, and a, b, and c are integers of at least 1. These embodiments include any of the above formulas and variations or combinations thereof. For example, the sequence of the polypeptides of the above formulas may include polypeptide 3, (c) -Polypeptide 1 (a) -Polypeptide 2 (b) , polypeptide 2 (b) -Polypeptide 3 (c) -Polypeptide 1 (a) , or variations or combinations thereof.

[0007] In some embodiments, the polypeptide 1 sequence includes a serpin polypeptide. Serpins are a group of proteins with similar structures that were first identified as a series of proteins capable of inhibiting 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 (SERPINB6), antithrombin (SERPINC1), plasminogen activator inhibitor 1 (SERPINE1), alpha 2 antiplasmin (SERPINF2), complement 1 inhibitor (SERPING1), and neuroserpin (SERPINI1).

[0008] In some embodiments, the polypeptide 1 sequence comprises an alpha 1 antitrypsin (AAT) polypeptide sequence or an amino acid sequence derived from AAT. In some embodiments, the polypeptide 1 sequence comprises a portion of an AAT protein. In some embodiments, the polypeptide 1 sequence comprises at least a reactive site loop portion of the AAT protein. In some embodiments, the reactive site loop portion of the AAT protein comprises at least the following amino acid sequence: [ka]

[0009] In a preferred embodiment, the AAT polypeptide sequence or the amino acid sequence derived from AAT is a human AAT polypeptide sequence or is derived from a human AAT polypeptide sequence.

[0010] In some embodiments, the fusion protein comprises a full-length human AAT polypeptide sequence having the following amino acid sequence: [ka]

[0011] In some embodiments, the fusion protein comprises 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.

[0012] In some embodiments, the AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide is selected from the group consisting of GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, N NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0013] In some embodiments, the fusion protein comprises one or more mutations. For example, the fusion protein comprises a mutation in at least one methionine (Met) residue in the serpin portion of the fusion protein. In these Met mutations, the Met residue is replaced by any amino acid. For example, the Met residue is replaced by an amino acid having a hydrophobic side chain, such as, for example, leucine (Leu, L). Without wishing to be bound by any theory, the Met mutation(s) prevents oxidation of the fusion protein of the invention and subsequent inactivation of the inhibitory activity. In some embodiments, the Met residue can be replaced by a charged residue, such as, for example, glutamic acid (Glu, E). In some embodiments, the Met mutation is at position 358 of the AAT polypeptide. For example, the Met mutation is Met358Leu (M358L). In some embodiments, the Met mutation is at position 351 of the AAT polypeptide. For example, the Met mutation is Met351Glu (M351E). In some embodiments, for example, the Met mutations are at positions 351 and 358 of the AAT polypeptide, e.g., the Met mutations are Met351Glu (M351E) and Met358Leu (M358L). For example, this variant of the reactive site loop of the fusion AAT polypeptide has the following sequence: [ka] In some embodiments, the Met mutations are at positions 351 and 358 of the AAT polypeptide, for example, the Met mutations are Met351Leu (M351L) and Met358Leu (M358L). For example, this variant of the reactive site loop of the fusion AAT polypeptide has the following sequence: [ka]

[0014] In some embodiments, the second polypeptide (polypeptide 2) of the serpin fusion protein is an Fc polypeptide or is derived from an Fc polypeptide. These embodiments are collectively referred to herein as "serpin-Fc fusion proteins." The serpin-Fc fusion proteins described herein contain at least a serpin polypeptide or an amino acid sequence derived from a serpin, and an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. In some embodiments, a serpin-Fc fusion protein contains one serpin polypeptide. In other embodiments, a serpin-Fc fusion protein contains two or more serpin polypeptides, and these embodiments are collectively referred to herein as "serpin-Fc fusion proteins." (a’) In some embodiments, the serpin (a’) Each serpin polypeptide in the -Fc fusion protein comprises the same amino acid sequence. (a’) Each serpin polypeptide in the -Fc fusion protein comprises a serpin polypeptide having a different amino acid sequence. (a’) The serpin polypeptide of the -Fc fusion protein can be positioned at any position within the fusion protein.

[0015] In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein comprises at least the amino acid sequence of a reactive site loop portion of an AAT protein. In some embodiments, the reactive site loop portion of an AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein comprises at least the amino acid sequence of a variant of a reactive site loop portion of an AAT protein. In some embodiments, the variant of a reactive site loop portion of an AAT protein comprises 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 comprises at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein comprises 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 or 32 or 33.

[0016] In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein comprises an AAT polypeptide sequence, and the amino acid sequence thereof or derived from the AAT polypeptide is set forth in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA3498. 2.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0017] In some embodiments, the Fc polypeptide of the fusion protein is a human Fc polypeptide, such as a human IgG Fc polypeptide sequence or an amino acid sequence derived from a human IgG Fc polypeptide sequence. For example, in some embodiments, the Fc polypeptide is a human IgG1 Fc polypeptide or an amino acid sequence derived from a human IgG1 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence derived from a human IgG2 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG3 Fc polypeptide or an amino acid sequence derived from a human IgG3 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG4 Fc polypeptide or an amino acid sequence derived from a human IgG4 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgM Fc polypeptide or an amino acid sequence derived from a human IgM Fc polypeptide sequence.

[0018] In some embodiments in which a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG1 Fc polypeptide sequence having the following amino acid sequence: [ka]

[0019] In some embodiments where a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG1 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.

[0020] In some embodiments where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn binding. In these embodiments, the mutated or modified Fc polypeptide comprises the following mutations: Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) or Met428Leu, and Asn434Ser (M428L, N434S) using the Kabat numbering system. In some embodiments, the Fc polypeptide moiety is mutated or otherwise modified to prevent Fc-mediated dimerization. In these embodiments, the fusion protein is monomeric in nature.

[0021] In some embodiments in which a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG2 Fc polypeptide sequence having the following amino acid sequence: [ka]

[0022] In some embodiments, where the fusion protein of the invention comprises an Fc polypeptide, The Fc polypeptide of the fusion protein comprises 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.

[0023] In some embodiments in which a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG3 Fc polypeptide sequence having the following amino acid sequence: [ka]

[0024] In some embodiments where a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises 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.

[0025] In some embodiments in which a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG4 Fc polypeptide sequence having the following amino acid sequence: [ka]

[0026] In some embodiments where a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises 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.

[0027] In some embodiments in which a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgM Fc polypeptide sequence having the following amino acid sequence: [ka]

[0028] In some embodiments where a fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises 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.

[0029] In some embodiments of the fusion proteins provided herein, the second polypeptide (polypeptide 2) of the serpin fusion protein is a cytokine target polypeptide or is derived from a cytokine target polypeptide. These embodiments are collectively referred to herein as "serpin-cytokine target polypeptide fusion proteins." The serpin-cytokine target polypeptide fusion proteins described herein contain at least a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide, and a cytokine target polypeptide, or derivatives thereof. In some embodiments, the serpin-cytokine target polypeptide fusion protein contains one serpin polypeptide. In other embodiments, the serpin-cytokine target polypeptide fusion protein contains two or more serpin polypeptides, and these embodiments are collectively referred to herein as "serpin-cytokine target polypeptide fusion proteins." (a’) In some embodiments, the serpin (a’) Each serpin polypeptide in the cytokine targeting polypeptide fusion protein comprises the same amino acid sequence. (a’) Each serpin polypeptide within the cytokine target polypeptide fusion protein includes a serpin polypeptide having a different amino acid sequence.

[0030] In some embodiments, the cytokine target polypeptide of the serpin-cytokine target polypeptide fusion protein is or is derived from a cytokine receptor. In preferred embodiments, the amino acid sequence derived from the cytokine target polypeptide or cytokine receptor is or is derived from a human cytokine receptor sequence. In other embodiments, the cytokine target polypeptide is an antibody or antibody fragment, such as an anti-cytokine antibody or an anti-cytokine antibody fragment. In preferred embodiments, the cytokine target polypeptide or the amino acid sequence derived from an antibody or antibody fragment is derived from a chimeric antibody sequence, a humanized antibody sequence, or a fully human antibody sequence. The term antibody fragment includes single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single domain heavy chain antibodies, and single domain light chain antibodies.

[0031] In other embodiments, the cytokine targeting polypeptide binds to a cytokine receptor and prevents binding of the cytokine to the receptor, hi other embodiments, the cytokine targeting polypeptide is an antibody or antibody fragment, e.g., an anti-cytokine receptor antibody or an anti-cytokine receptor antibody fragment.

[0032] In some embodiments, the serpin polypeptide of the serpin-cytokine target polypeptide fusion protein comprises at least the amino acid sequence of a reactive site loop portion of an AAT protein. In some embodiments, the reactive site loop portion of an AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the serpin-cytokine target fusion protein comprises at least the amino acid sequence of a variant of a reactive site loop portion of an AAT protein. In some embodiments, the variant of a reactive site loop portion of an AAT protein comprises 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-cytokine target fusion protein comprises at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2, or is derived from at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2. In some embodiments, the serpin polypeptide of the serpin-cytokine target fusion protein comprises 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 or 32 or 33.

[0033] In some embodiments, the serpin polypeptide of the serpin-cytokine target fusion protein is selected from the group consisting of those listed in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, The invention comprises an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide, which is or is derived from one or more of the human AAT polypeptide sequences set forth in NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0034] A serpin-cytokine targeting polypeptide fusion protein can incorporate a portion of a serpin-Fc fusion protein. For example, an antibody contains an Fc polypeptide. Thus, in some embodiments where the cytokine targeting polypeptide is a cytokine targeting antibody, the serpin-cytokine targeting polypeptide fusion protein incorporates a portion of a serpin-Fc fusion protein. Furthermore, most therapeutically useful receptor fusion proteins are Fc fusion proteins. Thus, in some embodiments where the serpin-cytokine targeting polypeptide fusion protein is a serpin-cytokine receptor fusion protein, the serpin-cytokine targeting polypeptide fusion protein can also incorporate an Fc polypeptide in addition to the serpin and cytokine receptor portions.

[0035] In some embodiments where the serpin-cytokine targeting polypeptide fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence comprises or is derived from the amino acid sequence of any one of SEQ ID NOs: 3, 4, 5, 6, or 7. In some embodiments where the serpin-cytokine targeting fusion protein comprises 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 sequences of SEQ ID NOs: 3, 4, 5, 6, or 7. In some embodiments, the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a linker region, e.g., a glycine-serine linker or a 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 target polypeptide are operably linked via a linker region and a hinge region, while in other embodiments, the serpin polypeptide and the cytokine target polypeptide are directly linked.

[0036] In some embodiments of the fusion proteins provided herein, the second polypeptide (polypeptide 2) of the serpin fusion protein is a whey acidic protein (WAP) domain-containing polypeptide, or an amino acid sequence derived from a WAP domain-containing polypeptide. These embodiments are collectively referred to herein as "serpin-WAP domain fusion proteins." A serpin-WAP domain fusion protein comprises at least a serpin polypeptide, or at least an amino acid sequence derived from a serpin, a WAP domain-containing polypeptide, or an amino acid sequence derived from a WAP domain-containing polypeptide. In some embodiments, a serpin-WAP domain fusion protein comprises one serpin polypeptide. In other embodiments, a serpin-WAP target polypeptide fusion protein comprises two or more serpin polypeptides. These embodiments are collectively referred to herein as "serpin (a’) In some embodiments, the serpin (a’) The serpin polypeptides of the -WAP domain fusion proteins comprise the same amino acid sequence. (a’) The serpin polypeptide of the cytokine target polypeptide fusion protein includes serpin polypeptides having different amino acid sequences.

[0037] These serpin-WAP domain fusion proteins contain a WAP domain-containing polypeptide or a 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 that includes eight cysteines found in a characteristic four-disulfide core arrangement (also called the four-disulfide core motif). The WAP domain sequence motif is a functional motif characterized by serine protease inhibitor activity in many proteins.

[0038] WAP domain-containing polypeptides suitable for use in the fusion proteins provided herein include, by way of non-limiting examples, secretory leukocyte protease inhibitor (SLPI), Elafin, and Eppin.

[0039] In some embodiments, the WAP domain-containing polypeptide sequence of the fusion protein comprises a secretory leukocyte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence derived from SLPI. These embodiments are referred to herein as "serpin-SLPI derived fusion proteins." In some embodiments, the SLPI polypeptide sequence comprises a portion of a SLPI protein, such as, for example, the WAP2 domain or a subportion thereof. In preferred embodiments, the SLPI polypeptide sequence or the amino acid sequence derived from SLPI is a human SLPI polypeptide sequence or is derived from a human SLPI polypeptide sequence.

[0040] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI or SLPI-derived sequence of the fusion protein comprises a full-length human SLPI polypeptide sequence having the following amino acid sequence: [ka]

[0041] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI or SLPI-derived sequence of the fusion protein comprises 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.

[0042] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI or SLPI-derived sequence of the fusion protein comprises a portion of a full-length human SLPI polypeptide sequence, where the portion has the following amino acid sequence: [ka]

[0043] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI or SLPI-derived sequence of the fusion protein comprises 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.

[0044] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI or SLPI-derived sequence of the fusion protein comprises the WAP2 domain of the full-length human SLPI polypeptide sequence, wherein the WAP2 domain has the following amino acid sequence: [ka]

[0045] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI or SLPI-derived sequence of the fusion protein comprises 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.

[0046] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI polypeptide sequence or an amino acid sequence derived from a SLPI polypeptide is or is derived from one or more of the human SLPI polypeptide sequences set forth 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.

[0047] In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI polypeptide sequence or the SLPI-derived sequence of the fusion protein comprises a human SLPI polypeptide sequence modified at a methionine (Met) residue. In these Met mutations, the Met residue can be replaced by any amino acid. For example, the Met residue can be replaced by an amino acid having a hydrophobic side chain, such as, for example, leucine (Leu, L) or valine (Val, V). Without wishing to be bound by any theory, the Met mutation(s) prevents oxidation of the fusion proteins of the invention and subsequent inactivation of inhibitory activity. In some embodiments, the Met mutation is present at position 98 of the SLPI polypeptide. For example, the modified SLPI polypeptide sequence of the serpin-SLPI comprises the mutation M98L or M98V in SEQ ID NO:8.

[0048] In other embodiments, the WAP domain-containing polypeptide sequence of the fusion protein comprises an elafin polypeptide sequence or an amino acid sequence derived from elafin. These embodiments are referred to herein as "serpin-elafin fusion proteins." In some embodiments, the elafin polypeptide sequence comprises a portion of an elafin protein, such as, for example, the WAP domain or a subportion thereof. In preferred embodiments, the elafin polypeptide sequence or an amino acid sequence derived from elafin is or is derived from a human elafin polypeptide sequence.

[0049] In some embodiments of the serpin-elafin fusion protein, the fusion protein comprises a full-length human elafin polypeptide sequence having the following amino acid sequence: [ka]

[0050] In some embodiments of the serpin-elafin fusion protein, the fusion protein comprises 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.

[0051] In some embodiments of a serpin-elafin fusion protein, the fusion protein comprises a portion of a full-length human elafin polypeptide sequence, where the portion has the following amino acid sequence: [ka]

[0052] In some embodiments of the serpin-elafin fusion protein, the fusion protein comprises 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.

[0053] In some embodiments of a serpin-elafin fusion protein, the fusion protein comprises the WAP domain of the full-length human elafin polypeptide sequence, where the WAP domain has the following amino acid sequence: [ka]

[0054] In some embodiments of the serpin-elafin fusion protein, the fusion protein comprises 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.

[0055] In some embodiments of the serpin-elafin fusion protein, the elafin polypeptide sequence or an amino acid sequence derived from an elafin polypeptide is derived from one or more of the human elafin polypeptide sequences set forth in GenBank Accession Nos. P19957.3, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, Q8IUB2.1, and / or NP_542181.1.

[0056] In other embodiments, the WAP domain-containing polypeptide sequence of the fusion protein comprises an Eppin polypeptide sequence or an amino acid sequence derived from Eppin. These embodiments are also referred to herein as "serpin (a’) Serpin-Eppin fusion proteins are referred to as "serpin-Eppin fusion proteins." In some embodiments, the Eppin polypeptide sequence of a serpin-Eppin fusion protein comprises a portion of an Eppin protein, such as, for example, the WAP domain or a small portion thereof. In preferred embodiments, the Eppin polypeptide sequence or the amino acid sequence derived from Eppin is a human Eppin polypeptide sequence or is derived from a human Eppin polypeptide sequence.

[0057] In some embodiments of the serpin-Eppin fusion protein, the Eppin polypeptide sequence or an amino acid sequence derived from an Eppin polypeptide is or is derived from one or more of the human Eppin polypeptide sequences set forth in GenBank Accession Nos. O95925.1, NP_065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and / or AAG00546.1.

[0058] In some embodiments, the serpin polypeptide of the serpin-WAP domain fusion protein comprises at least the amino acid sequence of a reactive site loop portion of an AAT protein. In some embodiments, the reactive site loop portion of an AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the serpin-WAP fusion protein comprises at least the amino acid sequence of a variant of a reactive site loop portion of an AAT protein. In some embodiments, the variant of a reactive site loop portion of an AAT protein comprises 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-WAP domain fusion protein comprises at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2. In some embodiments, the serpin polypeptide of the serpin-WAP domain fusion protein comprises 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, or 32 or 33.

[0059] In some embodiments, the serpin polypeptide of the serpin-WAP domain fusion protein is selected from the group consisting of serpin polypeptides having the following GenBank accession numbers: AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_0011211 78.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1, or an amino acid sequence derived from an AAT polypeptide derived from one or more of the human AAT polypeptide sequences set forth therein.

[0060] In some embodiments, a serpin-WAP domain fusion protein also includes an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. These embodiments are collectively referred to herein as "serpin-Fc-WAP domain fusion proteins." In these embodiments, no particular order should 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 of combinations thereof. The serpin-Fc-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence derived from a serpin, a WAP domain-containing polypeptide or an amino acid sequence derived from a WAP domain-containing polypeptide, and an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide.

[0061] In some embodiments, when a serpin-WAP domain fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence can have the amino acid sequence of SEQ ID NO: 3-7. In other embodiments, when a serpin-WAP domain fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence can be 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: 3-7. In some embodiments, a serpin-WAP domain fusion protein can also comprise an albumin polypeptide or an amino acid sequence derived from an albumin polypeptide. These embodiments are collectively referred to 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 any variation of serpin-albumin-WAP domain, serpin-WAP domain-albumin, or combinations thereof. The serpin-albumin-WAP domain fusion proteins described herein contain at least a serpin polypeptide or an amino acid sequence derived from a serpin, a WAP domain-containing polypeptide or an amino acid sequence derived from a WAP domain-containing polypeptide, and an albumin polypeptide or an amino acid sequence derived from an albumin polypeptide.

[0062] In some embodiments in which a serpin-WAP domain fusion protein comprises an albumin polypeptide sequence, the albumin polypeptide sequence comprises the amino acid sequence of SEQ ID NOs: 14-15 described herein. In other embodiments in which a serpin-WAP domain fusion protein comprises an albumin polypeptide sequence, 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 any one of the amino acid sequences having SEQ ID NOs: 14 or 15.

[0063] In some embodiments, the second polypeptide (polypeptide 2) of the serpin fusion protein is or is derived from an albumin polypeptide. These embodiments are collectively referred to herein as "serpin (a’) Serpin-albumin fusion proteins described herein contain at least a serpin polypeptide or an amino acid sequence derived from a serpin, and an albumin polypeptide or an amino acid sequence derived from an albumin polypeptide. Additionally, the present invention relates to serpin-albumin binding polypeptide fusion proteins, in which the albumin is operatively linked to a serpin via an intermediate linking molecule. As used herein, serpins are non-covalently or covalently bound to human serum albumin.

[0064] In embodiments in which a fusion protein of the invention comprises an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein is a human serum albumin (HSA) polypeptide or an amino acid sequence derived from HSA. In some embodiments, the fusion protein comprises an HSA polypeptide sequence having the following amino acid sequence: [ka]

[0065] In embodiments in which a fusion protein of the invention comprises an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein comprises 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.

[0066] In embodiments in which a fusion protein of the invention comprises an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein comprises domain 3 of a human serum albumin polypeptide sequence having the following amino acid sequence: [ka]

[0067] In embodiments in which a fusion protein of the invention comprises an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein comprises 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.

[0068] In some embodiments where the fusion protein of the invention comprises an albumin polypeptide sequence, the fusion protein is linked to human serum albumin via an intermediate albumin binding polypeptide. The albumin binding polypeptide may be an antibody or antibody fragment or may be derived from an antibody or antibody fragment. In a preferred embodiment, the albumin binding polypeptide or the amino acid sequence derived from an antibody or antibody fragment is derived from a chimeric antibody sequence, a humanized antibody sequence, or a fully human antibody sequence. The term antibody fragment includes single chains, Fab fragments, F(ab')2 fragments, scFvs, scAbs, dAbs, single domain heavy chain antibodies, and single domain light chain antibodies. In addition, the albumin binding polypeptide may be an albumin binding peptide. Another embodiment of the invention is a serpin-albumin binding polypeptide fusion, where the albumin binding polypeptide is domain 3 of the Streptococcal G protein or is a sequence derived from domain 3 of the Streptococcal G protein.

[0069] In some embodiments, the serpin (a’)The serpin polypeptide of the serpin-albumin fusion protein comprises at least the amino acid sequence of a reactive site loop portion of an AAT protein. In some embodiments, the reactive site loop portion of an AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the serpin-albumin fusion protein comprises at least the amino acid sequence of a variant of a reactive site loop portion of an AAT protein. In some embodiments, the variant of a reactive site loop portion of an AAT protein comprises 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-albumin fusion protein comprises at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2. In some embodiments, the serpin polypeptide of the serpin-albumin fusion protein comprises 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, or 32 or 33.

[0070] In some embodiments, the serpin polypeptide of the serpin-albumin fusion protein is selected from the group consisting of those listed in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121179.1, NP_001121179.2, NP_001121179.3, NP_001121179.4, NP_001121179.5, NP_001121179.6, NP_001121179.7, NP_001121179.8, NP_001121179.9 ... NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0071] In some embodiments, the fusion protein is modified to increase or otherwise inhibit proteolytic cleavage, for example, by mutating one or more proteolytic cleavage sites. In some embodiments, the fusion protein is modified to alter or otherwise modulate the Fc effector function of the fusion protein, while still retaining binding and inhibitory functions compared to the unmodified fusion protein. Fc effector functions include, by way of non-limiting example, Fc receptor binding, prevention of proinflammatory mediator release upon Fc receptor binding, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity (ADCC), modified complement-dependent cytotoxicity (CDC), and modified glycosylation at the Asn297 residue of the Fc polypeptide (EU index of Kabat numbering, Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some embodiments, the fusion protein is mutated or otherwise modified to affect Fc receptor binding. In some embodiments, the Fc polypeptide is modified to enhance FcRn binding. Examples of 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) (Kabat et al 1991 EU Index of Sequences of Proteins of Immunological Interest). In some embodiments, the Fc polypeptide portion is mutated or otherwise modified to prevent Fc-mediated dimerization (Ying et al 2012 J. Biol Chem 287(23): 19399-19408).In these embodiments, the fusion protein is monomeric in nature.

[0072] The fusion proteins and variants thereof provided herein provided inhibitory activity by inhibiting serine proteases, such as, for example, human neutrophil elastase (NE), a chemotrypsin-fold serine protease secreted by neutrophils during an inflammatory response. The fusion proteins provided herein completely or partially reduce or otherwise modulate serine protease expression or activity by binding to or otherwise interacting with a serine protease, such as a human serine protease. The reduction or modulation of the biological function of the serine protease is completely or partially due to the interaction of the fusion protein with a human serine protease protein, polypeptide, and / or peptide. A fusion protein is considered to completely inhibit the expression or activity of a serine protease when the level of serine protease expression or activity in the presence of the fusion protein is reduced by at least 95%, e.g., 96%, 97%, 98%, 99% or 100%, compared to the level of serine protease expression or activity in the absence of interaction, e.g., binding, with a fusion protein as described herein. A fusion protein is considered to partially inhibit the expression or activity of a serine protease when the level of serine protease expression or activity in the presence of the fusion protein is reduced by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%, compared to the level of serine protease expression or activity in the absence of interaction, e.g., binding, with a fusion protein as described herein.

[0073] The fusion proteins described herein are useful in a variety of therapeutic, diagnostic, and prophylactic indications, for example, the fusion proteins are useful for treating a variety of diseases and disorders in a subject. In some embodiments, serpin fusion proteins, including those fusion proteins described herein, are useful for treating, alleviating the symptoms of, ameliorating and / or slowing the progression of a disease or disorder in a subject suffering from or determined to be 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, lung cancer, ischemia-reperfusion injury, including ischemia / reperfusion injury following, for example, heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, Type I and / or Type II diabetes mellitus, bacterial infection, fungal infection, viral infection, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing, systemic lupus erythematosus, and multiple sclerosis.

[0074] Pharmaceutical compositions according to the invention include the fusion proteins of the invention, including modified fusion proteins and other variants, together with a suitable carrier, and may be included in a kit, such as, for example, a diagnostic kit. [Brief description of the drawings]

[0075] [Figure 1A] 1 is a schematic diagram of several embodiments of a serpin-Fc fusion protein according to the invention. The serpin can be located at any position within the fusion protein. Serpin-Fc fusion proteins incorporating two or more serpin polypeptides are also shown. [Figure 1B] FIG. 1 is a photograph of an SDS-PAGE gel showing serum-derived AAT (lane 1), AAT-Fc1 (lane 2, human IgG1 Fc), and AAT-EL-Fc1 (lane 3, Met351Glu, Met358Leu mutations in AAT, human IgG1 Fc). [Figure 1C] 1 is a graph showing inhibition of neutrophil elastase activity by AAT-Fc fusion protein. [Figure 1D] FIG. 1 is a photograph of an SDS-PAGE gel showing tetravalent AAT-Fc-AAT having two AAT polypeptides per Fc polypeptide. [Figure 1E] FIG. 1 is a graph showing inhibition of neutrophil elastase activity by tetravalent AAT-Fc-AAT fusion protein. [Figure 1F] FIG. 1 is a graph demonstrating the effect of low pH elution from Protein A resin, in which the NE inhibitory ability of AAT-Fc fusion protein eluted at low pH is significantly reduced. [Figure 1G] FIG. 13 is a graph showing that the double mutant AAT-EL-Fc (Met351Glu, Met358Leu mutation) is resistant to H2O2 inactivation (conc.) compared to wild-type AAT and the single mutant AAT-EM-Fc (Met351Glu). [Figure 1H] 1 is a graph showing the serum clearance rate of serum-derived AAT (sdAAT) compared to AAT-Fc in rats administered 10 mg / kg protein (3 rats / test protein). The half-life of AAT-Fc is substantially longer than that of sdAAT.

[0076] [Figure 2A] 1 is a schematic diagram of several embodiments of a serpin-cytokine target fusion protein of the invention. The serpin can be fused to an antibody heavy chain, a light chain, or both. A serpin-cytokine receptor fusion protein is also shown. [Figure 2B] Photograph of an SDS-PAGE gel showing D2E7 antibody (lane 1) and D2E7 antibody with -AAT fused to the heavy chain (lane 2). [Figure 2C] 1 is a graph showing inhibition of neutrophil elastase activity by D2E7 antibody fused to AAT. Serum-derived AAT is shown as a positive control, while D2E7 antibody alone is shown as a negative control for NE inhibition.

[0077] [Figure 3A]1 is a schematic diagram of several embodiments of a serpin-Fc-WAP fusion protein. [Figure 3B] FIG. 1 is a photograph of an SDS-PAGE gel showing AAT-Fc-ELAFIN (lane 1) and AAT-Fc-SLPI (lane 2). [Figure 3C] 1 is a graph showing inhibition of neutrophil elastase activity by AAT-Fc-ELAFIN and AAT-Fc-SLPI fusion proteins, with AAT-Fc fusion protein and serum-derived AAT included for comparison.

[0078] [Figure 4A] 1 is a schematic diagram of some embodiments of an AAT-HSA fusion protein. [Figure 4B] Photograph of an SDS-PAGE gel showing AAT-HSA fusions. [Figure 4C] FIG. 1 is a graph showing inhibition of neutrophil elastase activity by AAT-HSA compared to serum-derived AAT.

[0079] Human neutrophil elastase (NE) is a chymotrypsin-folding serine protease secreted by neutrophils during inflammation. Aberrant activity of NE leads to progressive degradation of elastin tissue and gradual destruction of the alveolar structure of the lung, leading to emphysema and pulmonary fibrosis (Lungarella et al 2008 Int. J. Biochem Cell Biol 40: 1287). In many cases, erroneous NE activity is due to an imbalance of the protease with its natural inhibitor, α1-antitrypsin (AAT). This imbalance can lead to high neutrophil infiltration into the lung, as observed in the lungs of smokers and patients suffering from cystic fibrosis (CF) or acute respiratory distress syndrome (ARDS). Conversely, deficiency of AAT as a result of a point mutation that normally causes ATT to aggregate and accumulate in the liver leaves the lungs exposed to unchecked NE activity. Individuals suffering from AAT deficiency are at increased risk for emphysema, COPD, liver disease, and a number of other medical conditions.

[0080] AAT deficiency affects approximately 100,000 Americans (according to estimates by the Alpha 1 Foundation), and many of those affected die in their 30s and 40s. Currently, there are only a few drugs approved by the FDA for the treatment of ATT deficiency (Prolastin®, Aralast™, Zemaira®, and Glassia™). Each drug is a natural AAT derived from pooled human plasma, which appears insufficient to meet the anticipated clinical demand. Furthermore, these products have a short serum half-life (a T of approximately 5 days). 1 / 2 ), necessitating weekly infusions at high doses (60 mg / kg body weight). The current market for these drugs is estimated to be approximately $400 million. Based on estimates that as many as 95% of individuals suffering from AAT deficiency remain undiagnosed, and the fact that these drugs have the potential to be effective treatments for conditions characterized by high NE activity in individuals who are not AAT deficient (e.g., cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), smoking-induced emphysema, and / or COPD), the market for AAT-like drugs should be substantially larger.

[0081] 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 282(12): 8573-8582, Nita et al 2007 Int J Biochem Cell Biol 39: 1 165 -1176). Recently, increasing evidence has suggested that AAT may be effective in treating many human pathologies in addition to the inflammatory pulmonary conditions for which it is commonly recommended. Human AAT has been shown to protect mice from clinical and histopathological signs of experimental autoimmune encephalomyelitis (EAE), suggesting it is a potential therapeutic candidate for autoimmune diseases such as multiple sclerosis or systemic lupus erythematosus (SLE) (Subramanian et al 201 1 Metab Brain Dis 26: 107-113). Serum AAT has shown activity in rodent models of graft-versus-host disease (GVHD) (Tawara et al 2011 Proc. Natl. Acad. Sci. USA 109: 564-569, Marcondes et al 2011 Blood Nov 3; 118(18):5031-9), which has led to human clinical trials using AAT to treat individuals with steroid-refractory acute GVHD (NCT01523821).Furthermore, AAT has been effective in animal models of type I and type II diabetes and has been shown to reduce inflammation, protect islet cells from apoptosis, and prolong the survival of islet cell allografts (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). There are currently a number of early human clinical trials for type I diabetes using serum-derived AAT products (NCT01183468, NCT01319331, NCT01304537).

[0082] Current serum-derived AAT products have undergone extensive purification and testing to ensure the removal of pathogenic viruses, however, the risk of transmission of infectious agents cannot be completely eliminated. Moreover, serum is limited, which limits the production capacity of serum-derived AAT. Attempts to address the concerns and production issues of serum-derived products have been directed toward the expression of recombinant AAT. However, even after 20 years of work, the creation of therapeutically viable recombinant AAT has not yet reached market (Karnaukhova et al 2006 Amino Acids 30: 317). Like serum-derived products, recombinant versions of AAT also suffer from short serum half-life, low production rate, and poor lung distribution.

[0083] The fusion proteins of the present invention have increased functionality compared to unmodified AAT molecules. The fusion of AAT polypeptide with a second polypeptide that interacts with neonatal Fc receptor (FcRn) serves to extend serum half-life and provide much needed medication benefits to patients. These FcRn-interacting polypeptides of the fusion proteins include immunoglobulin (Ig) Fc polypeptides derived from human IgG1, IgG2, IgG3, IgG4, or IgM, or derivatives of human albumin. In some embodiments, the fusion proteins incorporate AAT moieties with mutations that make the molecule more resistant to inactivation by oxidation. For example, Met351Glu, Met358Leu (AAT-EL-Fc) has demonstrated resistance to inactivation by H2O2 oxidation (Figure 1G). While AAT is a natural anti-inflammatory protein, some embodiments of the present invention provide increased inflammation-relieving capabilities by fusing AAT polypeptide with a cytokine targeting polypeptide. The coupling of dual anti-inflammatory functions from AAT and a second polypeptide provides a more potent therapeutic protein than either polypeptide on its own. Furthermore, coupling AAT with anti-infectious activity reduces the risk of infection in most cytokine target organisms. Some embodiments provide more potent anti-inflammatory and anti-infectious proteins by fusing the AAT polypeptide with a WAP domain-containing polypeptide. The fusion proteins of the present invention are expected to have great therapeutic utility and be superior to current serum-derived AAT products.

[0084] To extend the half-life of recombinant AAT, recombinant DNA technology was used to create the AAT gene fused to the Fc domain of human IgG1, IgG2, IgG3, IgG4, IgM, or HSA, such that the expected protein products would be AAT followed by the Fc domain (AAT-Fc(IgG1), AAT-Fc(IgG2), AAT-Fc(IgG3), AAT-Fc(IgG4), AAT-Fc(IgM)), or AAT followed by HSA. Although it was known that fusion of the Fc domain of HSA to some proteins, protein regions or peptides may extend their half-life (see, e.g., Jazayeri et al. BioDrugs 22, 11-26, Huang et al. (2009) Curr Opin Biotechnol 20, 692-699, Kontermann et al. (2009) BioDrugs 23, 93-109, Schmidt et al. (2009) Curr Opin Drug Discov Devel 12, 284-295), it was not known whether the Fc domain or HSA fused to AAT would allow proper folding and retention of NE inhibitory activity or extend the half-life of recombinant AAT. The fusion proteins of the invention have been shown to be potent inhibitors of NE, have extended serum half-lives, and in some embodiments are resistant to oxidation. In other embodiments, the fusion proteins described herein have distinct properties due to the incorporation of other functional polypeptides, including cytokine targeting polypeptides and WAP domain-containing polypeptides.

[0085] The fusion proteins described herein comprise at least a serpin polypeptide or an amino acid sequence derived from a serpin, and a second polypeptide. In some embodiments, for example, the invention provides a serpin polypeptide fused to human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or an HSA derivative. The serpin fusions described herein are expected to be useful in treating a variety of indications including, by way of non-limiting examples, alpha 1 antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, including ischemia / reperfusion injury following, for example, heart 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.

[0086] In some embodiments, the fusion proteins described herein comprise at least an alpha 1 antitrypsin (AAT) polypeptide or an amino acid sequence derived from AAT, and a second polypeptide. For example, the invention provides alpha 1 antitrypsin (AAT) fused to human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or an HSA derivative.

[0087] In some embodiments, the fusion proteins described herein comprise at least a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide, and a cytokine target polypeptide or an amino acid sequence derived from a cytokine target polypeptide. For example, the invention provides a serpin polypeptide or a sequence derived from a serpin polypeptide fused to a human cytokine receptor or a derivative thereof. Another embodiment of the invention provides a serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine target antibody, e.g., an anti-cytokine antibody, or a sequence derived from a cytokine target antibody, e.g., an anti-cytokine antibody, or a fragment of a cytokine target antibody, e.g., a sequence derived from a serpin polypeptide or a sequence derived from a serpin polypeptide fused to a fragment of an anti-cytokine antibody. For example, the invention provides a serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine target polypeptide, where the cytokine target polypeptide binds to any of the following human cytokines: TNFα, IgE, IL-12, IL-23, IL-6, IL-1α, IL-1β, IL-17, IL-13, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

[0088] For example, in some embodiments, the cytokine targeting polypeptide targets TNFα and includes any of the following TNFα targeting polypeptides or sequences derived from the following TNFα targeting polypeptides: Remicade®, Humira®, Simponi®, Cimiza®, Enbrel®, or ATN-103 and ATN-192.

[0089] For example, in some embodiments, the cytokine targeting polypeptide targets IgE and includes any of the following IgE targeting polypeptides or sequences derived from the following IgE targeting polypeptides: Xolair or FcεRI.

[0090] For example, in some embodiments, the cytokine targeting polypeptide targets the shared p40 subunit of IL-12 and IL-23 and comprises a Stelara® polypeptide or a sequence derived from a Stelara® polypeptide.

[0091] For example, the cytokine targeting polypeptide Stelara® targets IL-13 and contains a CDP7766 polypeptide or a sequence derived from a CDP7766 polypeptide.

[0092] In some embodiments, the fusion proteins described herein comprise at least an alpha 1 antitrypsin (AAT) polypeptide or an amino acid sequence derived from an AAT, and a cytokine target polypeptide or an amino acid sequence derived from a cytokine target polypeptide. For example, the present invention provides an alpha 1 antitrypsin inhibitor (AAT) fused to a cytokine target polypeptide, where the cytokine target polypeptide binds to any of the following human cytokines: TNFα, IgE, IL-6, IL-1α, IL-1β, IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

[0093] In some embodiments, the cytokine targeting polypeptide binds to a cytokine receptor and prevents cytokine binding. For example, the invention includes a serpin fused to a cytokine receptor targeting antibody. For example, the invention provides an alpha 1 antitrypsin inhibitor (AAT) fused to a cytokine targeting polypeptide, where the cytokine targeting polypeptide binds to a receptor for any of the following human cytokines: TNFα, IgE, IL-6, IL-1α, IL-1β, 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.

[0094] For example, in some embodiments, the cytokine targeting polypeptide targets the IL-6 receptor and comprises an Actemra® polypeptide (described in patent publication EP0628639) or an ALX-0061 polypeptide (described in WO2010 / 115998), or a sequence derived from an Actemra® polypeptide or an ALX-0061 polypeptide.

[0095] For example, the cytokine targeting polypeptide Actemra® targets the IL-6 receptor and contains a tocilizumab polypeptide or a sequence derived from a tocilizumab polypeptide.

[0096] Targeting inflammatory cytokines and immune stimulants with protein therapy has demonstrated clinical success in a number of inflammatory conditions. The most common proteins used for cytokine targeting agents are soluble cytokine receptors, as well as monoclonal antibodies and fragments thereof. A notable drawback of targeting cytokines is the increased risk of infection in these patients, as evidenced by the TNFα-targeting biologics, Remicade®, Humira®, Simponi®, Cimiza® and Enbrel®, and the IL-12 / 23p40-targeting antibody, Stelara®. This makes targeting inflammatory cytokines a common problem that leads to immunosuppression in patients. Interestingly, AAT and other serpin proteins have demonstrated both anti-infective and anti-inflammatory activity. This allows the serpin-cytokine targeting polypeptide fusion proteins of the present invention to suppress aberrant cytokine activity while mitigating the risk of infection.

[0097] In some embodiments, the fusion proteins described herein comprise a serpin polypeptide or an amino acid sequence derived from a serpin, a WAP domain-containing polypeptide or an amino acid sequence derived from a WAP domain-containing polypeptide, and an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. For example, the present invention provides a serpin polypeptide, a WAP domain-containing polypeptide, and a human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, or IgM-Fc derivative operatively linked together in any functional combination. In some embodiments, the WAP domain-containing protein is human SLPI or is derived from human SLPI. In other embodiments, the WAP domain-containing protein is human ELAFIN or is derived from human ELAFIN. In some embodiments, the fusion proteins described herein comprise at least an alpha 1 antitrypsin (AAT) polypeptide or an amino acid sequence derived from AAT, and an SLPI polypeptide or an amino acid sequence derived from SLPI. In some embodiments, the fusion proteins described herein comprise at least an AAT polypeptide or an amino acid sequence derived from AAT, and an ELAFIN polypeptide or an amino acid sequence derived from Elafin.

[0098] SPLI and Elafin are WAP domain-containing proteins that exhibit serine protease inhibitory activity. Both of these proteins are anti-inflammatory in function. In addition, these proteins have broad anti-infective potential against many strains of bacteria, viruses, and fungi.

[0099] In some embodiments, the fusion proteins described herein comprise at least a serpin polypeptide or an amino acid sequence derived from a serpin, and a human serum albumin (HSA) polypeptide or an amino acid sequence derived from an HSA polypeptide. Further embodiments of the invention include serpin-albumin binding polypeptide fusion proteins, in which the albumin binding polypeptide is involved in binding of the serpin to HSA. Thus, the invention includes both covalent and non-covalent associations of serpin polypeptides and HSA polypeptides, or sequences derived from serpin polypeptides or HSA polypeptides. For example, the invention provides serpin polypeptides fused to human HSA, HSA derivatives, or HSA binding peptides or polypeptides.

[0100] In some embodiments, the fusion proteins described herein comprise at least an alpha 1 antitrypsin (AAT) polypeptide or an amino acid sequence derived from an AAT, and an HSA polypeptide or an amino acid sequence derived from an HSA polypeptide. For example, the invention provides alpha 1 antitrypsin (AAT) fused to HSA, a fragment derived from HSA, or an albumin binding polypeptide.

[0101] In some embodiments, the fusion proteins described herein comprise: The fusion proteins described herein include a serpin polypeptide or an amino acid sequence derived from a serpin, an HSA polypeptide or an amino acid sequence derived from an HSA polypeptide, and a WAP domain-containing polypeptide or an amino acid sequence derived from a WAP domain-containing polypeptide. In some embodiments, the fusion proteins described herein include at least an alpha 1 antitrypsin (AAT) polypeptide or an amino acid sequence derived from an AAT, an HSA polypeptide or an amino acid sequence derived from an HSA polypeptide, and an SLPI polypeptide or an amino acid sequence derived from an SLPI. In other embodiments, the fusion proteins described herein include at least an alpha 1 antitrypsin (AAT) polypeptide or an amino acid sequence derived from an AAT, an HSA polypeptide or an amino acid sequence derived from an HSA polypeptide, and an Elafin polypeptide or an amino acid sequence derived from an Elafin.

[0102] The fusion proteins of the present invention can be readily produced in 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 be readily used for the expression of the serpin fusion proteins described herein. Importantly, mammalian cell expression systems generally yield proteins that are more optimal for therapeutic applications. In contrast to bacterial, insect, or yeast-based expression systems, mammalian cell expression systems yield proteins with glycosylation patterns similar or similar to those found in native human proteins. The native glycosylation of a protein greatly affects serum stability, pharmacokinetics, biodistribution, protein folding, and functionality. Therefore, the ability to create therapeutic proteins in mammalian expression systems has clear 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 functionality over native AAT and can be produced in mammalian expression systems for clinical and commercial use. Some embodiments of the invention include a purification system that allows for the isolation of serpin fusion proteins that maintain their ability to inhibit NE. Importantly, the purification process of the invention can be easily integrated into today's commercial mammalian cell-based manufacturing processes.

[0103] Unless otherwise specified, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms include plural entities and plural terms include singular entities. In general, the terms utilized in cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry, and hybridization described herein, and the techniques thereof, are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly practiced in the art, or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature utilized in, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0104] It will be appreciated that administration of therapeutic entities according to the present invention will be administered with suitable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transport, delivery, tolerance, and the like. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 therein by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous adsorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures that include carbowax. Any of the foregoing mixtures may be suitable in the treatment and therapy according to the present invention, provided that the active ingredients in the formulation are not inactivated by the formulation and are physiologically compatible and acceptable for the route of administration.See Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein for further information related to formulations, excipients, and carriers known to pharmacists.

[0105] The therapeutic formulations of the invention comprising the fusion proteins of the invention are used to treat or alleviate symptoms associated with a disease or disorder associated with abnormal serine protease activity in a subject. The invention also provides methods of treating or alleviating symptoms associated with a disease or disorder associated with abnormal serine protease activity in a subject. The 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 abnormal serine protease activity in the subject using standard methods, including any of a variety of clinical and / or experimental procedures. The term patient includes human and animal subjects. The term subject includes humans and other mammals.

[0106] Efficacy of treatment is determined in association with any known method for diagnosing or treating a disease or disorder associated with aberrant serine protease activity in a particular subject. Alleviation of one or more symptoms of a disease or disorder associated with aberrant serine protease activity in a subject indicates that the fusion protein provides a clinical benefit.

[0107] Methods for screening fusion proteins with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and immunologically-mediated techniques known in the art.

[0108] The fusion proteins described herein can be used in methods known in the art related to localization and / or quantification of targets such as serine proteases (e.g., for use in measuring the levels of those targets in an appropriate physiological sample, for use in diagnostic methods, for use in imaging the proteins, etc.). The terms "physiological sample" and "biological sample", as used interchangeably herein, are intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids that were present within a subject. Thus, included within the term "physiological sample" and "biological sample" are blood and fractions or components of blood, including serum, plasma, or lymph.

[0109] In certain embodiments, a fusion protein specific for a given target, or a derivative, fragment, analog or homolog thereof, including a target binding domain, is utilized as a pharmacologically active compound (hereinafter referred to as a "therapeutic agent").

[0110] The fusion proteins of the invention can be used to isolate specific targets using standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Detection can be facilitated by coupling (i.e., physically linking) the fusion protein to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 Examples include H.

[0111] The therapeutically effective amount of the fusion protein of the present invention generally relates to the amount required to achieve the therapeutic objective. As described above, this may be a binding interaction between the fusion protein and its target that in certain cases prevents the target from functioning. Furthermore, the amount required to be administered depends on the binding affinity of the fusion protein to its specific target, and the amount also depends on the rate at which the administered fusion protein is depleted from the free volume of the other target to which the antibody is administered. The general range for therapeutically effective dosing of the fusion protein of the present invention or fragments thereof may be, but is not limited to, about 0.1 mg / kg body weight to about 250 mg / kg body weight. The general dosing frequency may range, for example, from twice a day to once a month.

[0112] When fusion protein fragments are used, the smallest inhibitory fragment that specifically binds to the target is preferred. For example, peptide molecules that retain the ability to bind the target can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulations can also contain more than one active compound (preferably compounds with complementary activities that do not adversely affect each other) as necessary for the particular indication being treated. Alternatively, or in addition, the composition can contain agents that enhance its function (e.g., cytotoxic agents, cytokines, chemotherapeutic agents, growth inhibitors, anti-inflammatory agents, or anti-infective agents, etc.). Such molecules are stable in combination in amounts that are effective for the intended purpose.

[0113] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques, or interfacial polymerization, which are, for example, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, e.g., hydroxymethylcellulose- or gelatin-microcapsules, and poly-(methyl methacrylate) microcapsules, respectively).

[0114] The formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0115] 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 (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. Pharmaceutical Compositions

[0116] The fusion protein or soluble chimeric polypeptide of the present invention (also referred to herein as "active compound"), as well as its derivatives, fragments, analogs and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the fusion protein and a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically 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, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference textbook 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 solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles (e.g., fixed oils) can also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0117] The pharmaceutical composition of the present invention can be formulated to suit its intended route of administration. Examples of 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 contain the following components: a sterile diluent (e.g., water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solutions); antibacterial agents (e.g., benzyl alcohol or methylparaben); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetates, citrates, or phosphates) and agents for adjusting tonicity (e.g., sodium chloride or dextrose). pH can be adjusted using acids or bases (e.g., hydrochloric acid or sodium hydroxide). The parenteral preparation can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0118] Pharmaceutical 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 dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and the composition should be fluid so long as easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating agents (e.g., lecithin), by the maintenance of the required particle size (in the case of dispersions), and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferable to include isotonic agents in the composition (e.g., sugars, polyalcohols (e.g., mannitol), sorbitol, sodium chloride). Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption (e.g., aluminum monostearate and gelatin).

[0119] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of the above-listed components, and then sterilizing by filtration if necessary.Generally, dispersion is prepared by incorporating the above-listed active compound in a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed components.In the case of sterile powder for preparing sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces powder of active ingredient plus any other desired components from the solution that is previously sterilized by filtration.

[0120] 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 an excipient 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, in which 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 may contain any of the following ingredients, or compounds of a similar nature: a binder (e.g., microcrystalline cellulose, gum tragacanth or gelatin); a filler (e.g., starch or lactose); a disintegrant (e.g., alginic acid, Primogel, or corn starch); a lubricant (e.g., magnesium stearate or Sterotes; a glidant (e.g., colloidal silicon dioxide); a sweetening agent (e.g., sucrose or saccharin); or a flavoring agent (e.g., peppermint, methyl salicylate, or orange flavoring).

[0121] For administration by inhalation, 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.

[0122] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such 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 achieved by the use of nasal sprays or suppositories. For transdermal administration, the active compound is generally formulated into ointments, salves, gels, or creams, as are known in the art.

[0123] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0124] In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body (e.g., controlled release formulations, including implants and microencapsulated delivery systems). Biodegradable, biocompatible polymers can be used. Methods for the preparation of such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells with monoclonal antibodies against viral antigens, can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0125] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.As used herein, dosage unit form refers to a physically separate unit suitable as a uniform dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by the unique characteristics of said active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the field of formulating such active compound for individual treatment.

[0126] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0127] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0128] Example 1: AAT-Fc fusion proteins and variants Representative, but non-limiting, examples of AAT-Fc fusion proteins according to the present invention include the following sequences: Although these examples include hinge and / or linker sequences, the fusion proteins of the present invention can be made using any hinge and / or linker sequence that is suitable in terms of length and / or flexibility. Alternatively, the fusion proteins can be made without hinge and / or linker sequences. For example, the polypeptide components can be directly linked.

[0129] An exemplary AAT-Fc fusion protein is AAT-hFc1 (human IgG1 Fc) described herein, as shown below, with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2) and the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:3). AAT-hFc1 (human IgG1 Fc) [ka]

[0130] An exemplary AAT-Fc fusion protein is AAT-hFc2 (human IgG2 Fc) described herein, as shown below, with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2) and the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:4). AAT-hFc2 (human IgG2 Fc) [ka]

[0131] An exemplary AAT-Fc fusion protein is AAT-MM-EL-hFc1 (human IgG1 Fc, Met351Glu / Met358Leu) described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:34), the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:3), the Met351Glu mutation boxed, and the Met358Leu mutation shaded in gray. AAT-MM-EL-hFc1 (human IgG1 Fc, Met351Glu / Met358Leu) [ka]

[0132] An exemplary AAT-Fc fusion protein is AAT-MM-EL-hFc2 (human IgG2 Fc, Met351Glu / Met358Leu) described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:34), the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:4), the Met351Glu mutation boxed, and the Met358Leu mutation shaded in gray. AAT-MM-EL-hFc2 (human IgG2 Fc, Met351Glu / Met358Leu) [ka]

[0133] An exemplary AAT-Fc fusion protein is AAT-MM-LL-hFc1 (human IgG1 Fc, Met351Leu / Met358Leu) described herein, as shown below, with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:35), the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:3), the Met351Leu mutation shaded in black, and the Met358Leu mutation shaded in grey. AAT-MM-LL-hFc1 (human IgG1 Fc, Met351Leu / Met358Leu) [ka]

[0134] An exemplary AAT-Fc fusion protein is AAT-MM:LL-hFc2 (human IgG2 Fc, Met351Leu / Met358Leu) described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:35), the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:4), the Met351Leu mutation shaded in black, and the Met358Leu mutation shaded in grey. AAT-MM:LL-hFc2 (human IgG2 Fc, Met351Leu / Met358Leu) [ka]

[0135] An exemplary AAT-Fc fusion protein is AAT-hFc1-AAT (human IgG1) described herein, as shown below, with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2) and the IgG-Fc polypeptide portion of the fusion protein in italics (SEQ ID NO:3). AAT-hFc1-AAT (human IgG1) [ka]

[0136] These representative AAT-Fc fusion proteins were generated using the following techniques.

[0137] The gene encoding human AAT was PCR amplified from human liver cDNA (Zyagen). Specific point mutations in the gene encoding AAT or the Fc region were created by overlap PCR. The AAT-encoding gene was cloned in frame with the gene encoding the hinge region, followed by the CH2 domain and the CH3 domain of human IgG1, IgG2, IgG3, IgG4, or IgM into a mammalian expression vector containing a mammalian secretion signal sequence upstream of the AAT gene insertion site. These expression vectors were transfected into mammalian cells (specifically, HEK293 or CHO cells) and cultured at 37° C. in 8% CO2 for several days. The recombinant AAT-Fc fusion protein was purified from the expressing cell supernatant by Protein A chromatography. Importantly, a buffer with a near-neutral pH was used to elute the AAT-Fc fusion protein from the Protein A resin (Gentle Ag / Ab Elution Buffer, Thermo Scientific). AAT-Fc fusion protein cannot be eluted from Protein A resin using standard low pH elution because the ability of AAT to inhibit NE is reduced after low pH treatment, likely due to low pH mediated oligomerization of AAT. Figure 1F shows the effect of low pH elution on the ability of AAT to inhibit neutrophil elastase. AAT-Fc fusion protein is purified by Protein A and near neutral pH elution buffer or by CaptureSelect® Alpha-1 Antitrypsin affinity matrix (BAC BV).

[0138] The purified AAT-Fc fusion proteins were tested for activity by measuring their ability to inhibit neutrophil elastase (NE). Figures 1B and 1D show reducing SDS-PAGE gels of purified serum-derived AAT (sdAAT) and AAT-Fc fusion proteins (Figure 1B-lane 1: sdAAT, lane 2: AAT-Fc (SEQ ID NO: 16), lane 3: AAT-EL-Fc (SEQ ID NO: 18), Figure 1D AAT-Fc-AAT (SEQ ID NO: 20). Proteins were visualized by staining with Coomassie blue.

[0139] A fluorescent microplate assay was used to monitor human neutrophil elastase (NE) activity. Inhibitory activity was assessed by the concomitant reduction of residual NE activity using the following assay. The assay buffer consisted of 100 mM Tris pH 7.4, 500 mM NaCl, and 0.0005% Triton X-100. Human NE was used at a final concentration of 5 nM (but 1-20 nM can also be used). The fluorescent peptide substrate AAVP-AMC was used in the assay at a final concentration of 100 μM. The assay kinetics were read using a Gemini EM plate reader from Molecular Devices, using excitation and emission wavelengths of 370 nm and 440 nm, respectively, and a cutoff of 420 nm. The assay was read for 10 minutes at room temperature and scanned every 5-10 seconds. The Vmax per second represented the residual NE activity, which was plotted against each concentration of inhibitor. The intercept with the x-axis indicates the concentration of inhibitor required to completely 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 protein exhibits potent NE inhibitory activity, as shown in FIG. 1C. The fusion of two AAT polypeptides in one Fc polypeptide (AAT-Fc-AAT) shows higher potency than both sdAAT and AAT-Fc fusion proteins containing one AAT polypeptide (FIG. 1E). These findings presented here demonstrate for the first time that AAT can be fused to an Fc region and retain its ability to inhibit NE. Of particular interest, the AAT-Fc-AAT fusion protein was found to be a more potent NE inhibitor.

[0140] Figure 1F demonstrates the resistance of the AAT-EL-Fc(M351E, M358L) fusion protein to inactivation by oxidation. The AAT fusion proteins, AAT-Fc(wt), AAT-EL-Fc(M351E, M358L), and AAT-EM-Fc(M351E), were treated with 33 mM H2O2 and compared to untreated fusion protein in an NE inhibition assay. The inhibition of NE by AAT-EL-Fc was not constituted by oxidation and was the opposite of the other proteins tested.

[0141] Furthermore, compared to serum-derived AAT, the AAT-Fc fusion protein exhibited a longer serum half-life in rats (Figure 1H). In these studies, three rats per each test protein were injected IV with 10 mg / kg sdAAT or AAT-Fc. Serum samples were collected at various time points over a 48-hour period. ELISA was used for serum ATT concentrations. These findings demonstrate that the fusion proteins of the invention have improved pharmacokinetic properties and are a superior therapeutic format over serum-derived AAT for treating many human inflammatory conditions and infections. Example 2: AAT-TNFα targeting molecule fusion protein

[0142] The studies presented herein provide several non-limiting examples of recombinant AAT derivatives, including human AAT fused to anti-TNFα antibodies or derivatives of the TNFα receptor. These examples are provided below to further illustrate various features of the invention. The examples also illustrate methodologies useful for carrying out the invention. These examples do not, and are not intended to, limit the invention described in the claims.

[0143] The following fusion proteins contain a cytokine targeting polypeptide sequence from or derived from (i) the anti-TNFα antibody D2E7 (also known as Adalimumab or Humira®) or (ii) the extracellular domain of the TNFα receptor type 2 (TNFR2-ECD). The AAT polypeptide portion of the fusion protein is underlined, the antibody constant region (CH1-hinge-CH2-CH3, or CL) is italicized, and D2E7-VH, D2E7-VK, and TNFR2-ECD are bolded. While these examples include hinge and / or linker sequences, the fusion proteins of the invention can be made using any hinge and / or linker sequence of suitable length and / or flexibility. Alternatively, the fusion proteins can be made without the use of hinge and / or linker sequences.

[0144] An exemplary AAT-TNFα fusion protein is the D2E7-light chain-AAT(G3S)2 linker described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2), the D2E7-VK in bold (SEQ ID NO:37), and the antibody constant region in italics (SEQ ID NO:38). D2E7-light chain-AAT(G3S)2 linker [ka]

[0145] An exemplary AAT-TNFα fusion protein is the D2E7-light chain-AAT ASTGS linker described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2), the D2E7-VK in bold (SEQ ID NO:37), and the antibody constant region in italics (SEQ ID NO:38). D2E7-light chain-AAT ASTGS linker [ka]

[0146] An exemplary AAT-TNFα fusion protein is the D2E7-heavy chain-AAT(G3S)2 linker described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2), the D2E7-VH in bold (SEQ ID NO:39), and the antibody constant region in italics (SEQ ID NO:40). D2E7-heavy chain-AAT(G3S)2 linker [ka]

[0147] An exemplary AAT-TNFα fusion protein is the D2E7-heavy chain--AAT ASTGS linker described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2), the D2E7-VH in bold (SEQ ID NO:39), and the antibody constant region in italics (SEQ ID NO:40). D2E7-heavy chain-AAT ASTGS linker [ka]

[0148] An exemplary AAT-TNFα fusion protein is the TNFR2-ECD-Fc1-AAT(G3S)2 linker described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2), the TNFR2-ECD in bold (SEQ ID NO:41), and the antibody constant region in italics (SEQ ID NO:42). TNFR2-ECD-Fc1-AAT(G3S)2 linker [ka]

[0149] An exemplary AAT-TNFα fusion protein is the TNFR2-ECD-Fc1-AAT ASTGS linker described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2), the TNFR2-ECD in bold (SEQ ID NO:41), and the antibody constant region in italics (SEQ ID NO:42). TNFR2-ECD-Fc1-AAT ASTGS linker [ka]

[0150] These representative AAT-TNFα target molecule fusion proteins were generated using the following techniques.

[0151] Genes encoding the variable heavy (VH) and variable kappa (VK) regions of an anti-TNFα antibody (D2E7) were generated by gene synthesis. The D2E7-VH gene was cloned in frame with a gene encoding a human IgG1 antibody heavy chain constant region consisting of the CH1, hinge, CH2, and CH3 domains in a mammalian expression vector containing a mammalian secretory signal sequence upstream 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 in a mammalian expression vector containing a mammalian secretory signal sequence upstream of the VK domain insertion site (D2E7-LC). The AAT coding gene and adjacent 5' linker sequence were cloned in frame at the 3' end of the coding sequence for 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) in the mammalian expression vector described above. The extracellular domain of TNFα receptor 2 (TNFR2-ECD) was produced by gene synthesis and cloned in frame with a gene encoding a hinge region into a mammalian expression vector containing a mammalian secretory signal sequence upstream of the TNFR2-ECD insertion site, followed by the CH2 and CH3 domains of human IgG1 (hFc1). The AAT coding gene and adjacent 5' linker sequence were cloned in frame at the 3' end of the gene encoding TNFR2-ECD-hFc1 into a mammalian expression vector (TNFR2-ECD-hFc1-AAT).

[0152] The D2E7-HC-AAT expression vector was co-transfected into mammalian cells (specifically, HEK293 or CHO cells) with either the D2E7-LC or D2E7-LC-AAT expression vector to generate D2E7 antibodies with AAT fused to the C-terminus of the heavy chain or to the C-terminus of both the heavy and light chains, respectively. The D2E7-LC-AAT was co-transfected into mammalian cells with the D2E7-HC expression vector to generate D2E7 antibodies with AAT fused to the C-terminus of the light chain. The TNFR2-hFc1-AAT expression vector was transfected into mammalian cells. The transfected cells were cultured at 37° C. in 8% CO2 for several days.

[0153] The recombinant AAT-TNFα target fusion protein was purified from the expressing cell supernatant by Protein A chromatography. A near-neutral pH buffer was used to elute the AAT-TNFα target fusion protein from the Protein A resin (Gentle Ag / Ab Elution Buffer, Thermo Scientific).

[0154] Figure 2B shows an SDS-PAGE gel of the D2E7 antibody alone (lane 1) and a mutant in which AAT is fused to the heavy chain of D2E7 (lane 2). Proteins were visualized by staining with Coomassie blue.

[0155] Purified AAT-TNFα target molecule fusion proteins were tested for activity by measuring their ability to inhibit neutrophil elastase. In these assays, human serum-derived AAT (sdAAT) was used as a positive control. NE inhibition assays were performed as described previously. Figure 2C demonstrates that AAT-TNFα target molecule fusion proteins exhibit similar neutrophil elastase inhibition compared to sdAAT, suggesting that the inhibitory ability of AAT is not compromised by its fusion with an antibody. Example 3: AAT-Fc-SLPI and AAT-Fc-Elafin

[0156] The studies presented herein describe several non-limiting examples of recombinant AAT derivatives, including human AAT fused with a WAP domain-containing protein. These examples are provided below to further illustrate various features of the present invention. The examples also illustrate methodologies useful for carrying out the present 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 hinge and / or linker sequences, the fusion proteins of the present invention can be made using any hinge and / or linker sequence of suitable length and / or flexibility. Alternatively, the fusion proteins can be made without hinge and / or linker sequences. For example, the polypeptide components can be directly linked.

[0157] An exemplary AAT-Fc-SLPI fusion protein is AAT-hFc1SLPI (human IgG1 Fc) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO:2), the Fc portion is in italics (SEQ ID NO:3), and the WAP domain-containing polypeptide is in bold font (SEQ ID NO:9). AAT-hFc1-SLPI (human IgG1 Fc) [ka]

[0158] An exemplary AAT-Fc-SLPI fusion protein is AAT-hFc1SLPI (human IgG1 Fc) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO:2), the Fc portion is in italics (SEQ ID NO:3), and the WAP domain-containing polypeptide is in bold font (SEQ ID NO:12). AAT-hFc1-elafin (human IgG1 Fc) [ka]

[0159] Genes encoding SLPI and Elafin were PCR amplified from human spleen cDNA (Zyagen). These genes were cloned into the mammalian expression vectors of Example 1, in which the SLPI or Elafin genes were inserted in frame with the AAT-Fc gene. These expression vectors were transfected into mammalian cells (specifically, HEK293 or CHO cells) and cultured at 37°C in 8% CO2 for several days. The recombinant AAT-Fc-WAP domain fusion protein was purified from the expressing cell supernatant by Protein A chromatography. A buffer with near-neutral pH was used to elute the AAT-Fc-WAP domain fusion protein from the Protein A resin (Gentle Ag / Ab Elution Buffer, Thermo Scientific).

[0160] FIG. 3B shows an SDS-PAGE gel of AAT-Fc-WAP fusion proteins (lane 1: AAT-Fc-Elafin, lane 2: AAT-Fc-SLPI). Proteins were visualized by staining with Coomassie blue. Purified AAT-Fc-WAP domain fusion proteins were tested for activity by measuring their ability to inhibit neutrophil elastase. NE inhibition assays were performed as previously described. In these assays, human serum-derived AAT (sdAAT) and AAT-Fc fusion protein were used as positive controls. Compared to sdAAT, AAT-Fc-WAP target molecule fusion protein showed a high ability of NE inhibition of neutrophil elastase (FIG. 3C). Example 4: AAT-Albumin

[0161] The studies presented herein describe several non-limiting examples of recombinant AAT derivatives, including human AAT fused to an albumin polypeptide. These examples are provided below to further illustrate various features of the invention. The examples also illustrate methodologies useful for practicing the invention. These examples do not, and are not intended to, limit the invention as claimed. The AAT moiety is underlined and the albumin moiety is italicized. For example, the polypeptide components can be directly conjugated.

[0162] An exemplary AAT albumin fusion protein is AAT-HSA, described herein, as shown below with the AAT polypeptide portion of the fusion protein underlined (SEQ ID NO:2) and the albumin polypeptide in italics (SEQ ID NO:14). AAT-HSA [ka]

[0163] An exemplary AAT albumin fusion protein is the 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). AAT-HSA domain 3 [ka]

[0164] The gene encoding human serum albumin (HSA) was PCR amplified from human liver cDNA (Zyagen). Mammalian expression vectors were generated in which the gene encoding HSA or domain 3 of HSA was cloned in frame at the 3' end of the AAT-encoding gene, containing a mammalian secretory signal sequence upstream of AAT.

[0165] These expression vectors were transfected into mammalian cells (specifically, HEK293 or CHO cells) and cultured for several days at 37° C. in 8% CO2. Recombinant AAT-HSA fusion protein was purified from the expression cell supernatant using CaptureSelect® Alpha-1 Antitrypsin Affinity Matrix (BAC BV) with a binding buffer consisting of 20 mM Tris, 150 mM NaCl, pH 7.4, and an elution buffer consisting of 20 mM Tris, 2 M MgCl2, pH 7.4.

[0166] Figure 4B shows the SDS-PAGE gel of AAT-HSA fusion protein. Proteins were visualized by staining with Coomassie blue. Purified AAT-HSA fusion proteins were tested for activity by measuring their ability to inhibit neutrophil elastase. NE inhibition assays were performed as described above. In these assays, AAT derived from human serum (sdAAT) was used as a positive control. Compared to sdAAT, AAT-HS fusion protein showed similar NE inhibition ability, demonstrating that fusion to albumin does not suppress the ability of AAT to inhibit NE (Figure 4C). Other embodiments

[0167] While the present invention has been described with reference to its detailed description, the above description is for purposes of illustration and not of limitation of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A dimer comprising a fusion protein, the fusion protein comprises an alpha-1 antitrypsin (AAT) polypeptide operably linked to a modified immunoglobulin G (IgG) Fc polypeptide; The AAT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32; the modified IgG Fc polypeptide is derived from a human IgG polypeptide and comprises one or more mutations; At least one of the mutations comprises a mutation at one of the following positions according to the Kabat numbering system: Met252, Ser254, Thr256, Met428, or Asn434; Dimer.

2. A dimer comprising a fusion protein, the fusion protein comprises an alpha-1 antitrypsin (AAT) polypeptide operably linked to a modified immunoglobulin G (IgG) Fc polypeptide; The AAT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32; the modified IgG Fc polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO:6; Dimer.

3. The dimer of claim 1 or 2, wherein the AAT polypeptide comprises the amino acid sequence set forth as SEQ ID NO:

34.

4. The dimer according to claim 1 or 2, wherein the modified IgG Fc polypeptide is derived from a human IgG4 Fc polypeptide.

5. 3. The dimer of claim 1 or 2, wherein the modified IgG Fc polypeptide comprises at least one of the following mutations: Met252Tyr, Ser254Thr, Thr256Glu, Met428Leu, or Asn434Ser.

6. 3. The dimer of claim 1 or 2, wherein the AAT polypeptide is operably linked to the modified IgG Fc polypeptide via a hinge region, a linker region, or both a hinge region and a linker region.

7. 3. The dimer of claim 1 or 2, wherein the AAT polypeptide is operably linked to the modified IgG Fc polypeptide via a linker region and a hinge region.

8. 8. The dimer of claim 7, wherein the AAT polypeptide is operably linked to the N-terminus of the modified IgG Fc polypeptide via a linker region and a hinge region.

9. The dimer of claim 6 , wherein the linker region comprises a peptide sequence.

10. The dimer of claim 6 , wherein the linker region comprises a glycine-serine linker.

11. The dimer of claim 6 , wherein the hinge region comprises a peptide sequence.

12. A fusion protein comprising an alpha-1 antitrypsin (AAT) polypeptide comprising the amino acid sequence of SEQ ID NO:

34.

13. The fusion protein of claim 12 , wherein the AAT polypeptide is operably linked to a second polypeptide.

14. The fusion protein of claim 13, wherein the second polypeptide is a modified IgG Fc polypeptide.

15. The fusion protein of claim 14 , wherein the modified IgG Fc polypeptide is derived from a human IgG Fc polypeptide.

16. The fusion protein of claim 14 or 15, wherein the modified IgG Fc polypeptide is derived from a human IgG4 Fc polypeptide.

17. The fusion protein of claim 14 or 15, wherein the modified IgG Fc polypeptide is a mutated IgG Fc polypeptide.

18. The fusion protein of claim 14 or 15, wherein the modified IgG Fc polypeptide comprises at least one of the following mutations: Met252Tyr, Ser254Thr, Thr256Glu, Met428Leu, or Asn434Ser.

19. The fusion protein of any one of claims 13 to 15, wherein the AAT polypeptide is operably linked to the second polypeptide via a hinge region, a linker region, or both a hinge region and a linker region.

20. The fusion protein of any one of claims 13 to 15, wherein the AAT polypeptide is operably linked to the second polypeptide via a hinge region and a linker region.

21. 21. The fusion protein of claim 20, wherein the AAT polypeptide is operably linked to the N-terminus of the second polypeptide via a linker or hinge region.

22. The fusion protein of claim 19 , wherein the linker region comprises a peptide sequence.

23. The fusion protein of claim 19, wherein the linker region comprises a glycine-serine linker.

24. The fusion protein of claim 19 , wherein the hinge region comprises a peptide sequence.

25. The fusion protein according to any one of claims 12 to 15, wherein the fusion protein is a dimeric fusion protein.

26. 16. A pharmaceutical composition for inhibiting or downregulating aberrant serine protease expression or activity in a subject in need thereof, comprising a dimer according to any one of claims 1 or 2, or a fusion protein according to any one of claims 12 to 15.

27. 27. The pharmaceutical composition of claim 26, wherein the subject is a human.

28. 28. The pharmaceutical composition of claim 27, wherein the subject has 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, lung cancer, ischemia-reperfusion injury, ischemia / reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes mellitus, pneumonia, sepsis, graft-versus-host disease (GVHD), wound healing, systemic lupus erythematosus, and multiple sclerosis.

29. 28. The pharmaceutical composition of claim 27, wherein the subject has alpha-1 antitrypsin (AAT) deficiency.

30. 28. The pharmaceutical composition of claim 27, wherein the subject has an infection selected from the following: a bacterial infection, a fungal infection, or a viral infection.

31. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein, the fusion protein comprises an alpha-1 antitrypsin (AAT) polypeptide operably linked to a modified immunoglobulin G (IgG) Fc polypeptide; The AAT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32; the modified IgG Fc polypeptide is derived from a human IgG polypeptide and comprises one or more mutations; At least one of the mutations comprises a mutation at one of the following positions according to the Kabat numbering system: Met252, Ser254, Thr256, Met428, or Asn434; Nucleic acid molecule.

32. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein, the fusion protein comprises an alpha-1 antitrypsin (AAT) polypeptide operably linked to a modified immunoglobulin G (IgG) Fc polypeptide; The AAT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32; the modified IgG Fc polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO:6; Nucleic acid molecule.

33. 33. The nucleic acid molecule of claim 31 or 32, wherein the AAT polypeptide comprises the amino acid sequence set forth as SEQ ID NO:

34.

34. The nucleic acid molecule of claim 31 or 32, wherein the modified IgG Fc polypeptide is derived from a human IgG4 Fc polypeptide.

35. 33. The nucleic acid molecule of claim 31 or 32, wherein the modified IgG Fc polypeptide comprises at least one of the following mutations: Met252Tyr, Ser254Thr, Thr256Glu, Met428Leu, or Asn434Ser;

36. 33. The nucleic acid molecule of claim 31 or 32, wherein the AAT polypeptide is operably linked to the modified IgG Fc polypeptide via a hinge region, a linker region, or both a hinge region and a linker region.

37. 33. The nucleic acid molecule of claim 31 or 32, wherein the AAT polypeptide is operably linked to the modified IgG Fc polypeptide via a linker region and a hinge region.

38. 38. The nucleic acid molecule of claim 37, wherein the AAT polypeptide is operably linked to the N-terminus of the modified IgG Fc polypeptide via a linker region and a hinge region.

39. The nucleic acid molecule of claim 36 , wherein the linker region comprises a peptide sequence.

40. The nucleic acid molecule of claim 36, wherein the linker region comprises a glycine-serine linker.

41. 37. The nucleic acid molecule of claim 36, wherein the hinge region comprises a peptide sequence.

42. 33. A recombinant expression vector comprising the nucleic acid molecule of claim 31 or 32.

43. 43. A mammalian host cell transformed with the recombinant expression vector of claim 42.

44. 1. A method for producing a fusion protein, comprising: Providing a mammalian cell according to claim 43; and maintaining said cells under conditions in which said fusion protein is expressed; A method comprising:

45. 45. The method of claim 44, further comprising purifying the fusion protein.

Images